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Sample records for cyclic amp regulation

  1. Down-regulation of Cell Surface Cyclic AMP Receptors and Desensitization of Cyclic AMP-stimulated Adenylate Cyclase by Cyclic AMP in Dictyostelium discoideum. Kinetics and Concentration Dependence

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1987-01-01

    cAMP binds to Dictyostelium discoideum surface receptors and induces a transient activation of adenylate cyclase, which is followed by desensitization. cAMP also induces a loss of detectable surface receptors (down-regulation). Cells were incubated with constant cAMP concentrations, washed free of

  2. Discovery of a cAMP Deaminase That Quenches Cyclic AMP-Dependent Regulation

    Science.gov (United States)

    Goble, Alissa M.; Feng, Youjun; Raushel, Frank M.; Cronan, John E.

    2013-01-01

    An enzyme of unknown function within the amidohydrolase superfamily was discovered to catalyze the hydrolysis of the universal second messenger, cyclic-3’, 5’-adenosine monophosphate (cAMP). The enzyme, which we have named CadD, is encoded by the human pathogenic bacterium Leptospira interrogans. Although CadD is annotated as an adenosine deaminase, the protein specifically deaminates cAMP to cyclic-3’, 5’-inosine monophosphate (cIMP) with a kcat/Km of 2.7 ± 0.4 × 105 M−1 s−1 and has no activity on adenosine, adenine, or 5’-adenosine monophosphate (AMP). This is the first identification of a deaminase specific for cAMP. Expression of CadD in Escherichia coli mimics the loss of adenylate cyclase in that it blocks growth on carbon sources that require the cAMP-CRP transcriptional activator complex for expression of the cognate genes. The cIMP reaction product cannot replace cAMP as the ligand for CRP binding to DNA in vitro and cIMP is a very poor competitor of cAMP activation of CRP for DNA binding. Transcriptional analyses indicate that CadD expression represses expression of several cAMP-CRP dependent genes. CadD adds a new activity to the cAMP metabolic network and may be a useful tool in intracellular study of cAMP-dependent processes. PMID:24074367

  3. Cyclic di-AMP regulation of osmotic homeostasis is essential in Group B Streptococcus.

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    Laura Devaux

    2018-04-01

    Full Text Available Cyclic nucleotides are universally used as secondary messengers to control cellular physiology. Among these signalling molecules, cyclic di-adenosine monophosphate (c-di-AMP is a specific bacterial second messenger recognized by host cells during infections and its synthesis is assumed to be necessary for bacterial growth by controlling a conserved and essential cellular function. In this study, we sought to identify the main c-di-AMP dependent pathway in Streptococcus agalactiae, the etiological agent of neonatal septicaemia and meningitis. By conditionally inactivating dacA, the only diadenyate cyclase gene, we confirm that c-di-AMP synthesis is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable due to the accumulation of compensatory mutations. We identified several mutations restoring the viability of a ΔdacA mutant, in particular a loss-of-function mutation in the osmoprotectant transporter BusAB. Identification of c-di-AMP binding proteins revealed a conserved set of potassium and osmolyte transporters, as well as the BusR transcriptional factor. We showed that BusR negatively regulates busAB transcription by direct binding to the busAB promoter. Loss of BusR repression leads to a toxic busAB expression in absence of c-di-AMP if osmoprotectants, such as glycine betaine, are present in the medium. In contrast, deletion of the gdpP c-di-AMP phosphodiesterase leads to hyperosmotic susceptibility, a phenotype dependent on a functional BusR. Taken together, we demonstrate that c-di-AMP is essential for osmotic homeostasis and that the predominant mechanism is dependent on the c-di-AMP binding transcriptional factor BusR. The regulation of osmotic homeostasis is likely the conserved and essential function of c-di-AMP, but each species has evolved specific c-di-AMP mechanisms of osmoregulation to adapt to its environment.

  4. Transcriptional regulation of the tyrosine hydroxylase gene by glucocorticoid and cyclic AMP

    International Nuclear Information System (INIS)

    Lewis, E.J.; Harrington, C.A.; Chikaraishi, D.M.

    1987-01-01

    Glucocorticoid and cyclic AMP increase tyrosine hydroxylase (TH) activity and mRNA levels in pheochromocytoma cultures. The transcriptional activity of the TH gene, as measured by nuclear run-on assay, is also increased when cultures are treated with the synthetic glucocorticoid dexamethasone or agents that increase intracellular cyclic AMP, such as forskolin and 8-BrcAMP. Both inducers effect transcriptional changes within 10 min after treatment and are maximal after 30 min for forskolin and after 60 min for dexamethasone. The 5' flanking sequences of the TH gene were fused to the bacterial gene chloramphenicol acetyltransferase (CAT), and the hybrid gene was transfected into pheochromocytoma cultures and GH 4 pituitary cells. In both cell lines, a region of the TH gene containing bases -272 to +27 conferred induction of CAT by cyclic AMP, but not by glucocorticoid. The same results were found when a region of the TH gene containing -773 to + 27 was used. Thus, the sequences required for induction of TH by cyclic AMP are contained within 272 bases of 5' flanking sequence, but sequences sufficient for glucocorticoid regulation are not contained with 773 bases

  5. Cellular regulation of basal and FSH-stimulated cyclic AMP production in irradiated rat testes

    International Nuclear Information System (INIS)

    Kangasniemi, M.; Kaipia, A.; Toppari, J.; Mali, P.; Huhtaniemi, I.; Parvinen, M.

    1990-01-01

    Basal and follicle-stimulating hormone (FSH)-stimulated cyclic AMP (cAMP) productions by seminiferous tubular segments from irradiated adult rats were investigated at defined stages of the epithelial cycle when specific spermatogenic cells were low in number. Seven days post-irradiation, depletion of spermatogonia did not influence the basal cAMP production, but FSH response increased in stages II-VIII. Seventeen days post-irradiation when spermatocytes were low in number, there was a small increase in basal cAMP level in stages VII-VIII and FSH-stimulated cAMP production increased in stages VII-XII and XIII-I. At 38 days when pachytene spermatocytes and round spermatids (steps 1-6) were low in number, a decreased basal cAMP production was measured in stages II-VI and IX-XII. FSH-stimulated cAMP output increased in stages VII-XII but decreased in stages II-VI. At 52 days when all spermatids were low in number, basal cAMP levels decreased in all stages of the cycle, whereas FSH response was elevated only in stages VII-XII. All spermatogenic cell types seem to have an effect on cAMP production by the seminiferous tubule in a stage-specific fashion. Germ cells appear to regulate Sertoli cell FSH response in a paracrine way, and a part of cAMP may originate from spermatids stimulated by an unknown FSH-dependent Sertoli cell factor. The FSH-dependent functions may control such phenomena as spermatogonial proliferation, final maturation of spermatids, and onset of meiosis

  6. CRP-Cyclic AMP Regulates the Expression of Type 3 Fimbriae via Cyclic di-GMP in Klebsiella pneumoniae.

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    Ching-Ting Lin

    Full Text Available Klebsiella pneumoniae is the predominant pathogen isolated from liver abscesses of diabetic patients in Asian countries. However, the effects of elevated blood glucose levels on the virulence of this pathogen remain largely unknown. Type 3 fimbriae, encoded by the mrkABCDF genes, are important virulence factors in K. pneumoniae pathogenesis. In this study, the effects of exogenous glucose and the intracellular cyclic AMP (cAMP signaling pathway on type 3 fimbriae expression regulation were investigated. The production of MrkA, the major subunit of type 3 fimbriae, was increased in glucose-rich medium, whereas cAMP supplementation reversed the effect. MrkA production was markedly increased by cyaA or crp deletion, but slightly decreased by cpdA deletion. In addition, the mRNA levels of mrkABCDF genes and the activity of PmrkA were increased in Δcrp strain, as well as the mRNA levels of mrkHIJ genes that encode cyclic di-GMP (c-di-GMP-related regulatory proteins that influence type 3 fimbriae expression. Moreover, the activities of PmrkHI and PmrkJ were decreased in ΔlacZΔcrp strain. These results indicate that CRP-cAMP down-regulates mrkABCDF and mrkHIJ at the transcriptional level. Further deletion of mrkH or mrkI in Δcrp strain diminished the production of MrkA, indicating that MrkH and MrkI are required for the CRP regulation of type 3 fimbriae expression. Furthermore, the high activity of PmrkHI in the ΔlacZΔcrp strain was diminished in ΔlacZΔcrpΔmrkHI, but increased in the ΔlacZΔcrpΔmrkJ strain. Deletion of crp increased the intracellular c-di-GMP concentration and reduced the phosphodiesterase activity. Moreover, we found that the mRNA levels of multiple genes related to c-di-GMP metabolism were altered in Δcrp strain. These indicate that CRP regulates type 3 fimbriae expression indirectly via the c-di-GMP signaling pathway. In conclusion, we found evidence of a coordinated regulation of type 3 fimbriae expression by the CRP-cAMP

  7. Regulation of cyclic AMP by extracellular ATP in cultured brain capillary endothelial cells

    Science.gov (United States)

    Anwar, Zubeya; Albert, Jennifer L; Gubby, Sharon E; Boyle, John P; Roberts, Jonathon A; Webb, Tania E; Boarder, Michael R

    1999-01-01

    In primary unpassaged rat brain capillary endothelial cell cultures (RBECs), using reverse-transcriptase PCR with primers specific for P2Y receptor subtypes, we detected mRNA for P2Y2, P2Y4 and P2Y6, but not P2Y1 receptors.None of the various nucleotides tested reduced forskolin elevated cyclic AMP levels in RBECs. ATP and ATPγS, as well as adenosine, enhanced cyclic AMP accumulation in the presence of forskolin.Comparison of the concentration response curves to ATPγS with those for ATP and adenosine, at different incubation times, indicated that the response to purine nucleotides was not wholly dependent on conversion to adenosine. Adenosine deaminase abolished the response to adenosine but only reduced the response to ATP by about 50%. These results suggest the participation of a receptor responsive to nucleotides.Isobutylmethylxanthine and 8-sulphophenyltheophylline prevented the cyclic AMP response, while neither 8-cyclopentyl-1,3-dipropylxanthine nor SCH58261 were effective antagonists. 2-chloradenosine gave a robust response, but neither 2-chloro-N6-cyclopentyladenosine nor CGS 21680 were agonists.These results show that adenosine and ATP can elevate the cyclic AMP levels of brain endothelial cells by acting on receptors which have a pharmacology apparently distinct from known P2Y and adenosine receptors. PMID:10510459

  8. Cyclic-AMP mediated regulation of ABCB mRNA expression in mussel haemocytes.

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    Silvia Franzellitti

    Full Text Available BACKGROUND: The multixenobiotic resistance system (MXR allows aquatic organisms to cope with their habitat despite high pollution levels by over-expressing membrane and intracellular transporters, including the P-glycoprotein (Pgp. In mammals transcription of the ABCB1 gene encoding Pgp is under cAMP/PKA-mediated regulation; whether this is true in mollusks is not fully clarified. METHODOLOGY/PRINCIPAL FINDINGS: cAMP/PKA regulation and ABCB mRNA expression were assessed in haemocytes from Mediterranean mussels (Mytilus galloprovincialis exposed in vivo for 1 week to 0.3 ng/L fluoxetine (FX alone or in combination with 0.3 ng/L propranolol (PROP. FX significantly decreased cAMP levels and PKA activity, and induced ABCB mRNA down-regulation. FX effects were abolished in the presence of PROP. In vitro experiments using haemocytes treated with physiological agonists (noradrenaline and serotonin and pharmacological modulators (PROP, forskolin, dbcAMP, and H89 of the cAMP/PKA system were performed to obtain clear evidence about the involvement of the signaling pathway in the transcriptional regulation of ABCB. Serotonin (5-HT decreased cAMP levels, PKA activity and ABCB mRNA expression but increased the mRNA levels for a putative 5-HT1 receptor. Interestingly, 5-HT1 was also over-expressed after in vivo exposures to FX. 5-HT effects were counteracted by PROP. Forskolin and dbcAMP increased PKA activity as well as ABCB mRNA expression; the latter effect was abolished in the presence of the PKA inhibitor H89. CONCLUSIONS: This study provides the first direct evidence for the cAMP/PKA-mediated regulation of ABCB transcription in mussels.

  9. Cyclic AMP regulates the biosynthesis of cellobiohydrolase in Cellulomonas flavigena growing in sugar cane bagasse.

    Science.gov (United States)

    Herrera-Herrera, Jesús Antonio; Pérez-Avalos, Odilia; Salgado, Luis M; Ponce-Noyola, Teresa

    2009-10-01

    Cellulomonas flavigena produces a battery of cellulase components that act concertedly to degrade cellulose. The addition of cAMP to repressed C. flavigena cultures released catabolic repression, while addition of cAMP to induced C. flavigena cultures led to a cellobiohydrolase hyperproduction. Exogenous cAMP showed positive regulation on cellobiohydrolase production in C. flavigena grown on sugar cane bagasse. A C. flavigena cellobiohydrolase gene was cloned (named celA), which coded for a 71- kDa enzyme. Upstream, a repressor celR1, identified as a 38 kDa protein, was monitored by use of polyclonal antibodies.

  10. Cyclic AMP Receptor Protein Acts as a Transcription Regulator in Response to Stresses in Deinococcus radiodurans.

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    Su Yang

    Full Text Available The cyclic AMP receptor protein family of transcription factors regulates various metabolic pathways in bacteria, and also play roles in response to environmental changes. Here, we identify four homologs of the CRP family in Deinococcus radiodurans, one of which tolerates extremely high levels of oxidative stress and DNA-damaging reagents. Transcriptional levels of CRP were increased under hydrogen peroxide (H2O2 treatment during the stationary growth phase, indicating that CRPs function in response to oxidative stress. By constructing all CRP single knockout mutants, we found that the dr0997 mutant showed the lowest tolerance toward H2O2, ultraviolet radiation, ionizing radiation, and mitomycin C, while the phenotypes of the dr2362, dr0834, and dr1646 mutants showed slight or no significant differences from those of the wild-type strain. Taking advantage of the conservation of the CRP-binding site in many bacteria, we found that transcription of 18 genes, including genes encoding chromosome-partitioning protein (dr0998, Lon proteases (dr0349 and dr1974, NADH-quinone oxidoreductase (dr1506, thiosulfate sulfurtransferase (dr2531, the DNA repair protein UvsE (dr1819, PprA (dra0346, and RecN (dr1447, are directly regulated by DR0997. Quantitative real-time polymerase chain reaction (qRT-PCR analyses showed that certain genes involved in anti-oxidative responses, DNA repair, and various cellular pathways are transcriptionally attenuated in the dr0997 mutant. Interestingly, DR0997 also regulate the transcriptional levels of all CRP genes in this bacterium. These data suggest that DR0997 contributes to the extreme stress resistance of D. radiodurans via its regulatory role in multiple cellular pathways, such as anti-oxidation and DNA repair pathways.

  11. Gαs regulates Glucagon-Like Peptide 1 Receptor-mediated cyclic AMP generation at Rab5 endosomal compartment.

    Science.gov (United States)

    Girada, Shravan Babu; Kuna, Ramya S; Bele, Shilpak; Zhu, Zhimeng; Chakravarthi, N R; DiMarchi, Richard D; Mitra, Prasenjit

    2017-10-01

    Upon activation, G protein coupled receptors (GPCRs) associate with heterotrimeric G proteins at the plasma membrane to initiate second messenger signaling. Subsequently, the activated receptor experiences desensitization, internalization, and recycling back to the plasma membrane, or it undergoes lysosomal degradation. Recent reports highlight specific cases of persistent cyclic AMP generation by internalized GPCRs, although the functional significance and mechanistic details remain to be defined. Cyclic AMP generation from internalized Glucagon-Like Peptide-1 Receptor (GLP-1R) has previously been reported from our laboratory. This study aimed at deciphering the molecular mechanism by which internalized GLP-R supports sustained cyclic AMP generation upon receptor activation in pancreatic beta cells. We studied the time course of cyclic AMP generation following GLP-1R activation with particular emphasis on defining the location where cyclic AMP is generated. Detection involved a novel GLP-1 conjugate coupled with immunofluorescence using specific endosomal markers. Finally, we employed co-immunoprecipitation as well as immunofluorescence to assess the protein-protein interactions that regulate GLP-1R mediated cyclic AMP generation at endosomes. Our data reveal that prolonged association of G protein α subunit Gαs with activated GLP-1R contributed to sustained cyclic AMP generation at Rab 5 endosomal compartment. The findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

  12. Cyclic AMP in rat pancreatic islets

    International Nuclear Information System (INIS)

    Grill, V.; Borglund, E.; Cerasi, E.; Uppsala Univ.

    1977-01-01

    The incorporation of [ 3 H]adenine into cyclic AMP was studied in rat pancreatic islets under varying conditions of labeling. Prolonging the exposure to [ 3 H]adenine progressively augmented the islet cyclic [ 3 H]AMP level. Islets labeled for different periods of time and subsequently incubated (without adenine) in the presence of D-glucose or cholera toxin showed stimulations of intra-islet cyclic [ 3 H]AMP that were proportionate to the levels of radioactive nucleotide present under non-stimulatory conditions. Labeling the islets in a high glucose concentration (27.7 mM) did not modify the nucleotide responses to glucose or cholera toxin. The specific activity of cyclic [ 3 H]AMP, determined by simultaneous assay of cyclic [ 3 H]AMP and total cyclic AMP, was not influenced by glucose or cholera toxin. Glucose had no effect on the specific activity of labeled ATP

  13. 3' : 5'-Cyclic AMP-dependent 3'

    NARCIS (Netherlands)

    Mato, José M.; Krens, Frans A.; Haastert, Peter J.M. van; Konijn, Theo M.

    1977-01-01

    Suspensions of 3':5'-cyclic AMP (cAMP)-sensitive cells of Dictyostelium discoideum responded to a cAMP pulse with increased 3':5'-cyclic GMP (cGMP) levels. Under the assay conditions used (2 × 10^8 cells per ml in 10 mM phosphate buffer, pH 6.0) cAMP (5 × 10-8 M final concentration) increased cGMP

  14. Gαs regulates Glucagon-Like Peptide 1 Receptor-mediated cyclic AMP generation at Rab5 endosomal compartment

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    Shravan Babu Girada

    2017-10-01

    Conclusions: The findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex.

  15. Regulation of cyclic AMP metabolism by prostaglandins in rabbit cortical collecting tubule cells

    International Nuclear Information System (INIS)

    Sonnenburg, W.K.

    1987-01-01

    In the rabbit cortical collecting tubule (RCCT), prostaglandin E 1 (PGE 1 ) and prostaglandin E 2 (PGE 2 ) at 1 nM inhibit arginine-vasopressin (AVP)-induced water reabsorption, while 100 nM PGE 1 and PGE 2 alone stimulate water reabsorption. Reported here are studies designed to investigate the molecular basis for the biphasic physiological action of PGE 1 and PGE 2 in the collecting duct. In freshly isolated RCCT cells, PGE 1 , PGE 2 , and 16,16-dimethyl-PGE 2 (DM-PGE 2 ) stimulated cAMP synthesis at concentrations ranging from 0.1 to 10 M. Other prostaglandins including the synthetic PGE 2 analogue, sulprostone, failed to stimulate cAMP synthesis. Moreover, sulprostone did not antagonize PGE 2 -stimulated cAMP formation. In contrast, PGE 2 and sulprostone at concentrations ranging from 1 to 100 nM, inhibited AVP-induced cAMP accumulation in freshly isolated RCCT cells. PGE 2 , PGE 1 , DM-PGE 2 and sulprostone at 100 nM were equally effective in inhibiting AVP-induced cAMP formation. Moreover sulprostone inhibited AVP-stimulated adenylate cyclase activity. These results suggest that PGE derivatives mediate either inhibition or activation of adenylate cyclase by stimulating different PGE receptors. To further test this concept, PGE 2 binding to freshly isolated RCCT cell membranes was characterized. Two different classes of PGE 2 binding were detected. / 3 H/PGE 2 binding to the high affinity class of sites was increased by the GTP-analogue, GTP S, while pertussis toxin pretreatment blocked the stimulatory action. In contrast, / 3 H/ PGE 2 binding to the low affinity class of sites was decreased by GTP S; this inhibitory effect was not blocked by pertussis toxin pretreatment

  16. The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12

    DEFF Research Database (Denmark)

    Søgaard-Andersen, L; Martinussen, J; Møllegaard, N E

    1990-01-01

    We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor and the cyclic AMP (cAMP) receptor protein (CRP) complexed to cAMP. Promoter regions controlled by these two proteins characteristically contain tandem cAMP-CRP binding sites. Here we show that (i) Cyt...

  17. 21 CFR 862.1230 - Cyclic AMP test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cyclic AMP test system. 862.1230 Section 862.1230....1230 Cyclic AMP test system. (a) Identification. A cyclic AMP test system is a device intended to measure the level of adenosine 3′, 5′-monophosphate (cyclic AMP) in plasma, urine, and other body fluids...

  18. Cyclic AMP Regulates Bacterial Persistence through Repression of the Oxidative Stress Response and SOS-Dependent DNA Repair in Uropathogenic Escherichia coli.

    Science.gov (United States)

    Molina-Quiroz, Roberto C; Silva-Valenzuela, Cecilia; Brewster, Jennifer; Castro-Nallar, Eduardo; Levy, Stuart B; Camilli, Andrew

    2018-01-09

    Bacterial persistence is a transient, nonheritable physiological state that provides tolerance to bactericidal antibiotics. The stringent response, toxin-antitoxin modules, and stochastic processes, among other mechanisms, play roles in this phenomenon. How persistence is regulated is relatively ill defined. Here we show that cyclic AMP, a global regulator of carbon catabolism and other core processes, is a negative regulator of bacterial persistence in uropathogenic Escherichia coli , as measured by survival after exposure to a β-lactam antibiotic. This phenotype is regulated by a set of genes leading to an oxidative stress response and SOS-dependent DNA repair. Thus, persister cells tolerant to cell wall-acting antibiotics must cope with oxidative stress and DNA damage and these processes are regulated by cyclic AMP in uropathogenic E. coli IMPORTANCE Bacterial persister cells are important in relapsing infections in patients treated with antibiotics and also in the emergence of antibiotic resistance. Our results show that in uropathogenic E. coli , the second messenger cyclic AMP negatively regulates persister cell formation, since in its absence much more persister cells form that are tolerant to β-lactams antibiotics. We reveal the mechanism to be decreased levels of reactive oxygen species, specifically hydroxyl radicals, and SOS-dependent DNA repair. Our findings suggest that the oxidative stress response and DNA repair are relevant pathways to target in the design of persister-specific antibiotic compounds. Copyright © 2018 Molina-Quiroz et al.

  19. Embryonic cholesterol esterification is regulated by a cyclic AMP-dependent pathway in yolk sac membrane-derived endodermal epithelial cells.

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    Siou-Huei Wang

    Full Text Available During avian embryonic development, endodermal epithelial cells (EECs absorb yolk through the yolk sac membrane. Sterol O-acyltransferase (SOAT is important for esterification and yolk lipid utilization during development. Because the major enzyme for yolk sac membrane cholesteryl ester synthesis is SOAT1, we cloned the avian SOAT1 promoter and elucidated the cellular functions of SOAT1. Treatments with either glucagon, isobutylmethylxanthine (IBMX, an adenylate cyclase activator (forskolin, a cAMP analog (dibutyryl-cAMP, or a low glucose concentration all increased SOAT1 mRNA accumulation in EECs from Japanese quail, suggesting that SOAT1 is regulated by nutrients and hormones through a cAMP-dependent pathway. Activity of protein kinase A (PKA was increased by IBMX, whereas co-treatment with the PKA inhibitor, H89 negated the increase in PKA activity. Cyclic AMP-induced EECs had greater cholesterol esterification than untreated EECs. By promoter deletion and point-mutation, the cAMP-response element (-349 to -341 bp was identified as critical in mediating transcription of SOAT1. In conclusion, expression of SOAT1 was regulated by a cAMP-dependent pathway and factors that increase PKA will increase SOAT1 to improve the utilization of lipids in the EECs and potentially modify embryonic growth.

  20. Regulation of the sodium/potassium/chloride cotransporter by calcium and cyclic AMP in cultured vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Higgins, B.L.; Smith, L.; Smith, J.B.

    1987-01-01

    The activity of the Na/K/Cl cotransporter in smooth muscle cells cultured from rat aorta was assayed by measuring the initial rate of furosemide-inhibitable 86 Rb influx or efflux. Five uM furosemide or 0.2 uM bumetanide inhibited influx by 50%. Furosemide-inhibitable 86 Rb influx depended on the presence of all 3 ions in the external medium. The dependence on Na and K was hyperbolic with apparent Km values of 45 and 5 mM, respectively. The dependence on Cl was sigmoidal. Assuming a stoichiometry of 1:1:2 for Na:K:Cl, a Km for Cl of 60 mM was obtained from a Hofstee plot of the data. Rapidly growing cells had 3 fold higher cotransport activity than quiescent cells. Angiotensin II (ANG) stimulated furosemide-inhibitable 86 Rb efflux by 2 fold. An ANG receptor antagonist prevented ANG from increasing cotransport activity. Two calcium ionophores, A23187 and ionomycin, increased cotransport activity by 2 fold. Phorbol myristate acetate had no effect on cotransport activity. Isoproterenol, dibutyryl cyclic AMP, cholera toxin, or methylisobutylxanthine inhibited furosemide-sensitive 86 Rb influx by 35 to 50%. From these findings they conclude that increasing cytoplasmic free calcium stimulates cotransport activity, whereas increasing cellular cyclic AMP inhibits the cotransporter

  1. DNA-Mediated Cyclic GMP-AMP Synthase-Dependent and -Independent Regulation of Innate Immune Responses.

    Science.gov (United States)

    Motani, Kou; Ito, Shinji; Nagata, Shigekazu

    2015-05-15

    Cytoplasmic DNA activates cyclic GMP-AMP synthase (cGAS) to produce cyclic 2'-5'3'-5'GMP-AMP dinucleotide (2'5 'cGAMP). The binding of 2'5'cGAMP to an adaptor protein, stimulator of IFN genes (STING), activates a transcription factor, IFN regulatory factor 3, leading to the induction of IFN and chemokine gene expression. In this study, we found that the 2'5'cGAMP-dependent STING activation induced highly upregulated CXCL10 gene expression. Formation of a distinct STING dimer, which was detected by native PAGE, was induced by 2'5'cGAMP, but not 3'-5'3'-5'cGAMP. Analysis of DNase II(-/-) mice, which constitutively produce IFN-β and CXCL10, showed the accumulation of 2'5'cGAMP in their fetal livers and spleens, suggesting that the undigested DNA accumulating in DNase II(-/-) cells may have leaked from the lysosomes into the cytoplasm. The DNase II(-/-) mouse embryonic fibroblasts produced 2'5'cGAMP in a cGAS-dependent manner during apoptotic cell engulfment. However, cGAS deficiency did not impair the STING-dependent upregulation of CXCL10 in DNase II(-/-) mouse embryonic fibroblasts that was induced by apoptotic cell engulfment or DNA lipofection. These results suggest the involvement of a cGAS-independent additional DNA sensor(s) that induces the STING-dependent activation of innate immunity. Copyright © 2015 by The American Association of Immunologists, Inc.

  2. Cyclic di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in Staphylococcus aureus but dispensable for viability in anaerobic conditions.

    Science.gov (United States)

    Zeden, Merve S; Schuster, Christopher F; Bowman, Lisa; Zhong, Qiyun; Williams, Huw D; Gründling, Angelika

    2018-03-02

    Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such as Staphylococcus aureus However, the exact role of this molecule has not been identified. dacA , the S. aureus gene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that an S. aureus dacA mutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP-requiring pathways. Mutations bypassing the essentiality of dacA were identified in alsT and opuD, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine-uptake system in S. aureus. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained in hepS, hemB, ctaA, and qoxB, coding proteins required for respiration. Furthermore, we show that dacA is dispensable for growth in anaerobic conditions. Together, these findings reveal an essential role for the c-di-AMP signaling network in aerobic, but not anaerobic, respiration in S. aureus . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Regulation of rat hepatocyte function by P2Y receptors: focus on control of glycogen phosphorylase and cyclic AMP by 2-methylthioadenosine 5'-diphosphate.

    Science.gov (United States)

    Dixon, C Jane; Hall, John F; Webb, Tania E; Boarder, Michael R

    2004-10-01

    Hepatocyte function is regulated by several P2Y receptor subtypes. Here we report that 2-methylthioadenosine 5'-diphosphate (2-MeSADP), an agonist at P2Y(1), P2Y(12), and P2Y(13) receptors, potently (threshold 30 nM) stimulates glycogen phosphorylase in freshly isolated rat hepatocytes. Antagonism by N(6)-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS 2179) confirms that this response is mediated by P2Y(1) receptors. In addition, in these cells, both 2-MeSADP and UTP inhibited glucagon-stimulated cyclic AMP accumulation. This inhibitory effect of 2-MeSADP was not reversed by the P2Y(1) antagonists, adenosine-3'-phosphate-5'-phosphate (A3P5P) or MRS 2179, both in the range 1 to 300 microM, indicating that it was not mediated by P2Y(1) receptors. This contrasts with the increase in cytosolic free Ca(2+) concentration ([Ca(2+)](c)) induced by 2-MeSADP, which has shown to be inhibited by A3P5P. Pertussis toxin abolished the inhibitory effect of both UTP and 2-MeSADP. After culture of cells for 48 h, the ability of 2-MeSADP to inhibit cyclic AMP accumulation was greatly diminished. Reverse transcriptase-polymerase chain reaction analysis revealed that during this culture period, there was a decline in the ability to detect transcripts for P2Y(12) and P2Y(13) receptors, both of which are activated by 2-MeSADP and negatively coupled to adenylyl cyclase. However, in freshly isolated cells, the P2Y(12) and P2Y(13) receptor antagonist, 2-propylthio-beta,gamma-dichloromethylene-d-ATP (AR-C67085) (10 nM to 300 microM) did not alter the ability of 2-MeSADP to inhibit glucagon-stimulated cyclic AMP accumulation. We conclude that 2-MeSADP regulates rat hepatocyte glycogen phosphorylase by acting on P2Y(1) receptors coupled to raised [Ca(2+)](c), and by inhibiting cyclic AMP levels by an unknown G(i)-coupled receptor subtype, distinct from P2Y(1), P2Y(12), or P2Y(13) receptors.

  4. Regulation of Cancer Cell Responsiveness to Ionizing Radiation Treatment by Cyclic AMP Response Element Binding Nuclear Transcription Factor

    Directory of Open Access Journals (Sweden)

    Francesca D’Auria

    2017-05-01

    Full Text Available Cyclic AMP response element binding (CREB protein is a member of the CREB/activating transcription factor (ATF family of transcription factors that play an important role in the cell response to different environmental stimuli leading to proliferation, differentiation, apoptosis, and survival. A number of studies highlight the involvement of CREB in the resistance to ionizing radiation (IR therapy, demonstrating a relationship between IR-induced CREB family members’ activation and cell survival. Consistent with these observations, we have recently demonstrated that CREB and ATF-1 are expressed in leukemia cell lines and that low-dose radiation treatment can trigger CREB activation, leading to survival of erythro-leukemia cells (K562. On the other hand, a number of evidences highlight a proapoptotic role of CREB following IR treatment of cancer cells. Since the development of multiple mechanisms of resistance is one key problem of most malignancies, including those of hematological origin, it is highly desirable to identify biological markers of responsiveness/unresponsiveness useful to follow-up the individual response and to adjust anticancer treatments. Taking into account all these considerations, this mini-review will be focused on the involvement of CREB/ATF family members in response to IR therapy, to deepen our knowledge of this topic, and to pave the way to translation into a therapeutic context.

  5. Regulation of Cox-2 by Cyclic AMP Response Element Binding Protein in Prostate Cancer: Potential Role for Nexrutine

    Directory of Open Access Journals (Sweden)

    Rita Ghosh

    2007-11-01

    Full Text Available We recently showed that NexrutineR, a Phellodendron amurense bark extract, suppresses proliferation of prostate cancer cell lines and tumor development in the transgenic adenocarcinoma of mouse prostate (TRAMP model. Our data also indicate that the antiproliferative effects of NexrutineR are mediated in part by Akt and Cyclic AMP response element binding protein (CREB. Cyclooxygenase (Cox-2, a pro-inflammatory mediator, is a CREB target that induces prostaglandin E2 (PGE2 and suppresses apoptosis. Treatment of LNCaP cells with NexrutineR reduced tumor necrosis factor α-induced enzymatic as well as promoter activities of Cox-2. NexrutineR also reduced the expression and promoter activity of Cox-2 in PC-3 cells that express high constitutive levels of Cox-2. Deletion analysis coupled with mutational analysis of the Cox-2 promoter identified CRE as being sufficient for mediating NexrutineR response. Immunohistochemical analysis of human prostate tumors show increased expression of CREB and DNA binding activity in high-grade tumors (three-fold higher in human prostate tumors compared to normal prostate; P = .01. We have identified CREB-mediated activation of Cox-2 as a potential signaling pathway in prostate cancer which can be blocked with a nontoxic, cost-effective dietary supplement like NexrutineR, demonstrating a prospective for development of NexrutineR for prostate cancer management.

  6. Caffeine, cyclic AMP and postreplication repair of mammalian DNA

    International Nuclear Information System (INIS)

    Ehmann, U.K.

    1976-01-01

    The methylxanthines, caffeine and theophylline, inhibit postreplication repair of DNA in mammalian cells. Because they also inhibit cyclic AMP phosphodiesterase, it was thought that there might be some connection between concentrations of cyclic AMP and postreplication repair. This possibility was tested by performing DNA sedimentation experiments with a cyclic AMP-resistant mouse lymphoma cell mutant and its wild-type counterpart. The results show that there is no connection between cellular cyclic AMP concentrations and the rate of postreplication repair. Therefore, it is more likely that caffeine and theophylline inhibit postreplication repair by some other means, such as by binding to DNA

  7. Cyclic AMP is a key regulator of M1 to M2a phenotypic conversion of microglia in the presence of Th2 cytokines.

    Science.gov (United States)

    Ghosh, Mousumi; Xu, Yong; Pearse, Damien D

    2016-01-13

    Microglia and macrophages play a central role in neuroinflammation. Pro-inflammatory cytokines trigger their conversion to a classically activated (M1) phenotype, sustaining inflammation and producing a cytotoxic environment. Conversely, anti-inflammatory cytokines polarize the cells towards an alternatively activated (M2), tissue reparative phenotype. Elucidation of the signal transduction pathways involved in M1 to M2 phenotypic conversion may provide insight into how the innate immune response can be harnessed during distinct phases of disease or injury to mediate neuroprotection and neurorepair. Microglial cells (cell line and primary) were subjected to combined cyclic adenosine monophosphate (cyclic AMP) and IL-4, or either alone, in the presence of pro-inflammatory mediators, lipopolysaccharide (LPS), or tumor necrosis factor-α (TNF-α). Their effects on the expression of characteristic markers for M1 and M2 microglia were assessed. Similarly, the M1 and M2 phenotypes of microglia and macrophages within the lesion site were then evaluated following a contusive spinal cord injury (SCI) to the thoracic (T8) spinal cord of rats and mice when the agents were administered systemically. It was demonstrated that cyclic AMP functions synergistically with IL-4 to promote M1 to M2 conversion of microglia in culture. The combination of cyclic AMP and IL-4, but neither alone, induced an Arg-1(+)/iNOS(-)cell phenotype with concomitant expression of other M2-specific markers including TG2 and RELM-α. M2-converted microglia showed ameliorated production of pro-inflammatory cytokines (TNF-α and IP-10) and reactive oxygen species, with no alteration in phagocytic properties. M2a conversion required protein kinase A (PKA), but not the exchange protein directly activated by cyclic AMP (EPAC). Systemic delivery of cyclic AMP and IL-4 after experimental SCI also promoted a significant M1 to M2a phenotypic change in microglia and macrophage population dynamics in the lesion

  8. "cAMP sponge": a buffer for cyclic adenosine 3', 5'-monophosphate.

    Directory of Open Access Journals (Sweden)

    Konstantinos Lefkimmiatis

    Full Text Available BACKGROUND: While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP. METHODS/PRINCIPAL FINDINGS: Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIbeta of protein kinase A (PKA. Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named "cAMP sponge" was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets. CONCLUSIONS: This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.

  9. Presenilins Regulate Neurotrypsin Gene Expression and Neurotrypsin-dependent Agrin Cleavage via Cyclic AMP Response Element-binding Protein (CREB) Modulation*

    Science.gov (United States)

    Almenar-Queralt, Angels; Kim, Sonia N.; Benner, Christopher; Herrera, Cheryl M.; Kang, David E.; Garcia-Bassets, Ivan; Goldstein, Lawrence S. B.

    2013-01-01

    Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment. PMID:24145027

  10. Presenilins regulate neurotrypsin gene expression and neurotrypsin-dependent agrin cleavage via cyclic AMP response element-binding protein (CREB) modulation.

    Science.gov (United States)

    Almenar-Queralt, Angels; Kim, Sonia N; Benner, Christopher; Herrera, Cheryl M; Kang, David E; Garcia-Bassets, Ivan; Goldstein, Lawrence S B

    2013-12-06

    Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment.

  11. Regulation of the Vibrio vulnificus hupA gene by temperature alteration and cyclic AMP receptor protein and evaluation of its role in virulence.

    Science.gov (United States)

    Oh, Man Hwan; Lee, Sung Min; Lee, Dong Hwan; Choi, Sang Ho

    2009-03-01

    Availability of free iron is extremely limited in the mammalian host, and the acquisition of iron in the host is essential for successful infection by pathogenic bacteria. Expression of many genes involved in acquiring iron is regulated in response to the level of iron availability, and iron regulation is mediated by Fur. In this study, cellular levels of Vibrio vulnificus HupA, a heme receptor protein, and the hupA transcript were found to increase in cells grown at 40 degrees C compared to cells grown at 30 degrees C. The results suggested that change in growth temperature, in addition to iron availability, is an environmental cue controlling the expression of the hupA gene. The influence of global regulatory proteins on the expression of hupA was examined, and the cyclic AMP receptor protein (CRP) was found to activate the expression of hupA at the transcriptional level. CRP exerts its effects by directly binding to DNA upstream of the hupA promoter P(hupA), and a CRP binding site, centered at 174 bp upstream of the transcription start site, was identified by a DNase I protection assay. Finally, a hupA mutant showed reduced virulence in mice and in tissue cultures, in which growth of the hupA mutant was impaired, indicating that HupA of V. vulnificus is essential for survival and multiplication during infection.

  12. Regulation of the Vibrio vulnificus hupA Gene by Temperature Alteration and Cyclic AMP Receptor Protein and Evaluation of Its Role in Virulence▿

    Science.gov (United States)

    Oh, Man Hwan; Lee, Sung Min; Lee, Dong Hwan; Choi, Sang Ho

    2009-01-01

    Availability of free iron is extremely limited in the mammalian host, and the acquisition of iron in the host is essential for successful infection by pathogenic bacteria. Expression of many genes involved in acquiring iron is regulated in response to the level of iron availability, and iron regulation is mediated by Fur. In this study, cellular levels of Vibrio vulnificus HupA, a heme receptor protein, and the hupA transcript were found to increase in cells grown at 40°C compared to cells grown at 30°C. The results suggested that change in growth temperature, in addition to iron availability, is an environmental cue controlling the expression of the hupA gene. The influence of global regulatory proteins on the expression of hupA was examined, and the cyclic AMP receptor protein (CRP) was found to activate the expression of hupA at the transcriptional level. CRP exerts its effects by directly binding to DNA upstream of the hupA promoter PhupA, and a CRP binding site, centered at 174 bp upstream of the transcription start site, was identified by a DNase I protection assay. Finally, a hupA mutant showed reduced virulence in mice and in tissue cultures, in which growth of the hupA mutant was impaired, indicating that HupA of V. vulnificus is essential for survival and multiplication during infection. PMID:19139193

  13. The Interplay between Cyclic AMP, MAPK, and NF-κB Pathways in Response to Proinflammatory Signals in Microglia

    Directory of Open Access Journals (Sweden)

    Mousumi Ghosh

    2015-01-01

    Full Text Available Cyclic AMP is an important intracellular regulator of microglial cell homeostasis and its negative perturbation through proinflammatory signaling results in microglial cell activation. Though cytokines, TNF-α and IL-1β, decrease intracellular cyclic AMP, the mechanism by which this occurs is poorly understood. The current study examined which signaling pathways are responsible for decreasing cyclic AMP in microglia following TNF-α stimulation and sought to identify the role cyclic AMP plays in regulating these pathways. In EOC2 microglia, TNF-α produced a dramatic reduction in cyclic AMP and increased cyclic AMP-dependent PDE activity that could be antagonized by Rolipram, myristoylated-PKI, PD98059, or JSH-23, implicating a role for PDE4, PKA, MEK, and NF-κB in this regulation. Following TNF-α there were significant increases in iNOS and COX-2 immunoreactivity, phosphorylated ERK1/2 and NF-κB-p65, IκB degradation, and NF-κB p65 nuclear translocation, which were reduced in the presence of high levels of cyclic AMP, indicating that reductions in cyclic AMP during cytokine stimulation are important for removing its inhibitory action on NF-κB activation and subsequent proinflammatory gene expression. Further elucidation of the signaling crosstalk involved in decreasing cyclic AMP in response to inflammatory signals may provide novel therapeutic targets for modulating microglial cell activation during neurological injury and disease.

  14. Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection

    Directory of Open Access Journals (Sweden)

    Benjamin A. Diner

    2016-11-01

    Full Text Available The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS. Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1 and human cytomegalovirus (HCMV infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses.

  15. Cyclic AMP regulation of the human glycoprotein hormone α-subunit gene is mediated by an 18-base-pair element

    International Nuclear Information System (INIS)

    Silver, B.J.; Bokar, J.A.; Virgin, J.B.; Vallen, E.A.; Milsted, A.; Nilson, J.H.

    1987-01-01

    cAMP regulates transcription of the gene encoding the α-subunit of human chorionic gonadotropin (hCG) in the choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, the authors inserted fragments from the 5' flanking region of the α-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between position -146 and -111. In the absence of cAMP, the α-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. They localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element. The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the α-subunit gene

  16. Microgravity changes in heart structure and cyclic-AMP metabolism

    Science.gov (United States)

    Philpott, D. E.; Fine, A.; Kato, K.; Egnor, R.; Cheng, L.

    1985-01-01

    The effects of microgravity on cardiac ultrastructure and cyclic AMP metabolism in tissues of rats flown on Spacelab 3 are reported. Light and electron microscope studies of cell structure, measurements of low and high Km phosphodiesterase activity, cyclic AMP-dependent protein kinase activity, and regulatory subunit compartmentation show significant deviations in flight animals when compared to ground controls. The results indicate that some changes have occurred in cellular responses associated with catecholamine receptor interactions and intracellular signal processing.

  17. [Prognostic significance of the cyclic AMP concentration in acute leukemias].

    Science.gov (United States)

    Paietta, E; Mittermayer, K; Schwarzmeier, J D

    1979-01-01

    In patients with acute leukemia (myeloblastic, lymphoblastic, undifferentiated) proliferation kinetics and cyclic adenosine-3', 5'-monophosphate (cAMP) concentration of the leukemic cells were studied for their significance in the prediction of responsiveness to cytostatic therapy. Patients with good clinical response had significantly faster turnover and lower cAMP-levels than those who failed to respond to treatment.

  18. The Cyclic AMP-Vfr Signaling Pathway in Pseudomonas aeruginosa Is Inhibited by Cyclic Di-GMP

    DEFF Research Database (Denmark)

    Almblad, Henrik; Harrison, Joe J; Rybtke, Morten

    2015-01-01

    infection give rise to rugose small colony variants (RSCVs), which are hyper-biofilm-forming mutants that commonly possess mutations that increase production of the biofilm-promoting secondary messenger cyclic di-GMP (c-di-GMP). We show that RSCVs display a decreased production of acute virulence factors...... as a direct result of elevated c-di-GMP content. Overproduction of c-di-GMP causes a decrease in the transcription of virulence factor genes that are regulated by the global virulence regulator Vfr. The low level of Vfr-dependent transcription is caused by a low level of its coactivator, cyclic AMP (c......AMP), which is decreased in response to a high level of c-di-GMP. Mutations that cause reversion of the RSCV phenotype concomitantly reactivate Vfr-cAMP signaling. Attempts to uncover the mechanism underlying the observed c-di-GMP-mediated lowering of cAMP content provided evidence that it is not caused...

  19. Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection.

    Science.gov (United States)

    Diner, Benjamin A; Lum, Krystal K; Toettcher, Jared E; Cristea, Ileana M

    2016-11-15

    The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses. How mammalian cells detect and respond to DNA viruses that replicate in the nucleus is poorly understood. Here, we decipher the distinct functions of two viral DNA sensors, IFI16 and cGAS, during active immune signaling upon infection with two herpesviruses, herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV). We show that IFI16

  20. Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

    Science.gov (United States)

    Melmed, RN; Karanian, PJ; Berlin, RD

    1981-01-01

    We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J

  1. Cyclic AMP system in muscle tissue during prolonged hypokinesia

    Science.gov (United States)

    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.

  2. Cyclic GMP-AMP Displays Mucosal Adjuvant Activity in Mice

    OpenAIRE

    Škrnjug, Ivana; Guzmán, Carlos Alberto; Ruecker, Christine

    2014-01-01

    The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP) after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice....

  3. Differential regulation of human 3β-hydroxysteroid dehydrogenase type 2 for steroid hormone biosynthesis by starvation and cyclic AMP stimulation: studies in the human adrenal NCI-H295R cell model.

    Directory of Open Access Journals (Sweden)

    Sameer Udhane

    Full Text Available Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3β-hydroxysteroid dehydrogenases (HSD3Bs. Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute versus long-term (chronic stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.

  4. Earl Sutherland (1915-1974) [corrected] and the discovery of cyclic AMP.

    Science.gov (United States)

    Blumenthal, Stanley A

    2012-01-01

    In 1945, Earl Sutherland (1915-1974) [corrected] and associates began studies of the mechanism of hormone-induced glycogen breakdown in the liver. In 1956, their efforts culminated in the identification of cyclic AMP, an ancient molecule generated in many cell types in response to hormonal and other extracellular signals. Cyclic AMP, the original "second messenger," transmits such signals through pathways that regulate a diversity of cellular functions and capabilities: metabolic processes such as lipolysis and glycogenolysis; hormone secretion; the permeability of ion channels; gene expression; cell proliferation and survival. Indeed, it can be argued that the discovery of cyclic AMP initiated the study of intracellular signaling pathways, a major focus of contemporary biomedical inquiry. This review presents relevant details of Sutherland's career; summarizes key contributions of his mentors, Carl and Gerti Cori, to the knowledge of glycogen metabolism (contributions that were the foundation for his own research); describes the experiments that led to his identification, isolation, and characterization of cyclic AMP; assesses the significance of his work; and considers some aspects of the impact of cyclic nucleotide research on clinical medicine.

  5. Radioprotection of mouse intestine by inhibitors of cyclic amp phosphodiesterase

    International Nuclear Information System (INIS)

    Lehnert, S.

    1979-01-01

    The survival of colony-forming units of the jejunal crypt was used to assay the radioprotective capacity of various inhibitors of cyclic AMP phosphodiesterase. DL-152, RO-20-1724 and the methyl xanthines, caffeine, theophylline, and methyl isbutyl xanthine (MIX) were all found to have some radioprotective effect. The degree of radioprotecton depended on the route of administration of the drug and on the timing of administration with respect to irradiation. Optimum survival of crypt stem cells was found following intraperitoneal administration of DL-152 (60 min before irradiation) or MIX (30 min before irradiaton), and following intravenous administration of caffeine (60 to 120 min before irradiaton) or theophylline (60 min before irradiation). When these protocols were used, crypt stem cell survival could be enhanced by a factor of from 6 to 7. All the compounds investigated produced some elevation of cyclic AMP content of the whole jejunum; this was found to be simultaneous with or to precede the period of maximum radioprotection. Cyclic AMP was localized with immunofluorescent staining; following injection of DL-152 it was found to be elevated in all parts of the jejunum but to the greatest extent in the lower part of the crypt. Survival curves for crypt stem cells from MIX and DL-152 treated mice were found to have almost the same exponential slope as the saline-injected control, suggesting that the mechanism of protection does not depend on induction of hypoxia

  6. Somite chrondrogenesis: alterations in cyclic AMP levels and proteoglycan synthesis

    International Nuclear Information System (INIS)

    Vasan, Nagaswamistri; Lamb, K.M.; Heick, A.E.

    1985-01-01

    Cyclic AMP (cAMP) levels have been shown to have a positive influence on chondrogenesis in limb buds and pelvic cartilage. In the present study the level of cAMP was measured during somite chondrogenesis in vitro and found to decrease from 1.38 pmol/μg DNA on day 0 to 0.9 pmol/μg DNA on day 6. Inclusion of notochord with somites caused a marked recution, with levels decreasing from 1.41 pmol/μg DNA on day 0 to 0.36 pmol/μg DNA on day 6. Concurrently, the incorporation of radioactive sulfate into sulfated glycosaminoglycans increased from day 3 to day 6 by 38% in somite and 77% in somite-notochord explants. The aggregation of proteoglycans was analyzed by gel chromatography and found to increase with a corresponding decrease in cAMP levels. The result indicate that a decrease in cAMP levels may be necessary for chondrogenic expression in somites. (author)

  7. Cyclic GMP-AMP displays mucosal adjuvant activity in mice.

    Directory of Open Access Journals (Sweden)

    Ivana Škrnjug

    Full Text Available The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice. A characteristic of the cGAMP-induced immune response is the slightly reduced induction of interleukin-17 as a hallmark of Th17 activity--a distinct feature that is not observed with other cyclic di-nucleotide adjuvants. We further characterize the innate immune stimulation activity in vitro on murine bone marrow-derived dendritic cells and human dendritic cells. The observed results suggest the consideration of cGAMP as a candidate mucosal adjuvant for human vaccines.

  8. Cyclic GMP-AMP displays mucosal adjuvant activity in mice.

    Science.gov (United States)

    Škrnjug, Ivana; Guzmán, Carlos Alberto; Rueckert, Christine; Ruecker, Christine

    2014-01-01

    The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP) after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice. A characteristic of the cGAMP-induced immune response is the slightly reduced induction of interleukin-17 as a hallmark of Th17 activity--a distinct feature that is not observed with other cyclic di-nucleotide adjuvants. We further characterize the innate immune stimulation activity in vitro on murine bone marrow-derived dendritic cells and human dendritic cells. The observed results suggest the consideration of cGAMP as a candidate mucosal adjuvant for human vaccines.

  9. Chemotaxis to cyclic AMP and folic acid is mediated by different G proteins in Dictyostelium discoideum

    NARCIS (Netherlands)

    Kesbeke, Fanja; Haastert, Peter J.M. van; Wit, René J.W. de; Snaar-Jagalska, B. Ewa

    1990-01-01

    Mutant Frigid A (fgdA) of Dictyostelium discoideum is defective in a functional Gα2 subunit of a G protein and is characterized by a complete blockade of the cyclic AMP-mediated sensory transduction steps, including cyclic AMP relay, chemotaxis and the cyclic GMP response. Folic acid-mediated

  10. Regulatory action of catalpol from radix rehmanniae on β-adrenoceptor-cyclic AMP system

    International Nuclear Information System (INIS)

    Liu Qingfeng; Sun Qixiang; Hu Yaer; Xia Zongqin

    2004-01-01

    Objective: It has been proved in previous works from this laboratory that the Yin tonic effect of water extract of Radix Rehmanniae is closely related to its down-regulation effect on β adrenoceptor-cyclic AMP system. The purpose of this work is to investigate whether catalpol from Radix Rehmanniae is the effective active component of this plant drug. Methods: Catalpol was extracted from fresh Radix Rehmanniae by organic solvents and purified by low pressure silica gel column and recrystallization. Experimental mice were divided into 4 groups. Three groups of hyperthyroid mouse models were prepared with subcutaneous injection of T 3 and two of them were additionally fed oral catalpol at low and high dose respectively. One group receiving neither T 3 nor catalpol served as controls. Plasma cAMP levels in these mice were measured with RIA 10 min after subcutaneous injection of a single dose of isoproterenol. Results: After injection of catalpol, the plasma cAMP content rose markedly to above 700 pmol/ml in control mice while that of the hyperthyroid mice rose to a even higher levels. Simultaneous additional oral administration of catalpol with purity >97% for 6 days significantly lowered the cAMP level in hyperthyroid mice. With catalpol dose of 1.56 mg/mouse/d and 6.0 mg/mouse/d, the plasma cAMP levels were 978 ± 283 pmol/ml and 1070 ± 394 pmol/ml respectively, the values in the group without catalpol being 1408 ± 568 pmol/ml and 1491 ± 486 pmol/ml, both P<0.05. Conclusion: Catalpol can repeat the regulatory effect of water extract of Radix Rehmanniae on β adrenoceptor-cyclic AMP system, that is, down regulate the response of hyperthyroid mice to isoproterenol. This result suggests that catalpol is an effective active component of the Yin tonic effect of Radix Rehmanniae. (authors)

  11. Opposing actions of dibutyryl cyclic AMP and GMP on temperature in conscious guinea-pigs

    Science.gov (United States)

    Kandasamy, S. B.; Williaes, B. A.

    1983-01-01

    It is shown that the intracerebroventricular administration of dibutyryl cyclic AMP (Db-cAMP) induced hyperthermia in guinea pigs which was not mediated through prostaglandins or norepinephrine since a prostaglandin synthesis inhibitor and an alpha-adrenergic receptor blocking agent did not antagonize the hyperthermia. However, the hyperthermic response to Db-cAMP was attenuated by the central administration of a beta-adrenergic receptor antagonist, which indicates that cAMP may be involved, through beta-adrenergic receptors, in the central regulation of heat production and conservation. The central administration of Db-cGMP produced hypothermia which was not mediated via histamine H1 or H2 receptors and serotonin. The antagonism of hypothermia induced by Db-cGMP and acetylcholine + physostigmine by central administration of a cholinergic muscarine receptor antagonist and not by a cholinergic nicotinic receptor antagonist suggests that cholinoceptive neurons and endogenous cGMP may regulate heat loss through cholinergic muscarine receptors. It is concluded that these results indicate a regulatory role in thermoregulation provided by a balance between opposing actions of cAMP and cGMP in guinea pigs.

  12. Region-selective effects of long-term lithium and carbamazepine administration on cyclic AMP levels in rat brain

    International Nuclear Information System (INIS)

    Wiborg, Ove; Krueger, Tanja; Jakosen, Soeren N.

    1999-01-01

    The effect of lithium and carbamazepine in the treatment of bipolar affective disorder is well established. Althougt a number of biochemical effects have been found, the exact molecular mechanisms underlying their therapeutic actions have not been elucidated nor are the target regions in the brain identified. Taken into account the important role of the cyclic AMP second messenger system in the regulation of neuronal exitability and the indications of its involvement in the pathophysiology of bipolar affective disorder, we have focused on the drug effects on cyclic AMP levels. The objectives of this investigation were to measure the effects on basal cyclic AMP levels, and to locate target regions within the rat brain after long-term administration of lithium and carbamazepine. Drug treatments were carried out for a period of 28 days. After either drug treatment the cyclic AMP level was increased 3-4 times in frontal cortex but unchanged in hippocampus, hypothalamus, thalamus, amygdala and in cerebellum. In neostratum the cyclic AMP level was decreased to about 30% after treatment with lithium. We suggest the common region-selective effect, observed for both drugs in frontal cortex, to be essential for the therapeutic actions of lithium and carbamazepine. (au)

  13. Region-selective effects of long-term lithium and carbamazepine administration on cyclic AMP levels in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Wiborg, Ove; Krueger, Tanja; Jakosen, Soeren N. [Psychiatric Hospital, Dept. of Biological Psychiatry, Risskov (Denmark)

    1999-02-01

    The effect of lithium and carbamazepine in the treatment of bipolar affective disorder is well established. Althougt a number of biochemical effects have been found, the exact molecular mechanisms underlying their therapeutic actions have not been elucidated nor are the target regions in the brain identified. Taken into account the important role of the cyclic AMP second messenger system in the regulation of neuronal exitability and the indications of its involvement in the pathophysiology of bipolar affective disorder, we have focused on the drug effects on cyclic AMP levels. The objectives of this investigation were to measure the effects on basal cyclic AMP levels, and to locate target regions within the rat brain after long-term administration of lithium and carbamazepine. Drug treatments were carried out for a period of 28 days. After either drug treatment the cyclic AMP level was increased 3-4 times in frontal cortex but unchanged in hippocampus, hypothalamus, thalamus, amygdala and in cerebellum. In neostratum the cyclic AMP level was decreased to about 30% after treatment with lithium. We suggest the common region-selective effect, observed for both drugs in frontal cortex, to be essential for the therapeutic actions of lithium and carbamazepine. (au) 46 refs.

  14. Cyclic AMP-receptor protein activates aerobactin receptor IutA expression in Vibrio vulnificus.

    Science.gov (United States)

    Kim, Choon-Mee; Kim, Seong-Jung; Shin, Sung-Heui

    2012-04-01

    The ferrophilic bacterium Vibrio vulnificus can utilize the siderophore aerobactin of Escherichia coli for iron acquisition via its specific receptor IutA. This siderophore piracy by V. vulnificus may contribute to its survival and proliferation, especially in mixed bacterial environments. In this study, we examined the effects of glucose, cyclic AMP (cAMP), and cAMP-receptor protein (Crp) on iutA expression in V. vulnificus. Glucose dose-dependently repressed iutA expression. A mutation in cya encoding adenylate cyclase required for cAMP synthesis severely repressed iutA expression, and this change was recovered by in trans complementing cya or the addition of exogenous cAMP. Furthermore, a mutation in crp encoding Crp severely repressed iutA expression, and this change was recovered by complementing crp. Accordingly, glucose deprivation under iron-limited conditions is an environmental signal for iutA expression, and Crp functions as an activator that regulates iutA expression in response to glucose availability.

  15. Cyclic AMP Inhibits the Activity and Promotes the Acetylation of Acetyl-CoA Synthetase through Competitive Binding to the ATP/AMP Pocket.

    Science.gov (United States)

    Han, Xiaobiao; Shen, Liqiang; Wang, Qijun; Cen, Xufeng; Wang, Jin; Wu, Meng; Li, Peng; Zhao, Wei; Zhang, Yu; Zhao, Guoping

    2017-01-27

    The high-affinity biosynthetic pathway for converting acetate to acetyl-coenzyme A (acetyl-CoA) is catalyzed by the central metabolic enzyme acetyl-coenzyme A synthetase (Acs), which is finely regulated both at the transcriptional level via cyclic AMP (cAMP)-driven trans-activation and at the post-translational level via acetylation inhibition. In this study, we discovered that cAMP directly binds to Salmonella enterica Acs (SeAcs) and inhibits its activity in a substrate-competitive manner. In addition, cAMP binding increases SeAcs acetylation by simultaneously promoting Pat-dependent acetylation and inhibiting CobB-dependent deacetylation, resulting in enhanced SeAcs inhibition. A crystal structure study and site-directed mutagenesis analyses confirmed that cAMP binds to the ATP/AMP pocket of SeAcs, and restrains SeAcs in an open conformation. The cAMP contact residues are well conserved from prokaryotes to eukaryotes, suggesting a general regulatory mechanism of cAMP on Acs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Insulin alters the target size of the peripheral cyclic AMP phosphodiesterase but not the integral cyclic GMP-stimulated cyclic AMP phosphodiesterase in liver plasma membranes

    International Nuclear Information System (INIS)

    Wallace, A.V.; Martin, B.R.; Houslay, M.D.

    1990-01-01

    Radiation inactivation of the two high affinity cyclic AMP phosphodiesterases (PDE) found in liver plasma membranes afforded an estimation of their molecular target sizes in situ. The activity of the peripheral plasma membrane PDE decayed as a single exponential with a target size corresponding to a monomer of circa 54 kDa. The integral, cyclic GMP-stimulated PDE decayed as a dimer of circa 125 kDa. Preincubation of plasma membranes with insulin (10nM), prior to irradiation, caused the target size of only the peripheral plasma membrane PDE to increase. We suggest that insulin addition causes the peripheral plasma membrane PDE to alter its coupling to an integral plasma membrane protein with a target size of circa 90 kDa

  17. Cyclic AMP-specific phosphodiesterase, PDE8A1, is activated by protein kinase A-mediated phosphorylation

    OpenAIRE

    Brown, Kim M.; Lee, Louisa C.Y; Findlay, Jane E.; Day, Jonathan P.; Baillie, George S.

    2012-01-01

    The cyclic AMP-specific phosphodiesterase PDE8 has been shown to play a pivotal role in important processes such as steroidogenesis, T cell adhesion, regulation of heart beat and chemotaxis. However, no information exists on how the activity of this enzyme is regulated. We show that under elevated cAMP conditions, PKA acts to phosphorylate PDE8A on serine 359 and this action serves to enhance the activity of the enzyme. This is the first indication that PDE8 activity can be modulated by a kin...

  18. Regulation of the Vibrio vulnificus hupA Gene by Temperature Alteration and Cyclic AMP Receptor Protein and Evaluation of Its Role in Virulence▿

    OpenAIRE

    Oh, Man Hwan; Lee, Sung Min; Lee, Dong Hwan; Choi, Sang Ho

    2009-01-01

    Availability of free iron is extremely limited in the mammalian host, and the acquisition of iron in the host is essential for successful infection by pathogenic bacteria. Expression of many genes involved in acquiring iron is regulated in response to the level of iron availability, and iron regulation is mediated by Fur. In this study, cellular levels of Vibrio vulnificus HupA, a heme receptor protein, and the hupA transcript were found to increase in cells grown at 40°C compared to cells gr...

  19. Radioimmunoassays for cyclic AMP cross-react with phosphodiesterase inhibitors and buffer components

    NARCIS (Netherlands)

    Sinha, B; Semmler, J; Haen, E; Moeller, J; Endres, S

    We addressed the issue of cross-reactivity of several commonly used phosphodiesterase inhibitors with radioimmunoassays for cyclic AMP, after we had observed a considerably high cross-reactivity with a noncommercial antibody. Theophylline, pentoxifylline, penthydroxifylline (BL 194), albifylline

  20. Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

    Science.gov (United States)

    Pinkney, M; Hoggett, J G

    1988-03-15

    Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase.

  1. Aspirin effects on lymphocyte cyclic AMP levels in normal human subjects.

    Science.gov (United States)

    Snider, D E; Parker, C W

    1976-01-01

    In purified lymphocytes from the peripheral blood of healthy human subjects who had ingested therapeutic doses of aspirin, there was a significant decrease in resting cyclic AMP levels as well as a partial inhibition of the rise in cyclic AMP with isoproterenol or prostaglandin E1. These changes were seen as early as 30 min after aspirin ingestion and did not appear to result from aspirin effects on lymphocyte recovery, purity, viability, or relative number of thymus- or bone marrow-derived lymphocytes. In contrast, the direct addition of aspirin to suspensions of purified peripheral lymphocytes did not significantly alter their cyclic AMP levels. However, an effect of aspirin could be obtained in vitro if aspirin was added to unprocessed whole blood during the dextran sedimentation phase of the cell purification. Thus the effect of aspirin on lymphocyte cyclic AMP metabolism, may be indirect, through other cells present in the peripheral blood. PMID:182720

  2. The interplay between cyclic AMP and insulin during obesity development

    DEFF Research Database (Denmark)

    Borkowski, Kamil

    Insulin and cAMP signalling are related to two opposite metabolic responses. Insulin secretion is elicited in response to food availability and trigger catabolic processes like lipogenesis and glycogen synthesis with a purpose of energy storage. On the other hand cAMP signalling is associated...

  3. Relation of intracellular cyclic AMP to the shape of mammalian cell survival curves

    International Nuclear Information System (INIS)

    Lehnert, S.

    1975-01-01

    Results of experiments with V79 cells growing in tissue culture indicate that the reproductive survival of cells following irradiation is influenced by the level of intracellular 3', 5'-cyclic adenosine monophosphate (cyclic AMP) at the time of irradiation. Cells containing high levels of cyclic AMP induced by treatments with drugs show a characteristic survival curve in which the extent of the shoulder is increased so that the survival after low doses is enhanced. The exponential slope or D 0 , however, is decreased so that at high doses the survival of cells containing high levels of cyclic AMP may be less than that of controls. Naturally occurring changes in radiosensitivity such as those observed as cells pass through the division cycle, may also be related to parallel changes in cyclic AMP concentration occurring during the cycle. Injection of mice with compounds producing elevated cyclic AMP prior to whole-body irradiation increases survival at seven days post-irradiation. The shape of the survival curve for intestinal stem cells in these mice differs from that of the control in having an increased extrapolation number; no change in D 0 is observed in this in vivo situation. (author)

  4. Is the level of cyclic AMP in the pyloric caeca of the starfish Asterias rubens related to the reproductive cycle?

    NARCIS (Netherlands)

    Voogt, P.A.; Rheenen, J.W.A. van

    1984-01-01

    1. 1. Cyclic AMP levels were significantly higher (P = 0.01) in female than in male animals. The average difference found was 0.50 pmole/mg dry weight. 2. 2. Cyclic AMP levels were negatively correlated to the pyloric caeca-index. In females this correlation was −0.54 (P < 0.05). 3. 3. Cyclic

  5. Activation of the adenylyl cyclase/cyclic AMP/protein kinase A pathway in endothelial cells exposed to cyclic strain

    Science.gov (United States)

    Cohen, C. R.; Mills, I.; Du, W.; Kamal, K.; Sumpio, B. E.

    1997-01-01

    The aim of this study was to assess the involvement of the adenylyl cyclase/cyclic AMP/protein kinase A pathway (AC) in endothelial cells (EC) exposed to different levels of mechanical strain. Bovine aortic EC were seeded to confluence on flexible membrane-bottom wells. The membranes were deformed with either 150 mm Hg (average 10% strain) or 37.5 mm Hg (average 6% strain) vacuum at 60 cycles per minute (0.5 s strain; 0.5 s relaxation) for 0-60 min. The results demonstrate that at 10% average strain (but not 6% average strain) there was a 1.5- to 2.2-fold increase in AC, cAMP, and PKA activity by 15 min when compared to unstretched controls. Further studies revealed an increase in cAMP response element binding protein in EC subjected to the 10% average strain (but not 6% average strain). These data support the hypothesis that cyclic strain activates the AC/cAMP/PKA signal transduction pathway in EC which may occur by exceeding a strain threshold and suggest that cyclic strain may stimulate the expression of genes containing cAMP-responsive promoter elements.

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

    Science.gov (United States)

    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

  7. Cyclic AMP Pathway Activation and Extracellular Zinc Induce Rapid Intracellular Zinc Mobilization in Candida albicans

    Science.gov (United States)

    Kjellerup, Lasse; Winther, Anne-Marie L.; Wilson, Duncan; Fuglsang, Anja T.

    2018-01-01

    Zinc is an essential micronutrient, required for a range of zinc-dependent enzymes and transcription factors. In mammalian cells, zinc serves as a second messenger molecule. However, a role for zinc in signaling has not yet been established in the fungal kingdom. Here, we used the intracellular zinc reporter, zinbo-5, which allowed visualization of zinc in the endoplasmic reticulum and other components of the internal membrane system in Candida albicans. We provide evidence for a link between cyclic AMP/PKA- and zinc-signaling in this major human fungal pathogen. Glucose stimulation, which triggers a cyclic AMP spike in this fungus resulted in rapid intracellular zinc mobilization and this “zinc flux” could be stimulated with phosphodiesterase inhibitors and blocked via inhibition of adenylate cyclase or PKA. A similar mobilization of intracellular zinc was generated by stimulation of cells with extracellular zinc and this effect could be reversed with the chelator EDTA. However, zinc-induced zinc flux was found to be cyclic AMP independent. In summary, we show that activation of the cyclic AMP/PKA pathway triggers intracellular zinc mobilization in a fungus. To our knowledge, this is the first described link between cyclic AMP signaling and zinc homeostasis in a human fungal pathogen. PMID:29619016

  8. Mechanism for iron control of the Vibrio fischeri luminescence system: involvement of cyclic AMP and cyclic AMP receptor protein and modulation of DNA level.

    Science.gov (United States)

    Dunlap, P V

    1992-07-01

    Iron controls luminescence in Vibrio fischeri by an indirect but undefined mechanism. To gain insight into that mechanism, the involvement of cyclic AMP (cAMP) and cAMP receptor protein (CRP) and of modulation of DNA levels in iron control of luminescence were examined in V. fischeri and in Escherichia coli containing the cloned V. fischeri lux genes on plasmids. For V. fischeri and E. coli adenylate cyclase (cya) and CRP (crp) mutants containing intact lux genes (luxR luxICDABEG), presence of the iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid) (EDDHA) increased expression of the luminescence system like in the parent strains only in the cya mutants in the presence of added cAMP. In the E. coli strains containing a plasmid with a Mu dl(lacZ) fusion in luxR, levels of beta-galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the lux operon promoter) were both 2-3-fold higher in the presence of EDDHA in the parent strain, and for the mutants this response to EDDHA was observed only in the cya mutant in the presence of added cAMP. Therefore, cAMP and CRP are required for the iron restriction effect on luminescence, and their involvement in iron control apparently is distinct from the known differential control of transcription from the luxR and luxICDABEG promoters by cAMP-CRP. Furthermore, plasmid and chromosomal DNA levels were higher in E. coli and V. fischeri in the presence of EDDHA. The higher DNA levels correlated with an increase in expression of chromosomally encoded beta-galactosidase in E. coli and with a higher level of autoinducer in cultures of V. fischeri. These results implicate cAMP-CRP and modulation of DNA levels in the mechanism of iron control of the V. fischeri luminescence system.

  9. Cyclic GMP-AMP Containing Mixed Phosphodiester Linkages Is An Endogenous High Affinity Ligand for STING

    OpenAIRE

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J.

    2013-01-01

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2′-OH of GMP and 5′-phosphate of AMP, and the other between 3′-OH of AMP and 5′-phosphate of GMP. This mo...

  10. Theoretical Analysis of Allosteric and Operator Binding for Cyclic-AMP Receptor Protein Mutants

    Science.gov (United States)

    Einav, Tal; Duque, Julia; Phillips, Rob

    2018-02-01

    Allosteric transcription factors undergo binding events both at their inducer binding sites as well as at distinct DNA binding domains, and it is often difficult to disentangle the structural and functional consequences of these two classes of interactions. In this work, we compare the ability of two statistical mechanical models - the Monod-Wyman-Changeux (MWC) and the Koshland-N\\'emethy-Filmer (KNF) models of protein conformational change - to characterize the multi-step activation mechanism of the broadly acting cyclic-AMP receptor protein (CRP). We first consider the allosteric transition resulting from cyclic-AMP binding to CRP, then analyze how CRP binds to its operator, and finally investigate the ability of CRP to activate gene expression. In light of these models, we examine data from a beautiful recent experiment that created a single-chain version of the CRP homodimer, thereby enabling each subunit to be mutated separately. Using this construct, six mutants were created using all possible combinations of the wild type subunit, a D53H mutant subunit, and an S62F mutant subunit. We demonstrate that both the MWC and KNF models can explain the behavior of all six mutants using a small, self-consistent set of parameters. In comparing the results, we find that the MWC model slightly outperforms the KNF model in the quality of its fits, but more importantly the parameters inferred by the MWC model are more in line with structural knowledge of CRP. In addition, we discuss how the conceptual framework developed here for CRP enables us to not merely analyze data retrospectively, but has the predictive power to determine how combinations of mutations will interact, how double mutants will behave, and how each construct would regulate gene expression.

  11. Effect of drugs on lipid methylation, receptor-adenylate cyclase coupling and cyclic AMP secretion in Dictyostelium discoideum

    NARCIS (Netherlands)

    Van Waarde, Aren; Van Haastert, P.J.M.

    1986-01-01

    Intercellular communication in Dictyostelium discoldeum takes place by means of cyclic AMP-induced cyclic AMP-synthesis and secretion. Since phospholipid methylation has been suggested to play a role in receptor-adenylate cyclase coupling, we examined the effects of transmethylation inhibitors on

  12. The cyclic AMP cascade is altered in the fragile X nervous system.

    Directory of Open Access Journals (Sweden)

    Daniel J Kelley

    2007-09-01

    Full Text Available Fragile X syndrome (FX, the most common heritable cause of mental retardation and autism, is a developmental disorder characterized by physical, cognitive, and behavioral deficits. FX results from a trinucleotide expansion mutation in the fmr1 gene that reduces levels of fragile X mental retardation protein (FMRP. Although research efforts have focused on FMRP's impact on mGluR signaling, how the loss of FMRP leads to the individual symptoms of FX is not known. Previous studies on human FX blood cells revealed alterations in the cyclic adenosine 3', 5'-monophosphate (cAMP cascade. We tested the hypothesis that cAMP signaling is altered in the FX nervous system using three different model systems. Induced levels of cAMP in platelets and in brains of fmr1 knockout mice are substantially reduced. Cyclic AMP induction is also significantly reduced in human FX neural cells. Furthermore, cAMP production is decreased in the heads of FX Drosophila and this defect can be rescued by reintroduction of the dfmr gene. Our results indicate that a robust defect in cAMP production in FX is conserved across species and suggest that cAMP metabolism may serve as a useful biomarker in the human disease population. Reduced cAMP induction has implications for the underlying causes of FX and autism spectrum disorders. Pharmacological agents known to modulate the cAMP cascade may be therapeutic in FX patients and can be tested in these models, thus supplementing current efforts centered on mGluR signaling.

  13. Control of Saccharomyces cerevisiae catalase T gene (CTT1) expression by nutrient supply via the RAS-cyclic AMP pathway.

    Science.gov (United States)

    Bissinger, P H; Wieser, R; Hamilton, B; Ruis, H

    1989-03-01

    In Saccharomyces cerevisiae, lack of nutrients triggers a pleiotropic response characterized by accumulation of storage carbohydrates, early G1 arrest, and sporulation of a/alpha diploids. This response is thought to be mediated by RAS proteins, adenylate cyclase, and cyclic AMP (cAMP)-dependent protein kinases. This study shows that expression of the S. cerevisiae gene coding for a cytoplasmic catalase T (CTT1) is controlled by this pathway: it is regulated by the availability of nutrients. Lack of a nitrogen, sulfur, or phosphorus source causes a high-level expression of the gene. Studies with strains with mutations in the RAS-cAMP pathway and supplementation of a rca1 mutant with cAMP show that CTT1 expression is under negative control by a cAMP-dependent protein kinase and that nutrient control of CTT1 gene expression is mediated by this pathway. Strains containing a CTT1-Escherichia coli lacZ fusion gene have been used to isolate mutants with mutations in the pathway. Mutants characterized in this investigation fall into five complementation groups. Both cdc25 and ras2 alleles were identified among these mutants.

  14. The effect of in vitro procedures on cyclic AMP accumulation in human leucocytes

    DEFF Research Database (Denmark)

    Johansen, Torben; Friis, U G; Rasmussen, A K

    1994-01-01

    The aim of this study was to investigate the effect of various methodological procedures or protocols on cyclic AMP formation in human leucocytes. The data showed that: (1) ATP content and lactate production was unaffected by hypotonic lysis during leucocyte isolation; (2) there was a linear rela...

  15. Modification of radiation-induced division delay by caffeine analogues and dibutyryl cyclic AMP

    International Nuclear Information System (INIS)

    Kimler, B.F.; Leeper, D.B.; Snyder, M.H.; Rowley, R.; SChneiderman, M.H.

    1982-01-01

    The mitotic selection procedure for cell cycle analysis was utilized to investigate the concentration-dependent modification of x-radiation-induced division delay in Chinese hamster ovary (CHO) cells by methyl xanthines (caffeine, theophylline, and theobromine) and by dibutyryl cyclic AMP. The methyl xanthines (concentrations from 0.5 to 1000 μg/ml) all reduced radiation-induced division delay with the effect being linear between approximately 100 and 1000 μg/ml. After doses of 100-300 rad, delay was reduced by 75, 94 or 83 per cent at 1000 μg/ml for each drug, respectively. However, the addition of dibutyryl cyclic AMP had an opposite effect: radiation-induced delay was increased by the concentration range of 0.3 to 300 μg/ml. These results indicate that in mammalian cells the control of cell cycle progression and the modification of radiation-induced division delay are not simply related to intracellular levels of cyclic AMP. Rather, there appear to be at least two competing mechanisms which are differentially affected by caffeine analogues or by direct addition of dibutyryl cyclic AMP. The direct effect of caffeine and the methyl xanthines on membrane calcium permeability is considered. (author)

  16. Modification of radiation-induced division delay by caffeine analogues and dibutyryl cyclic AMP

    Energy Technology Data Exchange (ETDEWEB)

    Kimler, B.F.; Leeper, D.B.; Snyder, M.H.; Rowley, R.; Schneiderman, M.H. (Thomas Jefferson Univ., Philadelphia, PA (USA). Hospital)

    1982-01-01

    The mitotic selection procedure for cell cycle analysis was utilized to investigate the concentration-dependent modification of x-radiation-induced division delay in Chinese hamster ovary (CHO) cells by methyl xanthines (caffeine, theophylline, and theobromine) and by dibutyryl cyclic AMP. The methyl xanthines (concentrations from 0.5 to 1000 ..mu..g/ml) all reduced radiation-induced division delay with the effect being linear between approximately 100 and 1000 ..mu..g/ml. After doses of 100-300 rad, delay was reduced by 75, 94 or 83 per cent at 1000 ..mu..g/ml for each drug, respectively. However, the addition of dibutyryl cyclic AMP had an opposite effect: radiation-induced delay was increased by the concentration range of 0.3 to 300 ..mu..g/ml. These results indicate that in mammalian cells the control of cell cycle progression and the modification of radiation-induced division delay are not simply related to intracellular levels of cyclic AMP. Rather, there appear to be at least two competing mechanisms which are differentially affected by caffeine analogues or by direct addition of dibutyryl cyclic AMP. The direct effect of caffeine and the methyl xanthines on membrane calcium permeability is considered.

  17. Physiological and Molecular Effects of the Cyclic Nucleotides cAMP and cGMP on Arabidopsis thaliana

    KAUST Repository

    Herrera, Natalia M.

    2012-12-01

    The cyclic nucleotide monophosphates (CNs), cAMP and cGMP, are second messengers that participate in the regulation of development, metabolism and adaptive responses. In plants, CNs are associated with the control of pathogen responses, pollen tube orientation, abiotic stress response, membrane transport regulation, stomatal movement and light perception. In this study, we hypothesize that cAMP and cGMP promote changes in the transcription level of genes related to photosynthesis, high light and membrane transport in Arabidopsis thaliana leaves and, that these changes at the molecular level can have functional biological consequences. For this reason we tested if CNs modulate the photosynthetic rate, responses to high light and root ion transport. Real time quantitative PCR was used to assess transcription levels of selected genes and infrared gas analyzers coupled to fluorescence sensors were used to measure the photosynthetic parameters. We present evidence that both cAMP and cGMP modulate foliar mRNA levels early after stimulation. The two CNs trigger different responses indicating that the signals have specificity. A comparison of proteomic and transcriptional changes suggest that both transcriptional and post-transcriptional mechanisms are modulated by CNs. cGMP up-regulates the mRNA levels of components of the photosynthesis and carbon metabolism. However, neither cAMP nor cGMP trigger differences in the rate of carbon assimilation, maximum efficiency of the photosystem II (PSII), or PSII operating efficiency. It was also demonstrated that CN regulate the expression of its own targets, the cyclic nucleotide gated channels - CNGC. Further studies are needed to identify the components of the signaling transduction pathway that mediate cellular changes and their respective regulatory and/or signaling roles.

  18. Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes

    DEFF Research Database (Denmark)

    Petersen, Rasmus Koefoed; Madsen, Lise; Pedersen, Lone Møller

    2008-01-01

    AMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho......-kinase impaired proadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates a new mechanism of c......AMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response....

  19. Effects of hypokinesia on cyclic nucleotides and hormonal regulation ...

    African Journals Online (AJOL)

    PTH), calcitonin (CT), cyclic nucleotides (cAMP, cGMP) and calcium in the blood of rats, while in urine - phosphate, calcium and cyclic nucleotides. Design: Laboratory based experiment. Setting: Laboratory in the Department of Biochemistry, ...

  20. Role of coronary endothelium in cyclic AMP formation by the heart

    International Nuclear Information System (INIS)

    Kroll, K.; Schrader, J.

    1986-01-01

    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 μM), 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

  1. CHARACTERIZATION OF P2-PURINOCEPTOR MEDIATED CYCLIC-AMP FORMATION IN MOUSE C2C12 MYOTUBES

    NARCIS (Netherlands)

    HENNING, RH; DUIN, M; DENHERTOG, A; NELEMANS, A

    1 The formation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), induced by ATP and other nucleotides was investigated in mouse C2Cl2 myotubes. 2 ATP (100 muM) and ATPgammaS (100 muM) caused a sustained increase in cyclic AMP content of the cells,

  2. Dopamine D1 receptors and phosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein-32 in the medial preoptic area are involved in experience-induced enhancement of male sexual behavior in rats.

    Science.gov (United States)

    McHenry, Jenna A; Bell, Genevieve A; Parrish, Bradley P; Hull, Elaine M

    2012-08-01

    The medial preoptic area (MPOA) is an integral site for male sexual behavior. Dopamine is released in the MPOA before and during copulation and facilitates male rat sexual behavior. Repeated sexual experience and noncopulatory exposures to an estrous female facilitate subsequent copulation. However, the neurobiological mechanisms that mediate such enhancement remain unclear. Here, we examined the role of dopamine D₁ receptors in the MPOA in experience-induced enhancement of male sexual behavior in rats. In experiment 1, microinjections of the D₁ antagonist SCH-23390 into the MPOA before each of seven daily 30-min noncopulatory exposures to a receptive female impaired copulation on a drug-free test on Day 8, compared to vehicle-treated female-exposed animals. Copulatory performance in drug-treated animals was similar to that of vehicle-treated males that had not been preexposed to females. This effect was site specific. There were no group differences in locomotor activity in an open field on the copulation test day. In experiment 2, a separate cohort of animals was used to examine phosphorylation of dopamine- and cAMP-regulated phosphoprotein (DARPP-32) in the MPOA of animals with acute and/or chronic sexual experience. DARPP-32 is a downstream marker of D₁ receptor signaling and substrate of cAMP-dependent protein kinase (PKA). Western immunoblot analysis revealed that p-DARPP-32 expression was greatest in the MPOA of males that received both acute and chronic sexual experience, compared to all other mated conditions and naïve controls. These data suggest that D₁ receptors in the MPOA contribute to experience-induced enhancement of male sexual behavior, perhaps through a PKA regulated mechanism.

  3. Cyclic GMP-AMP Synthase is Activated by Double-stranded DNA-Induced Oligomerization

    OpenAIRE

    Li, Xin; Shu, Chang; Yi, Guanghui; Chaton, Catherine T.; Shelton, Catherine L.; Diao, Jiasheng; Zuo, Xiaobing; Kao, C Cheng; Herr, Andrew B.; Li, Pingwei

    2013-01-01

    Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor mediating innate antimicrobial immunity. It catalyzes the synthesis of a noncanonical cyclic dinucleotide 2′,5′ cGAMP that binds to STING and mediates the activation of TBK1 and IRF-3. Activated IRF-3 translocates to the nucleus and initiates the transcription of the IFN-β gene. The structure of mouse cGAS bound to an 18 bp dsDNA revealed that cGAS interacts with dsDNA through two binding sites, forming a 2:2 complex. Enzyme assays and ...

  4. Cyclic AMP signaling restricts activation and promotes maturation and antioxidant defenses in astrocytes.

    Science.gov (United States)

    Paco, Sonia; Hummel, Manuela; Plá, Virginia; Sumoy, Lauro; Aguado, Fernando

    2016-04-23

    cAMP signaling produces dramatic changes in astrocyte morphology and physiology. However, its involvement in phenotype acquisition and the transcriptionally mediated mechanisms of action are largely unknown. Here we analyzed the global transcriptome of cultured astroglial cells incubated with activators of cAMP pathways. A bulk of astroglial transcripts, 6221 annotated genes, were differentially regulated by cAMP signaling. cAMP analogs strongly upregulated genes involved in typical functions of mature astrocytes, such as homeostatic control, metabolic and structural support to neurons, antioxidant defense and communication, whereas they downregulated a considerable number of proliferating and immaturity-related transcripts. Moreover, numerous genes typically activated in reactive cells, such as scar components and immunological mediators, were repressed by cAMP. GSEA analysis contrasting gene expression profiles with transcriptome signatures of acutely isolated astrocytes and in situ evaluation of protein levels in these cells showed that cAMP signaling conferred mature and in vivo-like transcriptional features to cultured astrocytes. These results indicate that cAMP signaling is a key pathway promoting astrocyte maturation and restricting their developmental and activation features. Therefore, a positive modulation of cAMP signaling may promote the normal state of differentiated astrocytes and favor the protection and function of neuronal networks.

  5. Cyclic GMP-AMP Synthase is an Innate Immune Sensor of HIV and Other Retroviruses

    OpenAIRE

    Gao, Daxing; Wu, Jiaxi; Wu, You-Tong; Du, Fenghe; Aroh, Chukwuemika; Yan, Nan; Sun, Lijun; Chen, Zhijian J.

    2013-01-01

    Retroviruses, including HIV, can activate innate immune responses, but the host sensors for retroviruses are largely unknown. Here we show that HIV infection activates cyclic-GMP-AMP (cGAMP) synthase (cGAS) to produce cGAMP, which binds to and activates the adaptor protein STING to induce type-I interferons and other cytokines. Inhibitors of HIV reverse transcriptase, but not integrase, abrogated interferon-β induction by the virus, suggesting that the reverse transcribed HIV DNA triggers the...

  6. Cyclic AMP-receptor proteins in heart muscle of rats flown on Cosmos 1887

    Science.gov (United States)

    Mednieks, Maija I.; Popova, Irina A.; Grindeland, Richard E.

    1991-01-01

    The cellular compartmentalization of the cyclic AMP-receptor proteins in heart ventricular tissue obtained from rats flown on the Cosmos 1887 is determined. Photoaffinity labeling of soluble and particular cell fractions with a (32P)-8-azido analog of cyclic AMP is followed by electrophoretic separation of the proteins and by autoradiographic identification of the labeled isoforms of cAPK R subunits. It is shown that RII in the particulate subcellular fraction was significantly decreased in heart cells from rats in the flight group when compared to controls. Protein banding patterns in both the cytoplasmic fraction and in a fraction enriched in chromatin-bound proteins exhibited some variability in tissues of individual animals, but showed no changes that could be directly attributed to flight conditions. No significant change was apparent in the distribution of RI or RII cyclic AMP binding in the soluble fractions. It is inferred that the cardiac cell integrity or its protein content is not compromised under flight conditions.

  7. Prostaglandin A1 metabolism and inhibition of cyclic AMP extrusion by avian erythrocytes

    International Nuclear Information System (INIS)

    Heasley, L.E.; Brunton, L.L.

    1985-01-01

    Prostaglandins (PG) inhibit active cyclic AMP export from pigeon red cells, PGA1 and PGA2 most potently. To probe the mechanism of this action of PGA1, the authors have studied the interaction of [ 3 H]PGA1 with suspensions of pigeon red cells. The interaction of PGA1 with pigeon red cells is a multistep process of uptake, metabolism, and secretion. [ 3 H] PGA1 rapidly enters red cells and is promptly metabolized to a compound(s) that remains in the aqueous layer after ethylacetate extraction. The glutathione-depleting agent, diamide, inhibits formation of the PGA1 metabolite. The red cells secrete the polar metabolite of PGA1 by a saturable mechanism that lowered temperatures inhibit. Because uptake and metabolism progress with much greater rates than metabolite secretion, red cells transiently concentrate the polar compound intracellularly. Onset and reversal of inhibition of cyclic AMP export by PGA1 coincide with accumulation and secretion of PGA1 metabolite, suggesting that the polar metabolite acts at an intracellular site to inhibit cyclic AMP efflux

  8. Hepatitis C virus NS2 protein activates cellular cyclic AMP-dependent pathways

    International Nuclear Information System (INIS)

    Kim, Kyoung Mi; Kwon, Shi-Nae; Kang, Ju-Il; Lee, Song Hee; Jang, Sung Key; Ahn, Byung-Yoon; Kim, Yoon Ki

    2007-01-01

    Chronic infection of the hepatitis C virus (HCV) leads to liver cirrhosis and cancer. The mechanism leading to viral persistence and hepatocellular carcinoma, however, has not been fully understood. In this study, we show that the HCV infection activates cellular cAMP-dependent pathways. Expression of a luciferase reporter gene controlled by a basic promoter with the cAMP response element (CRE) was significantly elevated in human hepatoma Huh-7 cells infected with the HCV JFH1. Analysis with viral subgenomic replicons indicated that the HCV NS2 protein is responsible for the effect. Furthermore, the level of cellular transcripts whose stability is known to be regulated by cAMP was specifically reduced in cells harboring NS2-expressing replicons. These results allude to the HCV NS2 protein having a novel function of regulating cellular gene expression and proliferation through the cAMP-dependent pathway

  9. Proliferation kinetics and cyclic AMP as prognostic factors in adult acute leukemia.

    Science.gov (United States)

    Paietta, E; Mittermayer, K; Schwarzmeier, J

    1980-07-01

    In 41 adult patients with acute leukemia (myeloblastic, lymphoblastic, and undifferentiated), proliferation kinetics (as determined by double-label autoradiography) and cyclic adenosine 3',5'-monophosphate (cAMP) concentration were studied for their significance in the prediction of responsiveness to cytostatic therapy. Patients with good clinical response had significantly shorter turnover times and higher labeling indices in the bone marrow than did those who failed to respond to treatment. Cases for which cell kinetics did not correlate with clinical response were explained by variance in the distribution of leukemic blasts between the proliferative cell cycle and the resting pool. Good clinical response was also found to be associated with low levels of cAMP in leukemic cells prior to therapy, whereas high cAMP contents predicted failure. Low cAMP concentrations, however, did not necessarily correlate with short turnover times and vice versa. This might be due to fluctuations of the cAMP concentrations during the cell cycle.

  10. Ecklonia cava Polyphenol Has a Protective Effect against Ethanol-Induced Liver Injury in a Cyclic AMP-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Haruka Yamashita

    2015-06-01

    Full Text Available Previously, we showed that Ecklonia cava polyphenol (ECP treatment suppressed ethanol-induced increases in hepatocyte death by scavenging intracellular reactive oxygen species (ROS and maintaining intracellular glutathione levels. Here, we examined the effects of ECP on the activities of alcohol-metabolizing enzymes and their regulating mechanisms in ethanol-treated hepatocytes. Isolated hepatocytes were incubated with or without 100 mM ethanol. ECP was dissolved in dimethylsulfoxide. ECP was added to cultured cells that had been incubated with or without ethanol. The cells were incubated for 0–24 h. In cultured hepatocytes, the ECP treatment with ethanol inhibited cytochrome P450 2E1 (CYP2E1 expression and activity, which is related to the production of ROS when large quantities of ethanol are oxidized. On the other hand, ECP treatment with ethanol increased the activity of alcohol dehydrogenase (ADH and aldehyde dehydrogenase. These changes in activities of CYP2E1 and ADH were suppressed by treatment with H89, an inhibitor of protein kinase A. ECP treatment with ethanol enhanced cyclic AMP concentrations compared with those of control cells. ECP may be a candidate for preventing ethanol-induced liver injury via regulating alcohol metabolic enzymes in a cyclic AMP-dependent manner.

  11. Mechanisms of Altered Control of Proliferation by Cyclic Amp/Protein Kinase A During Mammary Tumor Progression

    National Research Council Canada - National Science Library

    Imagawa, Walter

    1999-01-01

    We hypothesize that alterations in the regulation of growth by growth factors and cAMP during mammary tumor progression are related to MAP kinase signaling pathways known to be affected by cAMP and pertussis toxin (PT...

  12. 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

    Science.gov (United States)

    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

  13. Dynamic fluctuations provide the basis of a conformational switch mechanism in apo cyclic AMP receptor protein.

    Directory of Open Access Journals (Sweden)

    Burcu Aykaç Fas

    Full Text Available Escherichia coli cyclic AMP Receptor Protein (CRP undergoes conformational changes with cAMP binding and allosterically promotes CRP to bind specifically to the DNA. In that, the structural and dynamic properties of apo CRP prior to cAMP binding are of interest for the comprehension of the activation mechanism. Here, the dynamics of apo CRP monomer/dimer and holo CRP dimer were studied by Molecular Dynamics (MD simulations and Gaussian Network Model (GNM. The interplay of the inter-domain hinge with the cAMP and DNA binding domains are pre-disposed in the apo state as a conformational switch in the CRP's allosteric communication mechanism. The hinge at L134-D138 displaying intra- and inter-subunit coupled fluctuations with the cAMP and DNA binding domains leads to the emergence of stronger coupled fluctuations between the two domains and describes an on state. The flexible regions at K52-E58, P154/D155 and I175 maintain the dynamic coupling of the two domains. With a shift in the inter-domain hinge position towards the N terminus, nevertheless, the latter correlations between the domains loosen and become disordered; L134-D138 dynamically interacts only with the cAMP and DNA binding domains of its own subunit, and an off state is assumed. We present a mechanistic view on how the structural dynamic units are hierarchically built for the allosteric functional mechanism; from apo CRP monomer to apo-to-holo CRP dimers.

  14. Reflections on: "A general role for adaptations in G-Proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function".

    Science.gov (United States)

    Nestler, Eric J

    2016-08-15

    -dependent protein kinase, and certain phosphoproteins in the rat locus coeruleus, but not in several other brain regions studied, and that chronic morphine decreases levels of Giα and increases levels of adenylate cyclase in dorsal root ganglion/spinal cord (DRG-SC) co-cultures. These findings led us to survey the effects of chronic morphine on the G-protein/cyclic AMP system in a large number of brain regions to determine how widespread such regulation might be. We found that while most regions showed no regulation in response to chronic morphine, nucleus accumbens (NAc) and amygdala did show increases in adenylate cyclase and cyclic AMP-dependent protein kinase activity, and thalamus showed an increase in cyclic AMP-dependent protein kinase activity only. An increase in cyclic AMP-dependent protein kinase activity was also observed in DRG-SC co-cultures. Morphine regulation of G-proteins was variable, with decreased levels of Giα seen in the NAc, increased levels of Giα and Goα amygdala, and no change in thalamus or the other brain regions studied. Interestingly, chronic treatment of rats with cocaine, but not with several non-abused drugs, produced similar changes compared to morphine in G-proteins, adenylate cyclase, and cyclic AMP-dependent protein kinase in the NAc, but not in the other brain regions studied. These results indicate that regulation of the G-protein/cyclic AMP system represents a mechanism by which a number of opiate-sensitive neurons adapt to chronic morphine and thereby develop aspects of opiate tolerance and/or dependence. The findings that chronic morphine and cocaine produce similar adaptations in the NAc, a brain region important for the reinforcing actions of many types of abused substances, suggest further that common mechanisms may underlie psychological aspects of drug addiction mediated by this brain region. © 1991. This article is part of a Special Issue entitled SI:50th Anniversary Issue. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

    International Nuclear Information System (INIS)

    Mukai, Atsushi; Hashimoto, Naohiro

    2008-01-01

    Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a 'myosheet,' was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells

  16. Suppression of Virulence of Toxigenic Vibrio cholerae by Anethole through the Cyclic AMP (cAMP-cAMP Receptor Protein Signaling System.

    Directory of Open Access Journals (Sweden)

    M Shamim Hasan Zahid

    Full Text Available Use of natural compounds as antivirulence drugs could be an alternative therapeutic approach to modify the outcome of bacterial infections, particularly in view of growing resistance to available antimicrobials. Here, we show that sub-bactericidal concentration of anethole, a component of sweet fennel seed, could suppress virulence potential in O1 El Tor biotype strains of toxigenic Vibrio cholerae, the causative agent of the ongoing 7th cholera pandemic. The expression of cholera toxin (CT and toxin coregulated pilus (TCP, the major virulence factors of V. cholerae, is controlled through a regulatory cascade involving activation of ToxT with synergistic coupling interaction of ToxR/ToxS with TcpP/TcpH. We present evidence that anethole inhibits in vitro expression of CT and TCP in a toxT-dependent but toxR/toxS-independent manner and through repression of tcpP/tcpH, by using bead-ELISA, western blotting and quantitative real-time RT-PCR assays. The cyclic AMP (cAMP-cAMP receptor protein (CRP is a well-studied global signaling system in bacterial pathogens, and this complex is known to suppress expression of tcpP/tcpH in V. cholerae. We find that anethole influences the virulence regulatory cascade by over-expressing cyaA and crp genes. Moreover, suppression of toxigenic V. cholerae-mediated fluid accumulation in ligated ileum of rabbit by anethole demonstrates its potentiality as an antivirulence drug candidate against the diseases caused by toxigenic V. cholerae. Taken altogether, these results revealing a mechanism of virulence inhibition in V. cholerae by the natural compound anethole, may have relevance in designing antivirulence compounds, particularly against multiple antibiotic resistant bacterial pathogens.

  17. Suppression of Virulence of Toxigenic Vibrio cholerae by Anethole through the Cyclic AMP (cAMP)-cAMP Receptor Protein Signaling System.

    Science.gov (United States)

    Zahid, M Shamim Hasan; Awasthi, Sharda Prasad; Asakura, Masahiro; Chatterjee, Shruti; Hinenoya, Atsushi; Faruque, Shah M; Yamasaki, Shinji

    2015-01-01

    Use of natural compounds as antivirulence drugs could be an alternative therapeutic approach to modify the outcome of bacterial infections, particularly in view of growing resistance to available antimicrobials. Here, we show that sub-bactericidal concentration of anethole, a component of sweet fennel seed, could suppress virulence potential in O1 El Tor biotype strains of toxigenic Vibrio cholerae, the causative agent of the ongoing 7th cholera pandemic. The expression of cholera toxin (CT) and toxin coregulated pilus (TCP), the major virulence factors of V. cholerae, is controlled through a regulatory cascade involving activation of ToxT with synergistic coupling interaction of ToxR/ToxS with TcpP/TcpH. We present evidence that anethole inhibits in vitro expression of CT and TCP in a toxT-dependent but toxR/toxS-independent manner and through repression of tcpP/tcpH, by using bead-ELISA, western blotting and quantitative real-time RT-PCR assays. The cyclic AMP (cAMP)-cAMP receptor protein (CRP) is a well-studied global signaling system in bacterial pathogens, and this complex is known to suppress expression of tcpP/tcpH in V. cholerae. We find that anethole influences the virulence regulatory cascade by over-expressing cyaA and crp genes. Moreover, suppression of toxigenic V. cholerae-mediated fluid accumulation in ligated ileum of rabbit by anethole demonstrates its potentiality as an antivirulence drug candidate against the diseases caused by toxigenic V. cholerae. Taken altogether, these results revealing a mechanism of virulence inhibition in V. cholerae by the natural compound anethole, may have relevance in designing antivirulence compounds, particularly against multiple antibiotic resistant bacterial pathogens.

  18. Cyclic GMP-AMP as an Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA.

    Science.gov (United States)

    Kato, Kazuki; Omura, Hiroki; Ishitani, Ryuichiro; Nureki, Osamu

    2017-06-20

    The innate immune system functions as the first line of defense against invading bacteria and viruses. In this context, the cGAS/STING [cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase/STING] signaling axis perceives the nonself DNA associated with bacterial and viral infections, as well as the leakage of self DNA by cellular dysfunction and stresses, to elicit the host's immune responses. In this pathway, the noncanonical cyclic dinucleotide 2',3'-cyclic GMP-AMP (2',3'-cGAMP) functions as a second messenger for signal transduction: 2',3'-cGAMP is produced by the enzyme cGAS upon its recognition of double-stranded DNA, and then the 2',3'-cGAMP is recognized by the receptor STING to induce the phosphorylation of downstream factors, including TBK1 (TANK binding kinase 1) and IRF3 (interferon regulatory factor 3). Numerous crystal structures of the components of this cGAS/STING signaling axis have been reported and these clarify the structural basis for their signal transduction mechanisms. In this review, we summarize recent progress made in the structural dissection of this signaling pathway and indicate possible directions of forthcoming research.

  19. A Novel Indirect Sequence Readout Component in the E. coli Cyclic AMP Receptor Protein Operator

    DEFF Research Database (Denmark)

    Lindemose, Søren; Nielsen, Peter Eigil; Valentin-Hansen, Poul

    2014-01-01

    binding sites in the E. coli genome, but the exact role of the N6 region in CRP interaction has not previously been systematic examined. Here we employ an in vitro selection system based on a randomized N6 spacer region to demonstrate that CRP binding to the lacP1 site may be enhanced up to 14-fold......The cyclic AMP receptor protein (CRP) from Escherichia coli has been extensively studied for several decades. In particular, a detailed characterization of CRP interaction with DNA has been obtained. The CRP dimer recognizes a consensus sequence AANTGTGANNNNNNTCACANTT through direct amino acid...

  20. Mechanical control of cyclic AMP signalling and gene transcription through integrins

    Science.gov (United States)

    Meyer, C. J.; Alenghat, F. J.; Rim, P.; Fong, J. H.; Fabry, B.; Ingber, D. E.

    2000-01-01

    This study was carried out to discriminate between two alternative hypotheses as to how cells sense mechanical forces and transduce them into changes in gene transcription. Do cells sense mechanical signals through generalized membrane distortion or through specific transmembrane receptors, such as integrins? Here we show that mechanical stresses applied to the cell surface alter the cyclic AMP signalling cascade and downstream gene transcription by modulating local release of signals generated by activated integrin receptors in a G-protein-dependent manner, whereas distortion of integrins in the absence of receptor occupancy has no effect.

  1. Regulation of cAMP on the first mitotic cell cycle of mouse embryos.

    Science.gov (United States)

    Yu, Aiming; Zhang, Zhe; Bi, Qiang; Sun, Bingqi; Su, Wenhui; Guan, Yifu; Mu, Runqing; Miao, Changsheng; Zhang, Jie; Yu, Bingzhi

    2008-03-01

    Mitosis promoting factor (MPF) plays a central role during the first mitosis of mouse embryo. We demonstrated that MPF activity increased when one-cell stage mouse embryo initiated G2/M transition following the decrease of cyclic adenosine 3', 5'-monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) activity. When cAMP and PKA activity increases again, MPF activity decreases and mouse embryo starts metaphase-anaphase transition. In the downstream of cAMP/PKA, there are some effectors such as polo-like kinase 1 (Plk1), Cdc25, Mos (mitogen-activated protein kinase kinase kinase), MEK (mitogen-activated protein kinase kinase), mitogen-activated protein kinase (MAPK), Wee1, anaphase-promoting complex (APC), and phosphoprotein phosphatase that are involved in the regulation of MPF activity. Here, we demonstrated that following activation of MPF, MAPK activity was steady, whereas Plk1 activity fluctuated during the first cell cycle. Plk1 activity was the highest at metaphase and decreased at metaphase-anaphase transition. Further, we established a mathematical model using Gepasi algorithm and the simulation was in agreement with the experimental data. Above all the evidences, we suggested that cAMP and PKA might be the upstream factors which were included in the regulation of the first cell cycle development of mouse embryo. Copyright 2007 Wiley-Liss, Inc.

  2. A simple electrostatic switch important in the activation of type I protein kinase A by cyclic AMP.

    Science.gov (United States)

    Vigil, Dominico; Lin, Jung-Hsin; Sotriffer, Christoph A; Pennypacker, Juniper K; McCammon, J Andrew; Taylor, Susan S

    2006-01-01

    Cyclic AMP activates protein kinase A by binding to an inhibitory regulatory (R) subunit and releasing inhibition of the catalytic (C) subunit. Even though crystal structures of regulatory and catalytic subunits have been solved, the precise molecular mechanism by which cyclic AMP activates the kinase remains unknown. The dynamic properties of the cAMP binding domain in the absence of cAMP or C-subunit are also unknown. Here we report molecular-dynamics simulations and mutational studies of the RIalpha R-subunit that identify the C-helix as a highly dynamic switch which relays cAMP binding to the helical C-subunit binding regions. Furthermore, we identify an important salt bridge which links cAMP binding directly to the C-helix that is necessary for normal activation. Additional mutations show that a hydrophobic "hinge" region is not as critical for the cross-talk in PKA as it is in the homologous EPAC protein, illustrating how cAMP can control diverse functions using the evolutionarily conserved cAMP-binding domains.

  3. Salivary gland ultrastructure and cyclic AMP-dependent reactions in Spacelab 3 rats

    International Nuclear Information System (INIS)

    Mednieks, M.I.; Hand, A.R.

    1987-01-01

    Environmental stimuli influencing catecholamine levels induce changes in cyclic AMP-dependent reactions and cell morphology in the rat parotid. Responses of salivary glands to spaceflight were determined by measurement of cyclic AMP-mediated reactions in fresh-frozen salivary glands and by microscopic evaluation of ultrastructure in fixed parotid glands. Decreased cell-free protein phosphorylation, determined by autoradiography, occurred in parotid glands in three of five flight animals. Protein kinase activity ratios were decreased in the soluble and increased in the particulate fractions of Spacelab 3 (SL-3) rat sublingual glands, compared with ground controls. Biochemical analyses show that effects of space flight on salivary glands are similar to those induced experimentally by physiological manipulation or alteration of catecholamine levels. Morphological evaluation of three SL-3 rat parotid glands showed increased numbers of lysosomes, autophagic vacuoles containing degenerating secretory product, and accumulation of lipid droplets. Since these animals lost weight, consistent with disruption of food and water consumption, morphological changes may in part be due to decreased masticatory stimulation, as occurs with reduced food intake or a liquid diet. The observed changes may reflect physiological responses of the gastrointestinal and autonomic systems to effects of spaceflight

  4. Cyclic GMP-AMP Containing Mixed Phosphodiester Linkages Is An Endogenous High Affinity Ligand for STING

    Science.gov (United States)

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J.

    2013-01-01

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2′-OH of GMP and 5′-phosphate of AMP, and the other between 3′-OH of AMP and 5′-phosphate of GMP. This molecule, termed 2′3′-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2′3′-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation. PMID:23747010

  5. The enzymatic preparation of [α-32P]nucleoside triphosphates, cyclic [32P]AMP, and cyclic [32P]GMP

    International Nuclear Information System (INIS)

    Walseth, T.F.; Johnson, R.A.

    1979-01-01

    A method has been developed for the enzymatic preparation of α- 32 P-labelled ribo- and deoxyribonucleoside triphosphates, cyclic [ 32 P]AMP, and cyclic [ 32 P]GMP of high specific radioactivity and in high yield from 32 Psub(i). The method also enables the preparation of [γ- 32 P]ATP, [γ- 32 P]GTP, [γ- 32 P]ITP, and [γ- 32 P]-dATP of very high specific activity and in high yield. (Auth.)

  6. Cyclic AMP-specific phosphodiesterase-4 as a target for the development of antidepressant drugs.

    Science.gov (United States)

    Zhang, Han-Ting

    2009-01-01

    Phosphodiesterase-4 (PDE4), one of eleven PDE enzyme families, specifically catalyzes hydrolysis of cyclic AMP (cAMP); it has four subtypes (PDE4A-D) with at least 25 splice variants. PDE4 plays a critical role in the control of intracellular cAMP concentrations. PDE4 inhibitors produce antidepressant actions in both animals and humans via enhancement of cAMP signaling in the brain. However, their clinical utility has been hampered by side effects, in particular nausea and emesis. While there is still a long way to go before PDE4 inhibitors with high therapeutic indices are available for treatment of depressive disorders, important advances have been made in the development of PDE4 inhibitors as antidepressants. First, limited, but significant studies point to PDE4D as the major PDE4 subtype responsible for antidepressant-like effects of PDE4 inhibitors, although the role of PDE4A cannot be excluded. Second, PDE4D may contribute to emesis, the major side effect of PDE4 inhibitors. For this reason, identification of roles of PDE4D splice variants in mediating antidepressant activity is particularly important. Recent studies using small interfering RNAs (siRNAs) have demonstrated the feasibility to identify cellular functions of individual PDE4 variants. Third, mixed inhibitors of PDE4 and PDE7 or PDE4 and serotonin reuptake have been developed and may be potential antidepressants with minimized side effects. Finally, relatively selective inhibitors of one or two PDE4 subtypes have been synthesized using structure- and scaffold-based design. This review also discusses the relationship between PDE4 and antidepressant activity based on structures, brain distributions, and pharmacological properties of PDE4 and its isoforms.

  7. Regulation of melanogenesis: the role of cAMP and MITF

    Directory of Open Access Journals (Sweden)

    Michał Otręba

    2012-01-01

    Full Text Available The article presents the melanogenesis pathway and the role of cyclic adenosine monophosphate (cAMP and microphthalmia transcription factor (MITF in regulation of this process. Products of melanogenesis are eu- and/or pheomelanins synthesized in a multistage process of tyrosine oxidation and polymerization. The conversions require the presence of tyrosinase (TYR, key enzyme, tyrosine hydroxylase isoform I (THI and tyrosinase related proteins (TRP1 and TRP2. Many types of signal molecules and transcription factors participate in regulation of melanin synthesis, but the most important are cAMP and MITF. cAMP is the second messenger in the intracellular signal cascade, which is synthesized from adenosine triphosphate (ATP by adenylyl cyclase, activated among others by the melanocortin receptor and the αS subunit of G protein. The signal molecule cAMP regulates MITF, TYR, THI, GTP-cyclohydroxylase I (GTP-CHI transcription and phenylalanine hydroxylase (PAH phosphorylation at Ser16 by protein kinase A (PKA. Mutations of genes encoding proteins belonging to the cAMP signal cascade may lead to McCune-Albright and Carney syndromes. MITF is one of the most important nuclear transcription factors regulating melanogenesis. Currently 10 isoforms of human MITF are known, but in melanocytes only MITF-M, MITF-Mdel, MITF-A and MITF-H occur. MITF transcription factor regulates melanogenesis by activation of tyrosinase, TRP1 and TRP2 transcription. It also affects expression of other factors regulating melanosome maturation, biogenesis and transport. Moreover, it regulates melanocyte proliferation and protection against apoptosis. Mutations of the MITF gene may lead to hereditary diseases: Waardenburg type II and Tietz syndromes.

  8. Effects of exogenous cyclic AMP on growth characteristics and radiation response of Reuber H35 hepatoma cells

    International Nuclear Information System (INIS)

    van Rijn, J.; van Den Berg, J.; van Meeteren, A.; van Wijk, R.

    1983-01-01

    Reuber H35 rat hepatoma cells, clone KRC, were used to study the effect of cyclic AMP on radiation-induced cell death. Treatment of logarithmically growing cultures with 0.5 mM cAMP for 17 hr prior to irradiation resulted in a decreased cell survival. Similar results were obtained with cultures irradiated after treatment with Bt 2 cAMP. Treatment of H35 cells with cAMP or Bt 2 cAMP caused inhibition of their proliferation and resulted in an accumulation of cells in early S phase and depletion of G2-phase cells. In synchronized cultures cells were relatively radioresistant during their S phase. In addition to single-dose treatment with X rays, the effect of Bt 2 cAMP on radiation-induced cell death was studied during fractionated irradiation wtih 2.5 Gy per day. This fractionated irradiation resulted in a dose-reduction factor of 1.6 at the 10% survival level and a 10-fold decrease in the surviving cell population due to the cooperative effects of Bt 2 cAMP on growth rate and radiation survival. The effect of cAMP on radiation-induced mitotic delay was also studied. It appeared that where cAMP had on effect on the progression of G2 cells into mitosis, it prevented cells from recovery from the X-ray mitotic delay in G2

  9. Structural Basis for the Catalytic Mechanism of DncV, Bacterial Homolog of Cyclic GMP-AMP Synthase.

    Science.gov (United States)

    Kato, Kazuki; Ishii, Ryohei; Hirano, Seiichi; Ishitani, Ryuichiro; Nureki, Osamu

    2015-05-05

    Cyclic dinucleotides (CDNs) play key roles as second messengers and signaling molecules in bacteria and metazoans. The newly identified dinucleotide cyclase in Vibrio cholerae (DncV) produces three different CDNs containing two 3'-5' phosphodiester bonds, and its predominant product is cyclic GMP-AMP, whereas mammalian cyclic GMP-AMP synthase (cGAS) produces only cyclic GMP-AMP containing mixed 2'-5' phosphodiester bonds. We report the crystal structures of V. cholerae and Escherichia coli DncV in complex with various nucleotides in the pre-reaction states. The high-resolution structures revealed that DncV preferably recognizes ATP and GTP as acceptor and donor nucleotides, respectively, in the first nucleotidyl transfer reaction. Considering the recently reported intermediate structures, our pre-reaction state structures provide the precise mechanism of 3'-5' linked cyclic AMP-GMP production in bacteria. A comparison with cGAS in the pre-reaction states suggests that the orientation of the acceptor nucleotide primarily determines the distinct linkage specificities between DncV and cGAS. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Synthesis of interleukin 6 (interferon-β2/B cell stimulatory factor 2) in human fibroblasts is triggered by an increase in intracellular cyclic AMP

    International Nuclear Information System (INIS)

    Zhange, Y.; Lin, J.X.; Vilcek, J.

    1988-01-01

    Interleukin 6 (IL-6; also referred to as interferon-β 2 , 26-kDa protein, and B cell stimulatory factor 2) is a cytokine whose actions include a stimulation of immunoglobulin synthesis, enhancement of B cell growth, and modulation of acute phase protein synthesis by hepatocytes. Synthesis of IL-6 is stimulated by interleukin 1 (IL-1), tumor necrosis factor (TNF), or platelet-derived growth factor. The authors examined the role of the cyclic AMP (cAMP)-dependent signal transduction pathway in IL-6 gene expression. Several activators of adenylate cyclase, including prostaglandin E1, forskolin, and cholera toxin, as well as the phosphodiesterase inhibitor isobutylmethylxanthine and the cAMP analog dibutyryl cAMP, shared the ability to cause a dramatic and sustained increase in IL-6 mRNA levels in human FS-4 fibroblasts. Actinomycin D treatment abolished this enhancement. Treatments that increased intracellular cAMP also stimulated the secretion of the IL-6 protein in a biologically active form. Increased intracellular cAMP appears to enhance IL-6 gene expression by a protein kinase C-independent mechanism because down-regulation of protein kinase C by a chronic exposure of cells to a high dose of 12-O-tetradecanoylphorbol 13-acetate did not abolish the enhancement of IL-6 expression by treatments that increase cAMP. IL-1 and TNF too increased IL-6 mRNA levels by a protein kinase C-independent mechanism. The results suggest a role for the cAMP-dependent pathway(s) in IL-6 gene activation by TNF and IL-1

  11. Early effects of synthetic bovine parathyroid hormone and synthetic salmon calcitonin on urinary excretion of cyclic AMP, phosphate and calcium in man.

    Science.gov (United States)

    Caniggia, A; Gennari, C; Vattimo, A; Nardi, P; Nuti, R; Galli, M

    1976-04-20

    Bovine synthetic parathyroid hormone infused intravenously in man increased both the urinary excretion of cyclic AMP and the urinary excretion of phosphate whereas a Salmon synthetic calcitonin infusion increased the urinary excretion of phosphate without change in urinary excretion of cyclic AMP. These data are consistent with the hypothesis that different renal mechanisms are involved in the response to each hormone.

  12. An AP-2 element acts synergistically with the cyclic AMP- and Phorbol ester-inducible enhancer of the human proenkephalin gene

    Energy Technology Data Exchange (ETDEWEB)

    Hyman, S.E.; Comb, M.; Pearlberg, J.; Goodman, H.M.

    1989-01-01

    An enhancer with two DNA elements, one containing the sequence CGTCA, is required for cyclic AMP-and phorbol ester-inducible transcription of the human proenkephalin gene. The authors report that an AP-2 element located adjacent to the enhancer acts synergistically with it to confer maximal response to cyclic AMP and phorbol esters.

  13. Angiotensin II potentiates prostaglandin stimulation of cyclic AMP levels in intact bovine adrenal medulla cells but not adenylate cyclase in permeabilized cells.

    Science.gov (United States)

    Boarder, M R; Plevin, R; Marriott, D B

    1988-10-25

    The level of cyclic AMP in primary cultures of bovine adrenal medulla cells is elevated by prostaglandin E1. Angiotensin II is commonly reported to act on receptors linked to phosphoinositide metabolism or to inhibition of adenylate cyclase. We have investigated the effect of angiotensin II on prostaglandin E1-stimulated cyclic AMP levels in these primary cultures. Rather than reducing cyclic AMP levels, we have found that angiotensin II powerfully potentiates prostaglandin E1-stimulated cyclic AMP accumulation in intact cells, both in the presence and absence of phosphodiesterase inhibitors. The 50% maximal response was similar to that for stimulation of phosphoinositide breakdown by angiotensin II in these cultures. The potentiation of stimulated cyclic AMP levels was seen, although to a smaller maximum, with the protein kinase C (Ca2+/phospholipid-dependent enzyme) activating phorbol ester tetradecanoyl phorbolacetate and with the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol; pretreatment (24 h) with active phorbol ester, which would be expected to diminish protein kinase C levels, attenuated the angiotensin II potentiation of cyclic AMP. Using digitonin-permeabilized cells we showed that adenylate cyclase activity was stimulated by prostaglandin E1 with the same dose-response relationship as was cyclic AMP accumulation in intact cells, but the permeabilized cells showed no response to angiotensin II. The results are discussed with respect to the hypothesis that the angiotensin II influence on cyclic AMP levels is mediated, in part, by diacylglycerol stimulation of protein kinase C.

  14. Dibutyryl cyclic AMP induces differentiation of human neuroblastoma SH-SY5Y cells into a noradrenergic phenotype.

    Science.gov (United States)

    Kume, Toshiaki; Kawato, Yuka; Osakada, Fumitaka; Izumi, Yasuhiko; Katsuki, Hiroshi; Nakagawa, Takayuki; Kaneko, Shuji; Niidome, Tetsuhiro; Takada-Takatori, Yuki; Akaike, Akinori

    2008-10-10

    Dibutyryl cyclic AMP (dbcAMP) and retinoic acid (RA) have been demonstrated to be the inducers of morphological differentiation in SH-SY5Y cells, a human catecholaminergic neuroblastoma cell line. However, it remains unclear whether morphologically differentiated SH-SY5Y cells by these compounds acquire catecholaminergic properties. We focused on the alteration of tyrosine hydroxylase (TH) expression and intracellular content of noradrenaline (NA) as the indicators of functional differentiation. Three days treatment with dbcAMP (1mM) and RA (10microM) induced morphological changes and an increase of TH-positive cells using immunocytochemical analysis in SH-SY5Y cells. The percentage of TH-expressing cells in dbcAMP (1mM) treatment was larger than that in RA (10microM) treatment. In addition, dbcAMP increased intracellular NA content, whereas RA did not. The dbcAMP-induced increase in TH-expressing cells is partially inhibited by KT5720, a protein kinase A (PKA) inhibitor. We also investigated the effect of butyrate on SH-SY5Y cells, because dbcAMP is enzymatically degraded by intracellular esterase, thereby resulting in the formation of butyrate. Butyrate induced the increase of NA content at lower concentrations than dbcAMP, although the increase in TH-expressing cells by butyrate was smaller than that by dbcAMP. The dbcAMP (1mM)- and butyrate (0.3mM)-induced increase in NA content was completely suppressed by alpha-methyl-p-tyrosine (1mM), an inhibitor of TH. These results suggest that dbcAMP induces differentiation into the noradrenergic phenotype through both PKA activation and butyrate.

  15. Essential role for cyclic-AMP responsive element binding protein 1 (CREB) in the survival of acute lymphoblastic leukemia

    NARCIS (Netherlands)

    van der Sligte, Naomi E.; Kampen, Kim R.; ter Elst, Arja; Scherpen, Frank J. G.; Meeuwsen-de Boer, Tiny G. J.; Guryev, Victor; van Leeuwen, Frank N.; Kornblau, Steven M.; de Bont, Eveline S. J. M.

    2015-01-01

    Acute lymphoblastic leukemia (ALL) relapse remains a leading cause of cancer related death in children, therefore, new therapeutic options are needed. Recently, we showed that a peptide derived from Cyclic-AMP Responsive Element Binding Protein (CREB) was highly phosphorylated in pediatric

  16. The effects of histamine and prostaglandin D2 on rat mast-cell cyclic AMP and mediator release

    International Nuclear Information System (INIS)

    Wescott, S.; Kaliner, M.

    1981-01-01

    The possibility that histamine may play a functional role in modulating mast-cell secretion, as has been suggested for basophil degranulation, has both physiologic and pharmacologic implications. Therefore the capacity of histamine to influence rat peritoneal mast-cell (RPMC) cyclic AMP levels and reversed anaphylatic degranulation as reflected in the release of 3H-serotonin (5-HT) was examined. To ascertain that RPMC were functionally responsive to exogenous hormonal stimulation, assessment of prostaglandin (PG) D2 effects on cyclic AMP and 5-HT release were determined in parallel. Although PGD2 (100 microM) increased cyclic AMP and inhibited 5-HT release in the presence of 50 microM aminophylline, histamine (up to 1000 microM) was ineffective was ineffective in both. However, 1000 microM histamine in the presence of 500 microM aminophylline was capable of transiently increasing RPMC cyclic AMP (for 15 to 30 sec) and under these conditions of suppressing 5-HT release. The receptor subtype involved in the suppressive actions of histamine appeared to be of the H-1 type as reflected in the capacity of specific H-1 agonists to reproduce the inhibition of 5-HT release, whereas neither H-2 agonists nor H-2 antagonists had any influence. Thus, under conditions in which phosphodiesterase enzymatic action is impaired, histamine in extremely high concentrations is able to modulate mast-cell secretion. However, it seems very unlikely that this action of histamine has any physiologic significance

  17. Cyclic GMP-AMP synthase is activated by double-stranded DNA-induced oligomerization.

    Science.gov (United States)

    Li, Xin; Shu, Chang; Yi, Guanghui; Chaton, Catherine T; Shelton, Catherine L; Diao, Jiasheng; Zuo, Xiaobing; Kao, C Cheng; Herr, Andrew B; Li, Pingwei

    2013-12-12

    Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor mediating innate antimicrobial immunity. It catalyzes the synthesis of a noncanonical cyclic dinucleotide, 2',5' cGAMP, that binds to STING and mediates the activation of TBK1 and IRF-3. Activated IRF-3 translocates to the nucleus and initiates the transcription of the IFN-β gene. The structure of mouse cGAS bound to an 18 bp dsDNA revealed that cGAS interacts with dsDNA through two binding sites, forming a 2:2 complex. Enzyme assays and IFN-β reporter assays of cGAS mutants demonstrated that interactions at both DNA binding sites are essential for cGAS activation. Mutagenesis and DNA binding studies showed that the two sites bind dsDNA cooperatively and that site B plays a critical role in DNA binding. The structure of mouse cGAS bound to dsDNA and 2',5' cGAMP provided insight into the catalytic mechanism of cGAS. These results demonstrated that cGAS is activated by dsDNA-induced oligomerization. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Phosphorylation of the protein kinase A catalytic subunit is induced by cyclic AMP deficiency and physiological stresses in the fission yeast, Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    McInnis, Brittney; Mitchell, Jessica; Marcus, Stevan

    2010-01-01

    Research highlights: → cAMP deficiency induces phosphorylation of PKA catalytic subunit (Pka1) in S. pombe. → Pka1 phosphorylation is further induced by physiological stresses. → Pka1 phosphorylation is not induced in cells lacking the PKA regulatory subunit. → Results suggest that cAMP-independent Pka1 phosphorylation is stimulatory in nature. -- Abstract: In the fission yeast, Schizosaccharomyces pombe, cyclic AMP (cAMP)-dependent protein kinase (PKA) is not essential for viability under normal culturing conditions, making this organism attractive for investigating mechanisms of PKA regulation. Here we show that S. pombe cells carrying a deletion in the adenylate cyclase gene, cyr1, express markedly higher levels of the PKA catalytic subunit, Pka1, than wild type cells. Significantly, in cyr1Δ cells, but not wild type cells, a substantial proportion of Pka1 protein is hyperphosphorylated. Pka1 hyperphosphorylation is strongly induced in cyr1Δ cells, and to varying degrees in wild type cells, by both glucose starvation and stationary phase stresses, which are associated with reduced cAMP-dependent PKA activity, and by KCl stress, the cellular adaptation to which is dependent on PKA activity. Interestingly, hyperphosphorylation of Pka1 was not detected in either cyr1 + or cyr1Δ S. pombe strains carrying a deletion in the PKA regulatory subunit gene, cgs1, under any of the tested conditions. Our results demonstrate the existence of a cAMP-independent mechanism of PKA catalytic subunit phosphorylation, which we propose could serve as a mechanism for inducing or maintaining specific PKA functions under conditions in which its cAMP-dependent activity is downregulated.

  19. Regulation of Maltodextrin Phosphorylase Synthesis in Escherichia coli by Cyclic Adenosine 3′, 5′-Monophosphate and Glucose1

    Science.gov (United States)

    Chao, Julie; Weathersbee, Carolyn J.

    1974-01-01

    Cyclic adenosine 3′, 5′-monophosphate (AMP) stimulates maltodextrin phosphorylase synthesis in Escherichia coli cells induced with maltose. A maximal effect occurs at 2 to 3 mM cyclic AMP. The action of cyclic AMP is specific, inasmuch as adenosine triphosphate, 3′-AMP, 5′-AMP, adenosine, and dibutyryl cyclic AMP are inactive. Glucose, α-methyl glucoside, 2-deoxyglucose, and pyridoxal 5′-phosphate repress maltodextrin phosphorylase synthesis. This repression is reversed by cyclic AMP. The action of cyclic AMP appears to be at the transcriptional level, since cyclic AMP fails to stimulate phosphorylase production in induced cells in which messenger ribonucleic acid synthesis has been arrested by rifampin or by inducer removal. The two other enzymes involved in the metabolism of maltose, amylomaltase and maltose permease, are also induced in this strain of E. coli and affected by glucose and cyclic AMP in a manner similar to phosphorylase. PMID:4358043

  20. Cooperative DNA binding of heterologous proteins: Evidence for contact between the cyclic AMP receptor protein and RNA polymerase

    International Nuclear Information System (INIS)

    Ren, Y.L.; Garges, S.; Adhya, S.; Krakow, J.S.

    1988-01-01

    Four cAMP-independent receptor protein mutants (designated CRP* mutants) isolated previously are able to activate in vivo gene transcription in the absence of cAMP and their activity can be enhanced by cAMP or cGMP. One of the four mutant proteins, CRP*598 (Arg-142 to His, Ala-144 to Thr), has been characterized with regard to its conformational properties and ability to bind to and support abortive initiation from the lac promoter. Binding of wild-type CRP to its site on the lac promoter and activation of abortive initiation by RNA polymerase on this promoter are effected by cAMP but not by cGMP. CRP*598 can activate lacP + -directed abortive initiation in the presence of cAMP and less efficiently in the presence of cGMP or in the absence of cyclic nucleotide. DNase I protection (footprinting) indicates that cAMP-CRP* binds to its site on the lac promoter whereas unliganded CRP* and cGMP-CRP* form a stable complex with the [ 32 P]lacP + fragment only in the presence of RNA polymerase, showing cooperative binding of two heterologous proteins. This cooperative binding provides strong evidence for a contact between CRP and RNA polymerase for activation of transcription. Although cGMP binds to CRP, it cannot replace cAMP in effecting the requisite conformational transition necessary for site-specific promoter binding

  1. Distribution and function of 3',5'-Cyclic-AMP phosphodiesterases in the human ovary

    DEFF Research Database (Denmark)

    Petersen, T S; Kristensen, S G; Jeppesen, J V

    2015-01-01

    The concentration of the important second messenger cAMP is regulated by phosphodiesterases (PDEs) and hence an attractive drug target. However, limited human data are available about the PDEs in the ovary. The aim of the present study was to describe and characterise the PDEs in the human ovary....... Results were obtained by analysis of mRNA microarray data from follicles and granulosa cells (GCs), combined RT-PCR and enzymatic activity analysis in GCs, immunohistochemical analysis of ovarian sections and by studying the effect of PDE inhibitors on progesterone production from cultured GCs. We found...

  2. Activation of PKA and Epac proteins by cyclic AMP depletes intracellular calcium stores and reduces calcium availability for vasoconstriction.

    Science.gov (United States)

    Cuíñas, Andrea; García-Morales, Verónica; Viña, Dolores; Gil-Longo, José; Campos-Toimil, Manuel

    2016-06-15

    We investigated the implication of PKA and Epac proteins in the endothelium-independent vasorelaxant effects of cyclic AMP (cAMP). Cytosolic Ca(2+) concentration ([Ca(2+)]c) was measured by fura-2 imaging in rat aortic smooth muscle cells (RASMC). Contraction-relaxation experiments were performed in rat aortic rings deprived of endothelium. In extracellular Ca(2+)-free solution, cAMP-elevating agents induced an increase in [Ca(2+)]c in RASMC that was reproduced by PKA and Epac activation and reduced after depletion of intracellular Ca(2+) reservoirs. Arginine-vasopressin (AVP)-evoked increase of [Ca(2+)]c and store-operated Ca(2+) entry (SOCE) were inhibited by cAMP-elevating agents, PKA or Epac activation in these cells. In aortic rings, the contractions induced by phenylephrine in absence of extracellular Ca(2+) were inhibited by cAMP-elevating agents, PKA or Epac activation. In these conditions, reintroduction of Ca(2+) induced a contraction that was inhibited by cAMP-elevating agents, an effect reduced by PKA inhibition and reproduced by PKA or Epac activators. Our results suggest that increased cAMP depletes intracellular, thapsigargin-sensitive Ca(2+) stores through activation of PKA and Epac in RASMC, thus reducing the amount of Ca(2+) released by IP3-generating agonists during the contraction of rat aorta. cAMP rise also inhibits the contraction induced by depletion of intracellular Ca(2+), an effect mediated by reduction of SOCE after PKA or Epac activation. Both effects participate in the cAMP-induced endothelium-independent vasorelaxation. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Expression of Cyclic GMP-AMP Synthase in Patients With Systemic Lupus Erythematosus.

    Science.gov (United States)

    An, Jie; Durcan, Laura; Karr, Reynold M; Briggs, Tracy A; Rice, Gillian I; Teal, Thomas H; Woodward, Joshua J; Elkon, Keith B

    2017-04-01

    Type I interferon (IFN) is implicated in the pathogenesis of systemic lupus erythematosus (SLE) and interferonopathies such as Aicardi-Goutières syndrome. A recently discovered DNA-activated type I IFN pathway, cyclic GMP-AMP synthase (cGAS), has been linked to Aicardi-Goutières syndrome and mouse models of lupus. The aim of this study was to determine whether the cGAS pathway contributes to type I IFN production in patients with SLE. SLE disease activity was measured by the Safety of Estrogens in Lupus Erythematosus National Assessment version of the Systemic Lupus Erythematosus Disease Activity Index. Expression of messenger RNA for cGAS and IFN-stimulated genes (ISGs) was determined by quantitative polymerase chain reaction analysis. Cyclic GMP-AMP (cGAMP) levels were examined by multiple reaction monitoring with ultra-performance liquid chromatography tandem mass spectrometry. Expression of cGAS in peripheral blood mononuclear cells (PBMCs) was significantly higher in SLE patients than in normal controls (n = 51 and n = 20 respectively; P < 0.01). There was a positive correlation between cGAS expression and the IFN score (P < 0.001). The expression of cGAS in PBMCs showed a dose response to type I IFN stimulation in vitro, consistent with it being an ISG. Targeted measurement of cGAMP by tandem mass spectrometry detected cGAMP in 15% of the SLE patients (7 of 48) but none of the normal (0 of 19) or rheumatoid arthritis (0 of 22) controls. Disease activity was higher in SLE patients with cGAMP versus those without cGAMP. Increased cGAS expression and cGAMP in a proportion of SLE patients indicates that the cGAS pathway should be considered as a contributor to type I IFN production. Whereas higher cGAS expression may be a consequence of exposure to type I IFN, detection of cGAMP in patients with increased disease activity indicates potential involvement of this pathway in disease expression. © 2016, American College of Rheumatology.

  4. A generalized allosteric mechanism for cis-regulated cyclic nucleotide binding domains.

    Directory of Open Access Journals (Sweden)

    Alexandr P Kornev

    2008-04-01

    Full Text Available Cyclic nucleotides (cAMP and cGMP regulate multiple intracellular processes and are thus of a great general interest for molecular and structural biologists. To study the allosteric mechanism of different cyclic nucleotide binding (CNB domains, we compared cAMP-bound and cAMP-free structures (PKA, Epac, and two ionic channels using a new bioinformatics method: local spatial pattern alignment. Our analysis highlights four major conserved structural motifs: 1 the phosphate binding cassette (PBC, which binds the cAMP ribose-phosphate, 2 the "hinge," a flexible helix, which contacts the PBC, 3 the beta(2,3 loop, which provides precise positioning of an invariant arginine from the PBC, and 4 a conserved structural element consisting of an N-terminal helix, an eight residue loop and the A-helix (N3A-motif. The PBC and the hinge were included in the previously reported allosteric model, whereas the definition of the beta(2,3 loop and the N3A-motif as conserved elements is novel. The N3A-motif is found in all cis-regulated CNB domains, and we present a model for an allosteric mechanism in these domains. Catabolite gene activator protein (CAP represents a trans-regulated CNB domain family: it does not contain the N3A-motif, and its long range allosteric interactions are substantially different from the cis-regulated CNB domains.

  5. Topoisomerase 1 Inhibition Promotes Cyclic GMP-AMP Synthase-Dependent Antiviral Responses

    Directory of Open Access Journals (Sweden)

    Geneviève Pèépin

    2017-10-01

    Full Text Available Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1 by low-dose camptothecin (CPT can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through cyclic GMP-AMP synthase (cGAS detection of cytoplasmic DNA. This argues against CPT having only anti-inflammatory activity. Furthermore, expression of the simian virus 40 (SV40 large T antigen was paramount to the proinflammatory antiviral activity of CPT, as it potentiated cytoplasmic DNA leakage and subsequent cGAS recruitment in human and mouse cell lines. This work suggests that the capacity of Top1 inhibitors to blunt inflammatory responses can be counteracted by viral oncogenes and that this should be taken into account for their therapeutic development.

  6. Molecular cloning and functional characterization of porcine cyclic GMP-AMP synthase.

    Science.gov (United States)

    Wang, Jiang; Chu, Beibei; Du, Lili; Han, Yingqian; Zhang, Xuemei; Fan, Shuangshuang; Wang, Yueying; Yang, Guoyu

    2015-06-01

    Cyclic GMP-AMP synthase (cGAS), which belongs to the nucleotidyltransferase family, recognizes cytosolic DNA and induces the type I interferon (IFN) pathway through the synthesis of the second messenger cGAMP. In this study, porcine cGAS (p-cGAS) was identified and its tissue distribution, subcellular localization, and functions in innate immunity were characterized. The coding sequence of p-cGAS is 1494 bp long, encodes 497 amino acids, and is most similar (74%) to Bos taurus cGAS. p-cGAS mRNA is abundant in the spleen, duodenum, jejunum, and ileum. The subcellular distribution of p-cGAS is not only in the cytosol, but also on the endoplasmic reticulum (ER) membrane. The overexpression of wild-type p-cGAS in porcine kidney epithelial cells, but not its catalytically inactive mutants, induced IFN-β expression, which was dependent on STING and IRF3. However, the downregulation of p-cGAS by RNA interference markedly reduced IFN-β expression after pseudorabies virus (PRV) infection or poly(dA:dT) transfection. These results demonstrate that p-cGAS is an important DNA sensor, required for IFN-β activation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Maintenance of cyclic GMP-AMP homeostasis by ENPP1 is involved in pseudorabies virus infection.

    Science.gov (United States)

    Wang, Jiang; Lu, Shao-Fang; Wan, Bo; Ming, Sheng-Li; Li, Guo-Li; Su, Bing-Qian; Liu, Jiao-Yang; Wei, Yu-Shuang; Yang, Guo-Yu; Chu, Bei-Bei

    2018-03-01

    In a previous study, we demonstrated that porcine cyclic GMP-AMP (cGAMP) synthase (cGAS) catalyzes cGAMP production and is an important DNA sensor for the pseudorabies virus (PRV)-induced activation of interferon β (IFN-β). Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) has recently been identified as the hydrolase of cGAMP in rodents, but its role in porcine cells is not clear. Our recent study demonstrated that porcine ENPP1 is responsible for the homeostasis of cGAMP and is critical for PRV infection. Porcine ENPP1 mRNA is predominantly expressed in muscle. PRV infection was enhanced by ENPP1 overexpression and attenuated by silencing of ENPP1. During PRV infection, the activation of IFN-β and NF-κB was reduced in ENPP1 overexpressed cells and promoted in ENPP1 knockdown cells. Investigation of the molecular mechanisms of ENPP1 during PRV infection showed that ENPP1 hydrolyzed cGAMP in PRV-infected or cGAMP-transfected cells and inhibited IRF3 phosphorylation, reducing IFN-β secretion. These results, combined with those for porcine cGAS, demonstrate that ENPP1 acts coordinately with cGAS to maintain the reservoir of cGAMP and participates in PRV infection. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. Regulation of phospholipid synthesis in Mycobacterium smegmatis by cyclic adenosine monophosphate

    International Nuclear Information System (INIS)

    Sareen, Monica; Kaur, Harpinder; Khuller, G.K.

    1993-01-01

    Forskolin, an adenylate cyclase activator and a cyclic AMP analogue, dibutyryl cyclic AMP have been used to examine the relationship between intracellular levels of cyclic AMP and lipid synthesis in Mycobacterium smegmatis. Total phospholipid content was found to be increased in forskolin grown cells as a result of increased cyclic AMP levels caused by activation of adenylate cyclase. Increased phospholipid content was supported by increased [ 14 C]acetate incorporation as well as increased activity of glycerol-3-phosphate acyltransferase. Pretreatment of cells with dibutyryl cyclic AMP had similar effects on lipid synthesis. Taking all these observations together it is suggested that lipid synthesis is being controlled by cyclic AMP in mycobacteria. (author). 14 refs., 4 tabs

  9. The reduction of radiation-induced mitotic delay by caffeine: a test of the cyclic AMP hypothesis

    International Nuclear Information System (INIS)

    Oleinick, N.L.; Brewer, E.N.; Rustad, R.C.

    1978-01-01

    A study has been made of the reduction in γ-radiation-induced mitotic delay by caffeine in the naturally-synchronous plasmodial slime mould. Physarum polycephalum during late G 2 and early prophase, and the results compared with those obtained with other compounds of similar structure and/or physiological function. The reduction of radiation-induced mitotic delay was related to increasing concentrations of caffeine over at least two orders of magnitude. Pre-irradiation treatment with caffeine had no detectable effect. Caffeine had to be present for most, if not all, of the post-irradiation pre-mitotic period. Other chemicals which are reported to inhibit cyclic AMP phosphodiesterase either reduce or increase radiation-induced mitotic delay. The results therefore indicate that the reduction of mitotic delay by caffeine is not a result of altered cyclic AMP levels. (UK)

  10. Reversal of propranolol blockade of adrenergic receptors and related toxicity with drugs that increase cyclic AMP.

    Science.gov (United States)

    Whitehurst, V E; Vick, J A; Alleva, F R; Zhang, J; Joseph, X; Balazs, T

    1999-09-01

    An overdose of propranolol, a widely used nonselective beta-adrenergic receptor blocking agent, can result in hypotension and bradycardia leading to irreversible shock and death. In addition, the blockade of adrenergic receptors can lead to alterations in neurotransmitter receptors resulting in the interruption of the activity of other second messengers and the ultimate cellular responses. In the present experiment, three agents, aminophylline, amrinone, and forskolin were tested in an attempt to reverse the potential lethal effects of a propranolol overdose in dogs. Twenty-two anesthetized beagle dogs were given a 10-min infusion of propranolol at a dose of 1 mg/kg/min. Six of the dogs, treated only with intravenous saline, served as controls. Within 15-30 min all six control dogs exhibited profound hypotension and severe bradycardia that led to cardiogenic shock and death. Seven dogs were treated with intravenous aminophylline 20 mg/kg 5 min after the end of the propranolol infusion. Within 10-15 min heart rate and systemic arterial blood pressure returned to near control levels, and all seven dogs survived. Intravenous amrinone (2-3 mg/kg) given to five dogs, and forskolin (1-2 mg/kg) given to four dogs, also increased heart rate and systemic arterial blood pressure but the recovery of these parameters was appreciably slower than that seen with aminophylline. All of these animals also survived with no apparent adverse effects. Histopathologic evaluation of the hearts of the dogs treated with aminophylline showed less damage (vacuolization, inflammation, hemorrhage) than the hearts from animals given propranolol alone. Results of this study showed that these three drugs, all of which increase cyclic AMP, are capable of reversing the otherwise lethal effects of a propranolol overdose in dogs.

  11. Topoisomerase 1 Inhibition Promotes Cyclic GMP-AMP Synthase-Dependent Antiviral Responses.

    Science.gov (United States)

    Pépin, Geneviève; Nejad, Charlotte; Ferrand, Jonathan; Thomas, Belinda J; Stunden, H James; Sanij, Elaine; Foo, Chwan-Hong; Stewart, Cameron R; Cain, Jason E; Bardin, Philip G; Williams, Bryan R G; Gantier, Michael P

    2017-10-03

    Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1) by low-dose camptothecin (CPT) can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through cyclic GMP-AMP synthase (cGAS) detection of cytoplasmic DNA. This argues against CPT having only anti-inflammatory activity. Furthermore, expression of the simian virus 40 (SV40) large T antigen was paramount to the proinflammatory antiviral activity of CPT, as it potentiated cytoplasmic DNA leakage and subsequent cGAS recruitment in human and mouse cell lines. This work suggests that the capacity of Top1 inhibitors to blunt inflammatory responses can be counteracted by viral oncogenes and that this should be taken into account for their therapeutic development. IMPORTANCE Recent studies suggest that low-dose DNA-damaging compounds traditionally used in cancer therapy can have opposite effects on antiviral responses, either suppressing (with the example of CPT) or potentiating (with the example of doxorubicin) them. Our work demonstrates that the minor DNA damage promoted by low-dose CPT can also trigger strong antiviral responses, dependent on the presence of viral oncogenes. Taken together, these results call for caution in the therapeutic use of low-dose chemotherapy agents to modulate antiviral responses in humans. Copyright © 2017 Pépin et al.

  12. Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases.

    Science.gov (United States)

    Gao, Daxing; Li, Tuo; Li, Xiao-Dong; Chen, Xiang; Li, Quan-Zhen; Wight-Carter, Mary; Chen, Zhijian J

    2015-10-20

    TREX1 is an exonuclease that digests DNA in the cytoplasm. Loss-of-function mutations of TREX1 are linked to Aicardi-Goutieres Syndrome (AGS) and systemic lupus erythematosus (SLE) in humans. Trex1(-/-) mice exhibit autoimmune and inflammatory phenotypes that are associated with elevated expression of interferon (IFN)-induced genes (ISGs). Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates the IFN pathway. Upon binding to DNA, cGAS is activated to catalyze the synthesis of cGAMP, which functions as a second messenger that binds and activates the adaptor protein STING to induce IFNs and other cytokines. Here we show that genetic ablation of cGas in Trex1(-/-) mice eliminated all detectable pathological and molecular phenotypes, including ISG induction, autoantibody production, aberrant T-cell activation, and lethality. Even deletion of just one allele of cGas largely rescued the phenotypes of Trex1(-/-) mice. Similarly, deletion of cGas in mice lacking DNaseII, a lysosomal enzyme that digests DNA, rescued the lethal autoimmune phenotypes of the DNaseII(-/-) mice. Through quantitative mass spectrometry, we found that cGAMP accumulated in mouse tissues deficient in Trex1 or DNaseII and that this accumulation was dependent on cGAS. These results demonstrate that cGAS activation causes the autoimmune diseases in Trex1(-/-) and DNaseII(-/-) mice and suggest that inhibition of cGAS may lead to prevention and treatment of some human autoimmune diseases caused by self-DNA.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ohnishi, Masatoshi, E-mail: ohnishi@fupharm.fukuyama-u.ac.jp [Department of Pharmacotherapeutics, Graduate School of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 985-1 Sanzo, Higashimura-cho, Fukuyama, Hiroshima, 729-0292 (Japan); Department of Pharmacotherapeutics, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 985-1 Sanzo, Higashimura-cho, Fukuyama, Hiroshima, 729-0292 (Japan); Urasaki, Tomoka [Department of Pharmacotherapeutics, Graduate School of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 985-1 Sanzo, Higashimura-cho, Fukuyama, Hiroshima, 729-0292 (Japan); Ochiai, Hiroyuki; Matsuoka, Kohei; Takeo, Shin; Harada, Tomoki; Ohsugi, Yoshihito [Department of Pharmacotherapeutics, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 985-1 Sanzo, Higashimura-cho, Fukuyama, Hiroshima, 729-0292 (Japan); Inoue, Atsuko [Department of Pharmacotherapeutics, Graduate School of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 985-1 Sanzo, Higashimura-cho, Fukuyama, Hiroshima, 729-0292 (Japan); Department of Pharmacotherapeutics, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 985-1 Sanzo, Higashimura-cho, Fukuyama, Hiroshima, 729-0292 (Japan)

    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. - Highlights: • Dibutyryl cAMP increased wnt4, but not wnt3a, 5a, 7a and 11, mRNA in mixed glia. • Wnt4 protein increased in astrocytes co-cultivated with microglia. • It took a long time to robustly increase wnt4 expression. • Rolipram

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    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. - Highlights: • Dibutyryl cAMP increased wnt4, but not wnt3a, 5a, 7a and 11, mRNA in mixed glia. • Wnt4 protein increased in astrocytes co-cultivated with microglia. • It took a long time to robustly increase wnt4 expression. • Rolipram

  15. New Insights into the Cyclic Di-adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic Dinucleotide for Acid Stress Resistance in Staphylococcus aureus.

    Science.gov (United States)

    Bowman, Lisa; Zeden, Merve S; Schuster, Christopher F; Kaever, Volkhard; Gründling, Angelika

    2016-12-30

    Nucleotide signaling networks are key to facilitate alterations in gene expression, protein function, and enzyme activity in response to diverse stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced by the human pathogen Staphylococcus aureus and is involved in regulating a number of physiological processes including potassium transport. S. aureus must ensure tight control over its cellular levels as both high levels of the dinucleotide and its absence result in a number of detrimental phenotypes. Here we show that in addition to the membrane-bound Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterase (PDE) GdpP, S. aureus produces a second cytoplasmic DHH/DHHA1 PDE Pde2. Although capable of hydrolyzing c-di-AMP, Pde2 preferentially converts linear 5'-phosphadenylyl-adenosine (pApA) to AMP. Using a pde2 mutant strain, pApA was detected for the first time in S. aureus, leading us to speculate that this dinucleotide may have a regulatory role under certain conditions. Moreover, pApA is involved in a feedback inhibition loop that limits GdpP-dependent c-di-AMP hydrolysis. Another protein linked to the regulation of c-di-AMP levels in bacteria is the predicted regulator protein YbbR. Here, it is shown that a ybbR mutant S. aureus strain has increased acid sensitivity that can be bypassed by the acquisition of mutations in a number of genes, including the gene coding for the diadenylate cyclase DacA. We further show that c-di-AMP levels are slightly elevated in the ybbR suppressor strains tested as compared with the wild-type strain. With this, we not only identified a new role for YbbR in acid stress resistance in S. aureus but also provide further insight into how c-di-AMP levels impact acid tolerance in this organism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Transporter function and cyclic AMP turnover in normal colonic mucosa from patients with and without colorectal neoplasia

    Directory of Open Access Journals (Sweden)

    Kleberg Karen

    2012-06-01

    Full Text Available Abstract Background The pathogenesis of colorectal neoplasia is still unresolved but has been associated with alterations in epithelial clearance of xenobiotics and metabolic waste products. The aim of this study was to functionally characterize the transport of cyclic nucleotides in colonic biopsies from patients with and without colorectal neoplasia. Methods Cyclic nucleotides were used as model substrates shared by some OATP- and ABC-transporters, which in part are responsible for clearance of metabolites and xenobiotics from the colonic epithelium. On colonic biopsies from patients with and without colorectal neoplasia, molecular transport was electrophysiologically registered in Ussing-chamber set-ups, mRNA level of selected transporters was quantified by rt-PCR, and subcellular location of transporters was determined by immunohistochemistry. Results Of four cyclic nucleotides, dibuturyl-cAMP induced the largest short circuit current in both patient groups. The induced short circuit current was significantly lower in neoplasia-patients (p = 0.024. The observed altered transport of dibuturyl-cAMP in neoplasia-patients could not be directly translated to an observed increased mRNA expression of OATP4A1 and OATP2B1 in neoplasia patients. All other examined transporters were expressed to similar extents in both patient groups. Conclusions OATP1C1, OATP4A1, OATP4C1 seem to be involved in the excretory system of human colon. ABCC4 is likely to be involved from an endoplasmic-Golgi complex and basolateral location in goblet cells. ABCC5 might be directly involved in the turnover of intracellular cAMP at the basolateral membrane of columnar epithelial cells, while OATP2B1 is indirectly related to the excretory system. Colorectal neoplasia is associated with lower transport or sensitivity to cyclic nucleotides and increased expression of OATP2B1 and OATP4A1 transporters, known to transport PGE2.

  17. Plasticity of regulation of mannitol phosphotransferase system operon by CRP-cAMP complex in Vibrio cholerae.

    Science.gov (United States)

    Zhou, Yan Yan; Zhang, Hong Zhi; Liang, Wei Li; Zhang, Li Juan; Zhu, Jun; Kan, Biao

    2013-10-01

    The complex of the cyclic AMP receptor protein (CRP) and cAMP is an important transcriptional regulator of numerous genes in prokaryotes. The transport of mannitol through the phosphotransferase systems (PTS) is regulated by the CRP-cAMP complex. The aim of the study is to investigate how the CRP-cAMP complex acting on the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype. The crp mutant strain was generated by homologous recombination to assess the need of CRP to activate the mannitol PTS operon of V. cholerae El Tor. Electrophoretic mobility shift assays (EMSA) and the reporter plasmid pBBRlux were used to confirm the role that the CRP-cAMP complex playing on the mannitol PTS operon mtl. In this study, we confirmed that CRP is strictly needed for the activation of the mtl operon. We further experimentally identified five CRP binding sites within the promoter region upstream of the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype and found that these sites display different affinities for CRP and provide different contributions to the activation of the operon. The five binding sites collectively confer the strong activation of mannitol transfer by CRP in V. cholerae, indicating an elaborate and subtle CRP activation mechanism. Copyright © 2013 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  18. Subcellular compartmentation, interdependency and dynamics of the cyclic AMP-dependent PKA subunits during pathogenic differentiation in rice blast.

    Science.gov (United States)

    Selvaraj, Poonguzhali; Tham, Hong Fai; Ramanujam, Ravikrishna; Naqvi, Naweed I

    2017-08-01

    The cAMP-dependent PKA signalling plays a central role in growth, asexual development and pathogenesis in fungal pathogens. Here, we functionally characterised RPKA, the regulatory subunit of cAMP/PKA and studied the dynamics and organisation of the PKA subunits in the rice blast pathogen Magnaporthe oryzae. The RPKA subunit was essential for proper vegetative growth, asexual sporulation and surface hydrophobicity in M. oryzae. A spontaneous suppressor mutation, SMR19, that restored growth and conidiation in the RPKA deletion mutant was isolated and characterised. SMR19 enhanced conidiation and appressorium formation but failed to suppress the pathogenesis defects in rpkAΔ. The PKA activity was undetectable in the mycelial extracts of SMR19, which showed a single mutation (val242leu) in the highly conserved active site of the catalytic subunit (CPKA) of cAMP/PKA. The two subunits of cAMP/PKA showed different subcellular localisation patterns with RpkA being predominantly nucleocytoplasmic in conidia, while CpkA was largely cytosolic and/or vesicular. The CpkA anchored RpkA in cytoplasmic vesicles, and localisation of PKA in the cytoplasm was governed by CpkA in a cAMP-dependant or independent manner. We show that there exists a tight regulation of PKA subunits at the level of transcription, and the cAMP signalling is differentially compartmentalised in a stage-specific manner in rice blast. © 2017 John Wiley & Sons Ltd.

  19. cAMP control of HCN2 channel Mg2+ block reveals loose coupling between the cyclic nucleotide-gating ring and the pore.

    Directory of Open Access Journals (Sweden)

    Alex K Lyashchenko

    Full Text Available Hyperpolarization-activated cyclic nucleotide-regulated HCN channels underlie the Na+-K+ permeable IH pacemaker current. As with other voltage-gated members of the 6-transmembrane KV channel superfamily, opening of HCN channels involves dilation of a helical bundle formed by the intracellular ends of S6 albeit this is promoted by inward, not outward, displacement of S4. Direct agonist binding to a ring of cyclic nucleotide-binding sites, one of which lies immediately distal to each S6 helix, imparts cAMP sensitivity to HCN channel opening. At depolarized potentials, HCN channels are further modulated by intracellular Mg2+ which blocks the open channel pore and blunts the inhibitory effect of outward K+ flux. Here, we show that cAMP binding to the gating ring enhances not only channel opening but also the kinetics of Mg2+ block. A combination of experimental and simulation studies demonstrates that agonist acceleration of block is mediated via acceleration of the blocking reaction itself rather than as a secondary consequence of the cAMP enhancement of channel opening. These results suggest that the activation status of the gating ring and the open state of the pore are not coupled in an obligate manner (as required by the often invoked Monod-Wyman-Changeux allosteric model but couple more loosely (as envisioned in a modular model of protein activation. Importantly, the emergence of second messenger sensitivity of open channel rectification suggests that loose coupling may have an unexpected consequence: it may endow these erstwhile "slow" channels with an ability to exert voltage and ligand-modulated control over cellular excitability on the fastest of physiologically relevant time scales.

  20. The DNA sensor, cyclic GMP-AMP synthase, is essential for induction of IFN-β during Chlamydia trachomatis infection.

    Science.gov (United States)

    Zhang, Yugen; Yeruva, Laxmi; Marinov, Anthony; Prantner, Daniel; Wyrick, Priscilla B; Lupashin, Vladimir; Nagarajan, Uma M

    2014-09-01

    IFN-β has been implicated as an effector of oviduct pathology resulting from genital chlamydial infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFN-β expression during chlamydial infection. We determined that three-prime repair exonuclease-1, a host 3' to 5' exonuclease, reduced IFN-β expression significantly during chlamydial infection using small interfering RNA and gene knockout fibroblasts, implicating cytosolic DNA as a ligand for this response. The DNA sensor cyclic GMP-AMP synthase (cGAS) has been shown to bind cytosolic DNA to generate cyclic GMP-AMP, which binds to the signaling adaptor stimulator of IFN genes (STING) to induce IFN-β expression. We determined that cGAS is required for IFN-β expression during chlamydial infection in multiple cell types. Interestingly, although infected cells deficient for STING or cGAS alone failed to induce IFN-β, coculture of cells depleted for either STING or cGAS rescued IFN-β expression. These data demonstrate that cyclic GMP-AMP produced in infected cGAS(+)STING(-) cells can migrate into adjacent cells via gap junctions to function in trans in cGAS(-)STING(+) cells. Furthermore, we observed cGAS localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane in association with STING, indicating that chlamydial DNA is most likely recognized outside the inclusion as infection progresses. These novel findings provide evidence that cGAS-mediated DNA sensing directs IFN-β expression during Chlamydia trachomatis infection and suggest that effectors from infected cells can directly upregulate IFN-β expression in adjacent uninfected cells during in vivo infection, contributing to pathogenesis. Copyright © 2014 by The American Association of Immunologists, Inc.

  1. Cyclic GMP-AMP Synthase Is Required for Cell Proliferation and Inflammatory Responses in Rheumatoid Arthritis Synoviocytes

    OpenAIRE

    Wang, Yan; Su, Guo-Hua; Zhang, Fang; Chu, Jing-Xue; Wang, Yun-Shan

    2015-01-01

    Rheumatoid arthritis (RA) is characterized by inflammatory cell infiltration, fibroblast-like synoviocytes (FLS) invasive proliferation, and joint destruction. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces immune activation. In this study, we examined whether cGAS plays a role in RA FLS. In this study, cGAS was overexpressed in RA-FLS compared with OA FLS. TNFα stimulation induced cGAS expression in RA FLS. Overexpression of cGAS promoted the proliferation and knockdow...

  2. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.

    Science.gov (United States)

    Sun, Lijun; Wu, Jiaxi; Du, Fenghe; Chen, Xiang; Chen, Zhijian J

    2013-02-15

    The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers host immune responses such as the production of type I interferons. Cytosolic DNA induces interferons through the production of cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, or cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced interferon-β in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and interferon-β induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.

  3. Cyclic [G(2',5')pA(3',5')p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase.

    Science.gov (United States)

    Gao, Pu; Ascano, Manuel; Wu, Yang; Barchet, Winfried; Gaffney, Barbara L; Zillinger, Thomas; Serganov, Artem A; Liu, Yizhou; Jones, Roger A; Hartmann, Gunther; Tuschl, Thomas; Patel, Dinshaw J

    2013-05-23

    Recent studies identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical, biochemical, and cellular assays to demonstrate that this second messenger contains G(2',5')pA and A(3',5')pG phosphodiester linkages, designated c[G(2',5')pA(3',5')p]. We show that, upon dsDNA binding, cGAS is activated through conformational transitions, resulting in formation of a catalytically competent and accessible nucleotide-binding pocket for generation of c[G(2',5')pA(3',5')p]. We demonstrate that cyclization occurs in a stepwise manner through initial generation of 5'-pppG(2',5')pA prior to cyclization to c[G(2',5')pA(3',5')p], with the latter positioned precisely in the catalytic pocket. Mutants of cGAS dsDNA-binding or catalytic pocket residues exhibit reduced or abrogated activity. Our studies have identified c[G(2',5')pA(3',5')p] as a founding member of a family of metazoan 2',5'-containing cyclic heterodinucleotide second messengers distinct from bacterial 3',5' cyclic dinucleotides. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Effects of subacute and chronic lead treatment on glucose homestasis and renal cyclic AMP metabolism in rats

    Energy Technology Data Exchange (ETDEWEB)

    Stevenson, A; Merali, Z; Kacew, S; Singhal, R L

    1976-01-01

    The effects of chronic oral ingestion of lead in doses ranging from 20 to 80 ppM were compared with those seen after the subacute exposure of rats to a 10 mg/kg daily dose of the heavy metal for 7 days. Irrespective of the treatment regimen used, lead treatment significantly increased the activities of renal pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose 1,6-diphosphatase and glucose 6-phosphatase. The observed enhancement of kidney gluconeogenic enzymes in chronically treated animals was associated with a stimulation of the adenylate cyclase-cyclic AMP system, a rise in blood glucose and urea as well as a depression in hepatic glycogen and serum immunoreactive insulin (IRI) levels. In contrast, subacute exposure to lead failed to significantly alter cyclic AMP metabolism and the concentrations of liver glycogen, blood glucose, serum urea or IRI. Whereas the insulinogenic index (the ratio of serum IRI to blood glucose concentration) was markedly suppressed in chronically treated rats, this ratio remained within normal limits following subacute exposure to the heavy metal. However, a marked decrease in the insulinogenic index was observed in subacutely treated rats 15 min after the administration of a glucose load. The data provide evidence to show that increased glucose synthesis as well as suppressed pancreatic function may be responsible for lead-induced disturbances in glucose homeostasis.

  5. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING.

    Science.gov (United States)

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J

    2013-07-25

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2'-OH of GMP and 5'-phosphate of AMP, and the other between 3'-OH of AMP and 5'-phosphate of GMP. This molecule, termed 2'3'-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2'3'-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. High glucose enhances cAMP level and extracellular signal-regulated kinase phosphorylation in Chinese hamster ovary cell: Usage of Br-cAMP in foreign protein β-galactosidase expression.

    Science.gov (United States)

    Lin, Hsiao-Hsien; Lee, Tsung-Yih; Liu, Ting-Wei; Tseng, Ching-Ping

    2017-07-01

    Glucose is a carbon source for Chinese hamster ovary (CHO) cell growth, while low growth rate is considered to enhance the production of recombinant proteins. The present study reveals that glucose concentrations higher than 1 g/L reduce the growth rate and substantially increase in cAMP (∼300%) at a high glucose concentration (10 g/L). High glucose also enhances the phosphorylation of extracellular signal-regulated kinase (ERK) and p27 kip by Western blot analysis. To determine whether the phosphorylation of ERK is involved in the mechanism, a cyclic-AMP dependent protein kinase A (PKA) inhibitor (H-8) or MEK (MAPKK) inhibitor (PD98059) was added to block ERK phosphorylation. We show that both the high glucose-induced ERK phosphorylation and growth rate return to baseline levels. These results suggest that the cAMP/PKA and MAP signaling pathways are involved in the abovementioned mechanism. Interestingly, the direct addition of 8-bromo-cAMP (Br-cAMP), a membrane-permeable cAMP analog, can mimic the similar effects produced by high glucose. Subsequently Br-cAMP could induce β-galactosidase (β-Gal) recombinant protein expression by 1.6-fold. Furthermore, Br-cAMP can additionally enhance the β-Gal production (from 2.8- to 4.5-fold) when CHO cells were stimulated with glycerol, thymidine, dimethyl sulfoxide, pentanoic acid, or sodium butyrate. Thus, Br-cAMP may be used as an alternative agent in promoting foreign protein expression for CHO cells. Copyright © 2017. Published by Elsevier B.V.

  7. An odor-specific threshold deficit implicates abnormal intracellular cyclic AMP signaling in schizophrenia.

    Science.gov (United States)

    Turetsky, Bruce I; Moberg, Paul J

    2009-02-01

    Although olfactory deficits are common in schizophrenia, their underlying pathophysiology remains unknown. Recent evidence has suggested that cAMP signaling may be disrupted in schizophrenia. Since cAMP mediates signal transduction in olfactory receptor neurons, this could contribute to the etiology of observed olfactory deficits. This study was designed to test this hypothesis by determining odor detection threshold sensitivities to two odorants that differ in their relative activations of this intracellular cAMP signaling cascade. Thirty schizophrenia patients, 25 healthy comparison subjects, and 19 unaffected first-degree relatives of schizophrenia patients were studied. Odor detection threshold sensitivities were measured for the two odorants citralva and lyral. Although both have fruity/floral scents, citralva strongly activates adenylyl cyclase to increase cAMP levels, while lyral is a very weak activator of adenylyl cyclase. There was a significant group-by-odor interaction. Both schizophrenia patients and unaffected first-degree relatives were impaired in their ability to detect lyral versus citralva. Comparison subjects were equally sensitive to both odorants. This selective deficit could not be explained by differences in age, sex, smoking, clinical symptom profile, or medication use. This study establishes the presence of an odor-specific hyposmia that may denote a disruption of cAMP-mediated signal transduction in schizophrenia. The presence of a parallel deficit in the patients' unaffected first-degree relatives suggests that this deficit is genetically mediated. Although additional physiological studies are needed to confirm the underlying mechanism, these results offer strong inferential support for the hypothesis that cAMP signaling is dysregulated in schizophrenia.

  8. Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases

    DEFF Research Database (Denmark)

    Issinger, O G; Beier, H; Speichermann, N

    1980-01-01

    Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were...... by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins...... from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates...

  9. Attempt to demonstrate an in vivo effect of mianserin hydrochloride on erythrocyte Na+-K+-ATPase activity and cyclic AMP concentration

    Science.gov (United States)

    Naylor, G. S.; Buckley, D. E.; Boardman, L. J.; Smith, A. H. W.; Moody, J. P.

    1978-01-01

    1 There is evidence that erythrocyte Na+-K+-ATPase activity and erythrocyte cyclic AMP change on recovery from a depressive illness. Mianserin is a recently introduced antidepressant but its mode of action is unknown. The present study was therefore designed to investigate in vivo the effect of mianserin on erythrocyte Na+-K+-ATPase and cyclic AMP. 2 Biochemical estimations were made on blood from depressed patients before beginning either mianserin or matched placebo treatment, after 1 week, and again after 2 weeks' treatment. 3 Neither the erythrocyte Na+-K+-ATPase, nor the erythrocyte cyclic AMP concentration, changed significantly in either the mianserin- or the placebo-treated group. 4 The study sheds no light on the possible mechanism of action of mianserin. PMID:203308

  10. Parathyroid hormone promotes the disassembly of cytoskeletal actin and myosin in cultured osteoblastic cells: Mediation by cyclic AMP

    International Nuclear Information System (INIS)

    Egan, J.J.; Gronowicz, G.; Rodan, G.A.

    1991-01-01

    Parathyroid hormone (PTH) alters the shape of osteoblastic cells both in vivo and in vitro. In this study, we examined the effect of PTH on cytoskeletal actin and myosin, estimated by polyacrylamide gel electrophoresis of Triton X-100 (1%) nonextractable proteins. After 2-5 minutes, PTH caused a rapid and transient decrease of 50-60% in polymerized actin and myosin associated with the Triton X-100 nonextractable cytoskeleton. Polymerized actin returned to control levels by 30 min. The PTH effect was dose-dependent with an IC50 of about 1 nM, and was partially inhibited by the (3-34) PTH antagonist. PTH caused a rapid transient rise in cyclic AMP (cAMP) in these cells that peaked at 4 min, while the nadir in cytoskeletal actin and myosin was recorded around 5 min. The intracellular calcium chelator Quin-2/AM (10 microM) also decreased cytoskeletal actin and myosin, to the same extent as did PTH (100 nM). To distinguish between cAMP elevation and Ca++ reduction as mediators of PTH action, we measured the phosphorylation of the 20 kD (PI 4.9) myosin light chain in cells preincubated with [32P]-orthophosphate. The phosphorylation of this protein decreased within 2-3 min after PTH addition and returned to control levels after 5 min. The calcium ionophore A-23187 did not antagonize this PTH effect. Visualization of microfilaments with rhodamine-conjugated phalloidin showed that PTH altered the cytoskeleton by decreasing the number of stress fibers. These changes in the cytoskeleton paralleled changes in the shape of the cells from a spread configuration to a stellate form with retracting processes. The above findings indicate that the alteration in osteoblast shape produced by PTH involve relatively rapid and transient changes in cytoskeletal organization that appear to be mediated by cAMP

  11. Export of cyclic AMP by avian red cells and inhibition by prostaglandin A1

    International Nuclear Information System (INIS)

    Heasley, L.E.

    1985-01-01

    The mechanism by which PGA 1 inhibits cAMP export by avian red cells was studied, to provide details on the molecular mechanism of a prostaglandin action and on the process of cAMP export itself. The interaction of PGA 1 with pigeon red cells is a multi-step process of uptake, metabolism and secretion. [ 3 H]PGA rapidly enters red cells and is promptly metabolized (V/sub max/ ≥ 1 nmol/min/10 7 cells) to a compound (5) that remains in the aqueous layer after ethyl acetate extraction. Chromatographic analyses, amino acid content and fast atom bombardment mass spectrometry reveal that the polar metabolite is conjugated with glutathione (PGA 1 -GSH) at C-11 via a thioether bond and is largely (80%) reduced to the C-9 hydroxyl derivative

  12. Protein kinases mediate increment of the phosphorylation of cyclic AMP -responsive element binding protein in spinal cord of rats following capsaicin injection

    Directory of Open Access Journals (Sweden)

    Li Junfa

    2005-09-01

    Full Text Available Abstract Background Strong noxious stimuli cause plastic changes in spinal nociceptive neurons. Intracellular signal transduction pathways from cellular membrane to nucleus, which may further regulate gene expression by critical transcription factors, convey peripheral stimulation. Cyclic AMP-responsive element binding protein (CREB is a well-characterized stimulus-induced transcription factor whose activation requires phosphorylation of the Serine-133 residue. Phospho-CREB can further induce gene transcription and strengthen synaptic transmission by the activation of the protein kinase cascades. However, little is known about the mechanisms by which CREB phosphorylation is regulated by protein kinases during nociception. This study was designed to use Western blot analysis to investigate the role of mitogen-activated protein (MAP/extracellular signal-regulated kinase (ERK kinase (MEK 1/2, PKA and PKC in regulating the phosphorylation of CREB in the spinal cord of rats following intraplantar capsaicin injection. Results We found that capsaicin injection significantly increased the phosphorylation level of CREB in the ipsilateral side of the spinal cord. Pharmacological manipulation of MEK 1/2, PKA and PKC with their inhibitors (U0126, H89 and NPC 15473, respectively significantly blocked this increment of CREB phosphorylation. However, the expression of CREB itself showed no change in any group. Conclusion These findings suggest that the activation of intracellular MAP kinase, PKA and PKC cascades may contribute to the regulation of phospho-CREB in central nociceptive neurons following peripheral painful stimuli.

  13. Interplay of the modified nucleotide phosphoadenosine 5'-phosphosulfate (PAPS) with global regulatory proteins in Escherichia coli: modulation of cyclic AMP (cAMP)-dependent gene expression and interaction with the HupA regulatory protein.

    Science.gov (United States)

    Longo, Francesca; Motta, Sara; Mauri, Pierluigi; Landini, Paolo; Rossi, Elio

    2016-11-25

    In the bacterium Escherichia coli, some intermediates of the sulfate assimilation and cysteine biosynthesis pathway can act as signal molecules and modulate gene expression. In addition to sensing and utilization of sulphur sources, these signaling mechanisms also impact more global cell processes, such as resistance to antimicrobial agents and biofilm formation. In a recent work, we have shown that inactivation of the cysH gene, encoding phosphoadenosine-phosphosulfate (PAPS) reductase, and the consequent increase in intracellular PAPS concentration, strongly affect production of several cell surface-associated structures, enhancing surface adhesion and cell aggregation. In order to identify the molecular mechanism relaying intracellular PAPS concentration to regulation of cell surface-associated structures, we looked for mutations able to suppress the effects of cysH inactivation. We found that mutations in the adenylate cyclase-encoding cyaA gene abolished the effects of PAPS accumulation; consistent with this result, cyclic AMP (cAMP)-dependent gene expression appears to be increased in the cysH mutant. Experiments aimed at the direct identification of proteins interacting with either CysC or CysH, i.e. the PAPS-related proteins APS kinase and PAPS reductase, allowed us to identify several regulators, namely, CspC, CspE, HNS and HupA. Protein-protein interaction between HupA and CysH was confirmed by a bacterial two hybrid system, and inactivation of the hupA gene enhanced the effects of the cysH mutation in terms of production of cell surface-associated factors. Our results indicate that PAPS can modulate different regulatory systems, providing evidence that this molecule acts as a global signal molecule in E. coli. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. Cyclic GMP-AMP Synthase is a Cytosolic DNA Sensor that Activates the Type-I Interferon Pathway

    Science.gov (United States)

    Sun, Lijun; Wu, Jiaxi; Du, Fenghe; Chen, Xiang; Chen, Zhijian J.

    2013-01-01

    The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers the host immune responses such as the production of type-I interferons (IFN). Cytosolic DNA induces IFN through the production of cyclic-GMP-AMP (cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced IFNβ in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and IFNβ induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP. PMID:23258413

  15. Leptin interferes with 3',5'-Cyclic Adenosine Monophosphate (cAMP signaling to inhibit steroidogenesis in human granulosa cells

    Directory of Open Access Journals (Sweden)

    HoYuen Basil

    2009-10-01

    Full Text Available Abstract Background Obesity has been linked to an increased risk of female infertility. Leptin, an adipocytokine which is elevated during obesity, may influence gonadal function through modulating steroidogenesis in granulosa cells. Methods The effect of leptin on progesterone production in simian virus 40 immortalized granulosa (SVOG cells was examined by Enzyme linked immunosorbent assay (ELISA. The effect of leptin on the expression of the steroidogenic enzymes (StAR, P450scc, 3betaHSD in SVOG cells was examined by real-time PCR and Western blotting. The mRNA expression of leptin receptor isoforms in SVOG cells were examined by using PCR. SVOG cells were co-treated with leptin and specific pharmacological inhibitors to identify the signaling pathways involved in leptin-reduced progesterone production. Silencing RNA against leptin receptor was used to determine that the inhibition of leptin on cAMP-induced steroidogenesis acts in a leptin receptor-dependent manner. Results and Conclusion In the present study, we investigated the cellular mechanisms underlying leptin-regulated steroidogenesis in human granulosa cells. We show that leptin inhibits 8-bromo cAMP-stimulated progesterone production in a concentration-dependent manner. Furthermore, we show that leptin inhibits expression of the cAMP-stimulated steroidogenic acute regulatory (StAR protein, the rate limiting de novo protein in progesterone synthesis. Leptin induces the activation of ERK1/2, p38 and JNK but only the ERK1/2 (PD98059 and p38 (SB203580 inhibitors attenuate the leptin-induced inhibition of cAMP-stimulated StAR protein expression and progesterone production. These data suggest that the leptin-induced MAPK signal transduction pathway interferes with cAMP/PKA-stimulated steroidogenesis in human granulosa cells. Moreover, siRNA mediated knock-down of the endogenous leptin receptor attenuates the effect of leptin on cAMP-induced StAR protein expression and progesterone

  16. Enhanced phosphorylation of cyclic AMP response element binding protein in Brain of mice following repetitive hypoxic exposure

    International Nuclear Information System (INIS)

    Gao Yanan; Gao Ge; Long Caixia; Han Song; Zu Pengyu; Fang Li; Li Junfa

    2006-01-01

    Cerebral ischemic/hypoxic preconditioning (I/HPC) is a phenomenon of endogenous protection that renders Brain tolerant to sustained ischemia/hypoxia. This profound protection induced by I/HPC makes it an attractive target for developing potential clinical therapeutic approaches. However, the molecular mechanism of I/HPC is unclear. Cyclic AMP (cAMP) response element binding protein (CREB), a selective nuclear transcriptional factor, plays a key role in the neuronal functions. Phosphorylation of CREB on Ser-133 may facilitate its transcriptional activity in response to various stresses. In the current study, we observed the changes in CREB phosphorylation (Ser-133) and protein expression in Brain of auto-hypoxia-induced HPC mice by using Western blot analysis. We found that the levels of phosphorylated CREB (Ser-133), but not protein expression of CREB, increased significantly (p < 0.05) in the hippocampus and the frontal cortex of mice after repetitive hypoxic exposure (H2-H4, n = 6 for each group), when compared to that of the normoxic (H0, n = 6) or hypoxic exposure once group (H1, n = 6). In addition, a significant enhancement (p < 0.05) of CREB phosphorylation (Ser-133) could also be found in the nuclear extracts from the whole hippocampus of hypoxic preconditioned mice (H2-H4, n = 6 for each group). These results suggest that the phosphorylation of CREB might be involved in the development of cerebral hypoxic preconditioning

  17. Adrenomedullin stimulates cyclic AMP production in the airway epithelial cells of guinea-pigs and in the human epithelial cell line

    Directory of Open Access Journals (Sweden)

    Takashi Kawaguchi

    1999-01-01

    Full Text Available This study was designed to examine the effects of adrenomedullin (AM on airway epithelial cells. Primary cultures of guinea-pig tracheal epithelial cells and the human bronchiolar epithelial cell line NCI-H441 were used. Intracellular cyclic adenosine monophosphate (cAMP, cyclic guanosine monophosphate (cGMP, prostaglandin E2 (PGE2, and stable end-products of nitric oxide were assayed. Adrenomedullin (10−6 mol/L stimulated cAMP production in guinea-pig epithelial cells. Indomethacin (10−5 mol/L significantly decreased the basal level of intracellular cAMP in guinea-pig epithelial cells, but not in NCI-H441 cells. However, AM did not stimulate production of PGE2, a major product that can increase cAMP formation. In the case of NCI-H441 cells, AM (10−8 – 10−6 mol/L did not significantly affect intracellular cGMP levels or nitrite content in conditioned medium. Adrenomedullin and calcitonin gene-related peptide (CGRP each stimulated cAMP production in NCI-H441 cells, but AM-stimulated cAMP production was antagonized by the CGRP fragment CGRP8–37. These findings suggest that AM stimulates cAMP production and functionally competes with CGRP for binding sites in airway epithelial cells, at least in human epithelial cells, but that it does not stimulate the release of PGE2 and nitric oxide. Though cyclooxygenase products contribute to some extent to cAMP formation in guinea-pigs, AM independently stimulates intracellular cAMP formation in airway epithelial cells.

  18. Influence of sulphate on effects of ADP, 3′,5′-cyclic amp and citrate on human platelet phosphofructokinase activity

    NARCIS (Netherlands)

    Akkerman, Jan Willem N.; Gorter, G.; Sixma, J.J.; Staal, Gerard E.J.

    1974-01-01

    1. 1. Sulphate ions activate partially-purified human platelet phosphofructokinase. This is caused by suppression of the cooperativity of the enzyme with respect to fructose 6-phosphate. 2. 2. Sulphate therefore markedly affects the influences of allosteric modifiers such as ADP, 3′,5′-cyclic AMP

  19. Dual effects of muscarinic M2 acetylcholine receptors on the synthesis of cyclic AMP in CHO cells: dependence on time, receptor density and receptor agonists

    Czech Academy of Sciences Publication Activity Database

    Michal, Pavel; Lysíková, Michaela; Tuček, Stanislav

    2001-01-01

    Roč. 132, č. 6 (2001), s. 1217-1228 ISSN 0007-1188 R&D Projects: GA ČR GA309/99/0214; GA AV ČR IAA7011910 Institutional research plan: CEZ:AV0Z5011922 Keywords : cyclic AMP * muscarinic receptors * CHO cells Subject RIV: ED - Physiology Impact factor: 3.502, year: 2001

  20. Characterization of the regulatory subunit from brain cyclic AMP-dependent protein kinase II

    International Nuclear Information System (INIS)

    Stein, J.C.

    1985-01-01

    Tryptic peptides derived from the regulatory subunits of brain and heart cAMP-dependent protein kinase II were mapped by reverse phase HPLC. At 280 nm, 15 unique peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. At 210 nm, 13 brain-RII specific and 15 heart-RII specific tryptic peptides were identified and resolved. Two-dimensional mapping analyses revealed that several 37 P-labeled tryptic fragments derived from the autophosphorylation and the photoaffinity labeled cAMP-binding sites of brain RII were separate and distinct from the 32 P-peptides isolated from similarly treated heart RII. The tryptic phosphopeptide containing the autophosphorylation site in brain RII was purified. The sequence and phosphorylation site is: Arg-Ala-Ser(P)-Val-Cys-Ala-Glu-Ala-Tyr-Asn-Pro-Asp-Glu-Glu-Glu-Asp-Asp-Ala-Glu. Astrocytes and neurons exhibit high levels of the brain RII enzyme, while oligodendrocytes contain the heart RII enzyme. Monoclonal antibodies to bovine cerebral cortex RII were made and characterized. The antibodies elucidated a subtle difference between membrane-associated and cytosolic RII from cerebral cortex

  1. Effect of Increased Cyclic AMP Concentration on Muscle Protein Synthesis and Beta-Adrenergic Receptor Expression in Chicken Skeletal Muscle Cells in Culture

    Science.gov (United States)

    Young, R. B.; Vaughn, J. R.; Bridge, K. Y.; Smith, C. K.

    1998-01-01

    Analogies of epinephrine are known to cause hypertrophy of skeletal muscle when fed to animals. These compounds presumably exert their physiological action through interaction with the P-adrenergic receptor. Since the intracellular signal generated by the Beta-adrenergic receptor is cyclic AMP (cAMP), experiments were initiated in cell culture to determine if artificial elevation of cAMP by treatment with forskolin would alter muscle protein metabolism and P-adrenergic receptor expression. Chicken skeletal muscle cells after 7 days in culture were treated with 0.2-30 micrometers forskolin for a total of three days. At the end of the treatment period, both the concentration of cAMP and the quantity of myosin heavy chain (MHC) were measured. Concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. In contrast, the quantity of MHC was increased approximately 50% above control cells at 0.2 micrometers forskolin, but exhibited a gradual decline at higher levels of forskolin so that the quantity of MHC in cells treated with 30 micrometers forskolin was not significantly different from controls. Curiously, the intracellular concentration of cAMP which elicited the maximum increase in the quantity of MHC was only 40% higher than cAMP concentration in control cells.

  2. PdeH, a high-affinity cAMP phosphodiesterase, is a key regulator of asexual and pathogenic differentiation in Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Ravikrishna Ramanujam

    2010-05-01

    Full Text Available Cyclic AMP-dependent pathways mediate the communication between external stimuli and the intracellular signaling machinery, thereby influencing important aspects of cellular growth, morphogenesis and differentiation. Crucial to proper function and robustness of these signaling cascades is the strict regulation and maintenance of intracellular levels of cAMP through a fine balance between biosynthesis (by adenylate cyclases and hydrolysis (by cAMP phosphodiesterases. We functionally characterized gene-deletion mutants of a high-affinity (PdeH and a low-affinity (PdeL cAMP phosphodiesterase in order to gain insights into the spatial and temporal regulation of cAMP signaling in the rice-blast fungus Magnaporthe oryzae. In contrast to the expendable PdeL function, the PdeH activity was found to be a key regulator of asexual and pathogenic development in M. oryzae. Loss of PdeH led to increased accumulation of intracellular cAMP during vegetative and infectious growth. Furthermore, the pdeHDelta showed enhanced conidiation (2-3 fold, precocious appressorial development, loss of surface dependency during pathogenesis, and highly reduced in planta growth and host colonization. A pdeHDelta pdeLDelta mutant showed reduced conidiation, exhibited dramatically increased (approximately 10 fold cAMP levels relative to the wild type, and was completely defective in virulence. Exogenous addition of 8-Br-cAMP to the wild type simulated the pdeHDelta defects in conidiation as well as in planta growth and development. While a fully functional GFP-PdeH was cytosolic but associated dynamically with the plasma membrane and vesicular compartments, the GFP-PdeL localized predominantly to the nucleus. Based on data from cAMP measurements and Real-Time RTPCR, we uncover a PdeH-dependent biphasic regulation of cAMP levels during early and late stages of appressorial development in M. oryzae. We propose that PdeH-mediated sustenance and dynamic regulation of cAMP signaling

  3. PKA and PDE4D3 anchoring to AKAP9 provides distinct regulation of cAMP signals at the centrosome

    Science.gov (United States)

    Terrin, Anna; Monterisi, Stefania; Stangherlin, Alessandra; Zoccarato, Anna; Koschinski, Andreas; Surdo, Nicoletta C.; Mongillo, Marco; Sawa, Akira; Jordanides, Niove E.; Mountford, Joanne C.

    2012-01-01

    Previous work has shown that the protein kinase A (PKA)–regulated phosphodiesterase (PDE) 4D3 binds to A kinase–anchoring proteins (AKAPs). One such protein, AKAP9, localizes to the centrosome. In this paper, we investigate whether a PKA–PDE4D3–AKAP9 complex can generate spatial compartmentalization of cyclic adenosine monophosphate (cAMP) signaling at the centrosome. Real-time imaging of fluorescence resonance energy transfer reporters shows that centrosomal PDE4D3 modulated a dynamic microdomain within which cAMP concentration selectively changed over the cell cycle. AKAP9-anchored, centrosomal PKA showed a reduced activation threshold as a consequence of increased autophosphorylation of its regulatory subunit at S114. Finally, disruption of the centrosomal cAMP microdomain by local displacement of PDE4D3 impaired cell cycle progression as a result of accumulation of cells in prophase. Our findings describe a novel mechanism of PKA activity regulation that relies on binding to AKAPs and consequent modulation of the enzyme activation threshold rather than on overall changes in cAMP levels. Further, we provide for the first time direct evidence that control of cell cycle progression relies on unique regulation of centrosomal cAMP/PKA signals. PMID:22908311

  4. Increase of Intracellular Cyclic AMP by PDE4 Inhibitors Affects HepG2 Cell Cycle Progression and Survival.

    Science.gov (United States)

    Massimi, Mara; Cardarelli, Silvia; Galli, Francesca; Giardi, Maria Federica; Ragusa, Federica; Panera, Nadia; Cinque, Benedetta; Cifone, Maria Grazia; Biagioni, Stefano; Giorgi, Mauro

    2017-06-01

    Type 4 cyclic nucleotide phosphodiesterases (PDE4) are major members of a superfamily of enzymes (PDE) involved in modulation of intracellular signaling mediated by cAMP. Broadly expressed in most human tissues and present in large amounts in the liver, PDEs have in the last decade been key therapeutic targets for several inflammatory diseases. Recently, a significant body of work has underscored their involvement in different kinds of cancer, but with no attention paid to liver cancer. The present study investigated the effects of two PDE4 inhibitors, rolipram and DC-TA-46, on the growth of human hepatoma HepG2 cells. Treatment with these inhibitors caused a marked increase of intracellular cAMP level and a dose- and time-dependent effect on cell growth. The concentrations of inhibitors that halved cell proliferation to about 50% were used for cell cycle experiments. Rolipram (10 μM) and DC-TA-46 (0.5 μM) produced a decrease of cyclin expression, in particular of cyclin A, as well as an increase in p21, p27 and p53, as evaluated by Western blot analysis. Changes in the intracellular localization of cyclin D1 were also observed after treatments. In addition, both inhibitors caused apoptosis, as demonstrated by an Annexin-V cytofluorimetric assay and analysis of caspase-3/7 activity. Results demonstrated that treatment with PDE4 inhibitors affected HepG2 cell cycle and survival, suggesting that they might be useful as potential adjuvant, chemotherapeutic or chemopreventive agents in hepatocellular carcinoma. J. Cell. Biochem. 118: 1401-1411, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  5. Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses.

    Science.gov (United States)

    Gao, Daxing; Wu, Jiaxi; Wu, You-Tong; Du, Fenghe; Aroh, Chukwuemika; Yan, Nan; Sun, Lijun; Chen, Zhijian J

    2013-08-23

    Retroviruses, including HIV, can activate innate immune responses, but the host sensors for retroviruses are largely unknown. Here we show that HIV infection activates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) to produce cGAMP, which binds to and activates the adaptor protein STING to induce type I interferons and other cytokines. Inhibitors of HIV reverse transcriptase, but not integrase, abrogated interferon-β induction by the virus, suggesting that the reverse-transcribed HIV DNA triggers the innate immune response. Knockout or knockdown of cGAS in mouse or human cell lines blocked cytokine induction by HIV, murine leukemia virus, and simian immunodeficiency virus. These results indicate that cGAS is an innate immune sensor of HIV and other retroviruses.

  6. Acute morphine alters GABAergic transmission in the central amygdala during naloxone-precipitated morphine withdrawal: role of cyclic AMP

    Directory of Open Access Journals (Sweden)

    Michal eBajo

    2014-06-01

    Full Text Available The central amygdala (CeA plays an important role in opioid addiction. Therefore, we examined the effects of naloxone-precipitated morphine withdrawal (WD on GABAergic transmission in rat CeA neurons using whole-cell recordings with naloxone in the bath. The basal frequency of miniature inhibitory postsynaptic currents (mIPSCs increased in CeA neurons from WD compared to placebo rats. Acute morphine (10 M had mixed effects (> 20% change from baseline on mIPSCs in placebo and WD rats. In most CeA neurons (64% from placebo rats, morphine significantly decreased mIPSC frequency and amplitude. In 32% of placebo neurons, morphine significantly increased mIPSC amplitudes but had no effect on mIPSC frequency. In WD rats, acute morphine significantly increased mIPSC frequency but had no effect on mIPSC amplitude in 41% of CeA neurons. In 45% of cells, acute morphine significantly decreased mIPSC frequency and amplitude. Pre-treatment with the cyclic AMP inhibitor (R-adenosine, cyclic 3’,5’-(hydrogenphosphorothioate triethylammonium (RP, prevented acute morphine-induced potentiation of mIPSCs. Pre-treatment of slices with the Gi/o G-protein subunit inhibitor pertussis toxin (PTX did not prevent the acute morphine-induced enhancement or inhibition of mIPSCs. PTX and RP decreased basal mIPSC frequencies and amplitudes only in WD rats. The results suggest that inhibition of GABAergic transmission in the CeA by acute morphine is mediated by PTX-insensitive mechanisms, although PTX-sensitive mechanisms cannot be ruled out for non-morphine responsive cells; by contrast, potentiation of GABAergic transmission is mediated by activated cAMP signaling that also mediates the increased basal GABAergic transmission in WD rats. Our data indicate that during the acute phase of WD, the CeA opioid and GABAergic systems undergo neuroadaptative changes conditioned by a previous chronic morphine exposure and dependence.

  7. Cyclic nucleotide dependent dephosphorylation of regulator of G-protein signaling 18 in human platelets.

    LENUS (Irish Health Repository)

    Gegenbauer, Kristina

    2013-11-01

    Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets.

  8. Cyclic GMP-AMP Synthase Is an Innate Immune DNA Sensor for Mycobacterium tuberculosis.

    Science.gov (United States)

    Collins, Angela C; Cai, Haocheng; Li, Tuo; Franco, Luis H; Li, Xiao-Dong; Nair, Vidhya R; Scharn, Caitlyn R; Stamm, Chelsea E; Levine, Beth; Chen, Zhijian J; Shiloh, Michael U

    2015-06-10

    Activation of the DNA-dependent cytosolic surveillance pathway in response to Mycobacterium tuberculosis infection stimulates ubiquitin-dependent autophagy and inflammatory cytokine production, and plays an important role in host defense against M. tuberculosis. However, the identity of the host sensor for M. tuberculosis DNA is unknown. Here we show that M. tuberculosis activated cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) in macrophages to produce cGAMP, a second messenger that activates the adaptor protein stimulator of interferon genes (STING) to induce type I interferons and other cytokines. cGAS localized with M. tuberculosis in mouse and human cells and in human tuberculosis lesions. Knockdown or knockout of cGAS in human or mouse macrophages blocked cytokine production and induction of autophagy. Mice deficient in cGAS were more susceptible to lethality caused by infection with M. tuberculosis. These results demonstrate that cGAS is a vital innate immune sensor of M. tuberculosis infection. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. The small molecule triclabendazole decreases the intracellular level of cyclic AMP and increases resistance to stress in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Yong Joo Lee

    Full Text Available The Ras-adenylyl cyclase-protein kinase A nutrient-sensing pathway controls metabolism, proliferation and resistance to stress in Saccharomyces cerevisiae. The genetic disruption of this pathway increases resistance to a variety of stresses. We show here that the pharmacological inhibition of this pathway by the drug triclabendazole increases resistance to oxidants, heat stress and extends the chronological life. Evidence is presented that triclabendazole decreases the intracellular level of cyclic AMP by inhibiting adenylyl cyclase and triggers the parallel rapid translocation of the stress-resistance transcription factor Msn2 from the cytosol into the nucleus, as deduced from experiments employing a strain in which MSN2 is replaced with MSN2-GFP (GFP, green fluorescent protein. Msn2 and Msn4 are responsible for activating the transcription of numerous genes that encode proteins that protect cells from stress. The results are consistent with triclabendazole either inhibiting the association of Ras with adenylyl cyclase or directly inhibiting adenylyl cyclase, which in turn triggers Msn2/4 to enter the nucleus and activate stress-responsible element gene expression.

  10. Cyclic GMP-AMP Synthase Is Required for Cell Proliferation and Inflammatory Responses in Rheumatoid Arthritis Synoviocytes

    Directory of Open Access Journals (Sweden)

    Yan Wang

    2015-01-01

    Full Text Available Rheumatoid arthritis (RA is characterized by inflammatory cell infiltration, fibroblast-like synoviocytes (FLS invasive proliferation, and joint destruction. Cyclic GMP-AMP synthase (cGAS is a cytosolic DNA sensor that induces immune activation. In this study, we examined whether cGAS plays a role in RA FLS. In this study, cGAS was overexpressed in RA-FLS compared with OA FLS. TNFα stimulation induced cGAS expression in RA FLS. Overexpression of cGAS promoted the proliferation and knockdown of cGAS inhibited the proliferation of RA FLS. cGAS overexpression enhanced the production of proinflammatory cytokines and matrix metalloproteinases (MMPs as well as AKT and ERK phosphorylation in TNFα-stimulated FLS. In contrast, cGAS silencing inhibited production of proinflammatory cytokines and matrix metalloproteinases (MMPs as well as AKT and ERK phosphorylation in TNFα-stimulated FLS. These results suggest that cGAS activates the AKT and ERK pathways to promote the inflammatory response of RA FLS, and the development of strategies targeting cGAS may have therapeutic potential for human RA.

  11. The catalytic mechanism of cyclic GMP-AMP synthase (cGAS) and implications for innate immunity and inhibition.

    Science.gov (United States)

    Hall, Justin; Ralph, Erik C; Shanker, Suman; Wang, Hong; Byrnes, Laura J; Horst, Reto; Wong, Jimson; Brault, Amy; Dumlao, Darren; Smith, James F; Dakin, Leslie A; Schmitt, Daniel C; Trujillo, John; Vincent, Fabien; Griffor, Matt; Aulabaugh, Ann E

    2017-12-01

    Cyclic GMP-AMP synthase (cGAS) is activated by ds-DNA binding to produce the secondary messenger 2',3'-cGAMP. cGAS is an important control point in the innate immune response; dysregulation of the cGAS pathway is linked to autoimmune diseases while targeted stimulation may be of benefit in immunoncology. We report here the structure of cGAS with dinucleotides and small molecule inhibitors, and kinetic studies of the cGAS mechanism. Our structural work supports the understanding of how ds-DNA activates cGAS, suggesting a site for small molecule binders that may cause cGAS activation at physiological ATP concentrations, and an apparent hotspot for inhibitor binding. Mechanistic studies of cGAS provide the first kinetic constants for 2',3'-cGAMP formation, and interestingly, describe a catalytic mechanism where 2',3'-cGAMP may be a minor product of cGAS compared with linear nucleotides. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

  12. Cyclic GMP-AMP Synthase Is Required for Cell Proliferation and Inflammatory Responses in Rheumatoid Arthritis Synoviocytes.

    Science.gov (United States)

    Wang, Yan; Su, Guo-Hua; Zhang, Fang; Chu, Jing-Xue; Wang, Yun-Shan

    2015-01-01

    Rheumatoid arthritis (RA) is characterized by inflammatory cell infiltration, fibroblast-like synoviocytes (FLS) invasive proliferation, and joint destruction. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces immune activation. In this study, we examined whether cGAS plays a role in RA FLS. In this study, cGAS was overexpressed in RA-FLS compared with OA FLS. TNFα stimulation induced cGAS expression in RA FLS. Overexpression of cGAS promoted the proliferation and knockdown of cGAS inhibited the proliferation of RA FLS. cGAS overexpression enhanced the production of proinflammatory cytokines and matrix metalloproteinases (MMPs) as well as AKT and ERK phosphorylation in TNFα-stimulated FLS. In contrast, cGAS silencing inhibited production of proinflammatory cytokines and matrix metalloproteinases (MMPs) as well as AKT and ERK phosphorylation in TNFα-stimulated FLS. These results suggest that cGAS activates the AKT and ERK pathways to promote the inflammatory response of RA FLS, and the development of strategies targeting cGAS may have therapeutic potential for human RA.

  13. Cutting edge: Antimalarial drugs inhibit IFN-β production through blockade of cyclic GMP-AMP synthase-DNA interaction.

    Science.gov (United States)

    An, Jie; Woodward, Joshua J; Sasaki, Tomikazu; Minie, Mark; Elkon, Keith B

    2015-05-01

    Type I IFN is strongly implicated in the pathogenesis of systemic autoimmune diseases, such as lupus, and rare monogenic IFNopathies, including Aicardi-Goutières syndrome. Recently, a new DNA-activated pathway involving the enzyme cyclic GMP-AMP synthase (cGAS) was described and potentially linked to Aicardi-Goutières syndrome. To identify drugs that could potentially inhibit cGAS activity, we performed in silico screening of drug libraries. By computational analysis, we identified several antimalarial drugs (AMDs) that were predicted to interact with the cGAS/dsDNA complex. Our studies validated that several AMDs were effective inhibitors of IFN-β production and that they functioned by inhibiting dsDNA stimulation of cGAS. Because AMDs have been widely used in human diseases and have an excellent safety profile, our findings suggest new therapeutic strategies for the treatment of severe debilitating diseases associated with type I IFNs due to cGAS activation. Copyright © 2015 by The American Association of Immunologists, Inc.

  14. [Effects of cytosolic bacteria on cyclic GMP-AMP synthase expression in human gingival tissues and periodontal ligament cells].

    Science.gov (United States)

    Xiaojun, Yang; Yongmei, Tan; Zhihui, Tian; Ting, Zhou; Wanghong, Zhao; Jin, Hou

    2017-04-01

    This work aims to determine the effect of cytosolic bacteria on the expression of cyclic GMP-AMP synthase (cGAS) in human periodontal ligament cells (hPDLCs) and gingival tissues. The ability of Porphyromonas gingivalis (P. gingivalis) to invade hPDLCs was detected using laser scanning confocal microscope assay at a multiplicity of infection of 10. P. gingivalis-infected cells were sorted by fluorescence-activated cell sorting (FACS). Then, quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect cGAS expression in infected cells. Finally, the location and expression of cGAS in inflammatory and normal gingival tissues were investigated by immunohistochemistry. P. gingivalis actively invaded hPDLCs. Moreover, cGAS expression significantly increased in P. gingivalis-infected cells. Although cGAS was expressed in the epithelial and subepithelial cells of both inflamed and normal gingival tissues, cGAS expression significantly increased in inflamed gingival tissues. Cytosolic bacteria can upregulate cGAS expression in infected cells. These data suggest that cGAS may act as pattern-recognition receptors and participate in recognizing cytosolic nucleic acid pathogen-associated molecular patterns.
.

  15. A family of octopamine [corrected] receptors that specifically induce cyclic AMP production or Ca2+ release in Drosophila melanogaster.

    Science.gov (United States)

    Balfanz, Sabine; Strünker, Timo; Frings, Stephan; Baumann, Arnd

    2005-04-01

    In invertebrates, the biogenic-amine octopamine is an important physiological regulator. It controls and modulates neuronal development, circadian rhythm, locomotion, 'fight or flight' responses, as well as learning and memory. Octopamine mediates its effects by activation of different GTP-binding protein (G protein)-coupled receptor types, which induce either cAMP production or Ca(2+) release. Here we describe the functional characterization of two genes from Drosophila melanogaster that encode three octopamine receptors. The first gene (Dmoa1) codes for two polypeptides that are generated by alternative splicing. When heterologously expressed, both receptors cause oscillatory increases of the intracellular Ca(2+) concentration in response to applying nanomolar concentrations of octopamine. The second gene (Dmoa2) codes for a receptor that specifically activates adenylate cyclase and causes a rise of intracellular cAMP with an EC(50) of approximately 3 x 10(-8) m octopamine. Tyramine, the precursor of octopamine biosynthesis, activates all three receptors at > or = 100-fold higher concentrations, whereas dopamine and serotonin are non-effective. Developmental expression of Dmoa genes was assessed by RT-PCR. Overlapping but not identical expression patterns were observed for the individual transcripts. The genes characterized in this report encode unique receptors that display signature properties of native octopamine receptors.

  16. Cyclic adenosine 3',5'-monophosphate (cAMP) enhances cAMP-responsive element binding (CREB) protein phosphorylation and phospho-CREB interaction with the mouse steroidogenic acute regulatory protein gene promoter.

    Science.gov (United States)

    Clem, Brian F; Hudson, Elizabeth A; Clark, Barbara J

    2005-03-01

    Steroidogenic acute regulatory protein (StAR) transcription is regulated through cAMP-protein kinase A-dependent mechanisms that involve multiple transcription factors including the cAMP-responsive element binding protein (CREB) family members. Classically, binding of phosphorylated CREB to cis-acting cAMP-responsive elements (5'-TGACGTCA-3') within target gene promoters leads to recruitment of the coactivator CREB binding protein (CBP). Herein we examined the extent of CREB family member phosphorylation on protein-DNA interactions and CBP recruitment with the StAR promoter. Immunoblot analysis revealed that CREB, cAMP-responsive element modulator (CREM), and activating transcription factor (ATF)-1 are expressed in MA-10 mouse Leydig tumor cells, yet only CREB and ATF-1 are phosphorylated. (Bu)2cAMP treatment of MA-10 cells increased CREB phosphorylation approximately 2.3-fold within 30 min but did not change total nuclear CREB expression levels. Using DNA-affinity chromatography, we now show that CREB and ATF-1, but not CREM, interact with the StAR promoter, and this interaction is dependent on the activator protein-1 (AP-1) cis-acting element within the cAMP-responsive region. In addition, (Bu)2cAMP-treatment increased phosphorylated CREB (P-CREB) association with the StAR promoter but did not influence total CREB interaction. In vivo chromatin immunoprecipitation assays demonstrated CREB binding to the StAR proximal promoter is independent of (Bu)2cAMP-treatment, confirming our in vitro analysis. However, (Bu)2cAMP-treatment increased P-CREB and CBP interaction with the StAR promoter, demonstrating for the first time the physical role of P-CREB:DNA interactions in CBP recruitment to the StAR proximal promoter.

  17. Kinetics of activation of the P4 promoter of pBR322 by the Escherichia coli cyclic AMP receptor protein.

    Science.gov (United States)

    Hoggett, J G; Brierley, I

    1992-11-01

    The activation of transcription initiation from the P4 promoter of pBR322 by the Escherichia coli cyclic AMP receptor protein (CRP) has been investigated using a fluorescence abortive initiation assay. The effect of the cyclic-AMP/CRP complex on the linear P4 promoter was to increase the initial binding (KB) of RNA polymerase to the promoter by about a factor of 10, but the rate of isomerization of closed to open complex (kf) was unaffected. One molecule of CRP per promoter was required for activation, and the concentration of cyclic AMP producing half-maximal stimulation was about 7-8 microM. Supercoiling caused a 2-3-fold increase in the rate of isomerization of the CRP-activated promoter, but weakened the initial binding of polymerase by about one order of magnitude. The unactivated supercoiled promoter was too weak to allow reliable assessment of kinetic parameters against the high background rate originating from the rest of the plasmid.

  18. Cyclic 3',5'-adenosine monophosphate (cAMP) signaling in the anterior pituitary gland in health and disease.

    Science.gov (United States)

    Hernández-Ramírez, Laura C; Trivellin, Giampaolo; Stratakis, Constantine A

    2018-03-05

    The cyclic 3',5'-adenosine monophosphate (cAMP) was the first among the so-called "second messengers" to be described. It is conserved in most organisms and functions as a signal transducer by mediating the intracellular effects of multiple hormones and neurotransmitters. In this review, we first delineate how different members of the cAMP pathway ensure its correct compartmentalization and activity, mediate the terminal intracellular effects, and allow the crosstalk with other signaling pathways. We then focus on the pituitary gland, where cAMP exerts a crucial function by controlling the responsiveness of the cells to hypothalamic hormones, neurotransmitters and peripheral factors. We discuss the most relevant physiological functions mediated by cAMP in the different pituitary cell types, and summarize the defects affecting this pathway that have been reported in the literature. We finally discuss how a deregulated cAMP pathway is involved in the pathogenesis of pituitary disorders and how it affects the response to therapy. Copyright © 2017. Published by Elsevier B.V.

  19. Induction of dopamine biosynthesis by l-DOPA in PC12 cells: implications of L-DOPA influx and cyclic AMP.

    Science.gov (United States)

    Jin, Chun Mei; Yang, Yoo Jung; Huang, Hai Shan; Lim, Sung Cil; Kai, Masaaki; Lee, Myung Koo

    2008-09-04

    The effects of 3,4-dihydroxyphenylalanine (l-DOPA) on dopamine biosynthesis and cytotoxicity were investigated in PC12 cells. l-DOPA treatment (20-200 microM) increased the levels of dopamine by 226%-504% after 3-6 h of treatment and enhanced the activities of tyrosine hydroxylase (TH) and aromatic l-amino acid decarboxylase (AADC). l-DOPA (20-200 muM) treatment led to a 562%-937% increase in l-DOPA influx at 1 h, which inhibited the activity of TH, but not AADC, during the same period. The extracellular releases of dopamine were also increased by 231%-570% after treatment with 20 and 200 microM l-DOPA for 0.5-3 h. l-DOPA at a concentration of 100-200 microM, but not 20 microM, exerted apoptotic cytotoxicity towards PC12 cells for 24-48 h. l-DOPA (20-200 microM) increased the intracellular cyclic AMP levels by 318%-557% after 0.5-1 h in a concentration-dependent manner. However, the elevated cyclic AMP levels by l-DOPA could not protect against l-DOPA (100-200 microM)-induced cytotoxicity after 24-48 h. In addition, l-DOPA (20-200 microM)-induced increases in cyclic AMP and dopamine were significantly reduced by treatment with SCH23390 (dopamine D(1) receptor antagonist). The increased levels of dopamine by l-DOPA were also reduced by H89 (protein kinase A, PKA, inhibitor) and GF109203X (protein kinase C inhibitor); however, the reduction by GF109203X was not significant. l-DOPA at 20-200 microM stimulated the phosphorylation of PKA and cyclic AMP-response element binding protein and induced the biosynthesis of the TH protein. These results indicate that 20-200 microM l-DOPA induces dopamine biosynthesis by two pathways. One pathway involves l-DOPA directly entering the cells to convert dopamine through AADC activity (l-DOPA decarboxylation). The other pathway involves l-DOPA and/or released dopamine activating TH to enhance dopamine biosynthesis by the dopamine D(1) receptor-cyclic AMP-PKA signaling system (dopamine biosynthesis by TH).

  20. Inhibition of cyclic AMP response element-directed transcription by decoy oligonucleotides enhances tumor-specific radiosensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Park, Serk In, E-mail: serkin@korea.edu [Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of); The BK21 Plus Program for Biomedical Sciences, Korea University College of Medicine, Seoul (Korea, Republic of); Department of Medicine and Center for Bone Biology, Vanderbilt University School of Medicine, Nashville, TN (United States); Park, Sung-Jun [Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of); Laboratory of Obesity and Aging Research, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD (United States); Lee, Junghan; Kim, Hye Eun; Park, Su Jin; Sohn, Jeong-Won [Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of); Park, Yun Gyu, E-mail: parkyg@korea.ac.kr [Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of)

    2016-01-15

    The radiation stress induces cytotoxic responses of cell death as well as cytoprotective responses of cell survival. Understanding exact cellular mechanism and signal transduction pathways is important in improving cancer radiotherapy. Increasing evidence suggests that cyclic AMP response element binding protein (CREB)/activating transcription factor (ATF) family proteins act as a survival factor and a signaling molecule in response to stress. We postulated that CREB inhibition via CRE decoy oligonucleotide increases tumor cell sensitization to γ-irradiation-induced cytotoxic stress. In the present study, we demonstrate that CREB phosphorylation and CREB DNA-protein complex formation increased in time- and radiation dose-dependent manners, while there was no significant change in total protein level of CREB. In addition, CREB was phosphorylated in response to γ-irradiation through p38 MAPK pathway. Further investigation revealed that CREB blockade by decoy oligonucleotides functionally inhibited transactivation of CREB, and significantly increased radiosensitivity of multiple human cancer cell lines including TP53- and/or RB-mutated cells with minimal effects on normal cells. We also demonstrate that tumor cells ectopically expressing dominant negative mutant CREB (KCREB) and the cells treated with p38 MAPK inhibitors were more sensitive to γ-irradiation than wild type parental cells or control-treated cells. Taken together, we conclude that CREB protects tumor cells from γ-irradiation, and combination of CREB inhibition plus ionizing radiation will be a promising radiotherapeutic approach. - Highlights: • γ-Irradiation induced CREB phosphorylation and CRE-directed transcription in tumor. • γ-Irradiation-induced transcriptional activation of CREB was via p38 MAPK pathway. • CRE blockade increased radiosensitivity of tumor cells but not of normal cells. • CRE decoy oligonucleotides or p38 MAPK inhibitors can be used as radiosensitizers.

  1. Inhibition of cyclic AMP response element-directed transcription by decoy oligonucleotides enhances tumor-specific radiosensitivity

    International Nuclear Information System (INIS)

    Park, Serk In; Park, Sung-Jun; Lee, Junghan; Kim, Hye Eun; Park, Su Jin; Sohn, Jeong-Won; Park, Yun Gyu

    2016-01-01

    The radiation stress induces cytotoxic responses of cell death as well as cytoprotective responses of cell survival. Understanding exact cellular mechanism and signal transduction pathways is important in improving cancer radiotherapy. Increasing evidence suggests that cyclic AMP response element binding protein (CREB)/activating transcription factor (ATF) family proteins act as a survival factor and a signaling molecule in response to stress. We postulated that CREB inhibition via CRE decoy oligonucleotide increases tumor cell sensitization to γ-irradiation-induced cytotoxic stress. In the present study, we demonstrate that CREB phosphorylation and CREB DNA-protein complex formation increased in time- and radiation dose-dependent manners, while there was no significant change in total protein level of CREB. In addition, CREB was phosphorylated in response to γ-irradiation through p38 MAPK pathway. Further investigation revealed that CREB blockade by decoy oligonucleotides functionally inhibited transactivation of CREB, and significantly increased radiosensitivity of multiple human cancer cell lines including TP53- and/or RB-mutated cells with minimal effects on normal cells. We also demonstrate that tumor cells ectopically expressing dominant negative mutant CREB (KCREB) and the cells treated with p38 MAPK inhibitors were more sensitive to γ-irradiation than wild type parental cells or control-treated cells. Taken together, we conclude that CREB protects tumor cells from γ-irradiation, and combination of CREB inhibition plus ionizing radiation will be a promising radiotherapeutic approach. - Highlights: • γ-Irradiation induced CREB phosphorylation and CRE-directed transcription in tumor. • γ-Irradiation-induced transcriptional activation of CREB was via p38 MAPK pathway. • CRE blockade increased radiosensitivity of tumor cells but not of normal cells. • CRE decoy oligonucleotides or p38 MAPK inhibitors can be used as radiosensitizers.

  2. Presence of cyclic adenosine-3':5'-monophosphate (cAMP) in primary shoots of Zea mays L

    International Nuclear Information System (INIS)

    Edlich, W.; Graeser, H.

    1978-01-01

    The concentration of cAMP was determined in extracts of crude homogenates of maize seedlings, 3800 . g supernatants and isolated chloroplasts by the isotope dilution test. After extractpurification by precipitation with BaSO 4 and by chromatography on aluminium oxide column, a factor by means of which the binding of [8- 3 H]-cAMP with cAMP-dependent protein kinase was inhibited. The inhibitor was found inactive after treatment of the extracts with phosphodiesterase. In conclusion, this factor was identical with cAMP. It is suggested that cAMP-synthesis is localized in chloroplasts. Microbial contaminations which might disturb the detection of cAMP was excluded at least in the preparations of chloroplasts. (author)

  3. Regulation of AMP-activated protein kinase by LKB1 and CaMKK in adipocytes

    DEFF Research Database (Denmark)

    Gormand, Amélie; Henriksson, Emma; Ström, Kristoffer

    2011-01-01

    AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates cellular and whole body energy homeostasis. In adipose tissue, activation of AMPK has been demonstrated in response to a variety of extracellular stimuli. However, the upstream kinase that activates AMPK in adipocytes...

  4. Redox regulation of the AMP-activated protein kinase.

    Directory of Open Access Journals (Sweden)

    Yingying Han

    2010-11-01

    Full Text Available Redox state is a critical determinant of cell function, and any major imbalances can cause severe damage or death.The aim of this study is to determine if AMP-activated protein kinase (AMPK, a cellular energy sensor, is activated by oxidants generated by Berberine in endothelial cells (EC.Bovine aortic endothelial cells (BAEC were exposed to Berberine. AMPK activity and reactive oxygen species were monitored after the incubation.In BAEC, Berberine caused a dose- and time-dependent increase in the phosphorylation of AMPK at Thr172 and acetyl CoA carboxylase (ACC at Ser79, a well characterized downstream target of AMPK. Concomitantly, Berberine increased peroxynitrite, a potent oxidant formed by simultaneous generation of superoxide and nitric oxide. Pre-incubation of BAEC with anti-oxidants markedly attenuated Berberine-enhanced phosphorylation of both AMPK and ACC. Consistently, adenoviral expression of superoxide dismutase and pretreatment of L-N(G-Nitroarginine methyl ester (L-NAME; a non-selective NOS inhibitor blunted Berberine-induced phosphorylation of AMPK. Furthermore, mitochondria-targeted tempol (mito-tempol pretreatment or expression of uncoupling protein attenuated AMPK activation caused by Berberine. Depletion of mitochondria abolished the effects of Berberine on AMPK in EC. Finally, Berberine significantly increased the phosphorylation of LKB1 at Ser307 and gene silencing of LKB1 attenuated Berberine-enhanced AMPK Thr172 phosphorylation in BAEC.Our results suggest that mitochondria-derived superoxide anions and peroxynitrite are required for Berberine-induced AMPK activation in endothelial cells.

  5. Physiological desensitization of carbohydrate permeases and adenylate cyclase to regulation by the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli and Salmonella typhimurium. Involvement of adenosine cyclic 3',5'-phosphate and inducer.

    Science.gov (United States)

    Saier, M H; Keeler, D K; Feucht, B U

    1982-03-10

    Adenylate cyclase and a number of carbohydrate transport systems are subject to regulation by the phosphoenolpyruvate:sugar phosphotransferase system. These sensitive carbohydrate transport systems are desensitized to regulation by the phosphotransferase system, and adenylate cyclase is deactivated when cells are grown in medium containing cyclic AMP. These effects are specific for cyclic AMP and are potentiated by the genetic loss of cyclic AMP phosphodiesterase. Inclusion in the growth medium of an inducer of a sensitive transport system also promotes desensitization of that particular transport system. Inducer-promoted desensitization is specific for the particular target transport system, while cyclic AMP-promoted desensitization is general and affects several systems. Desensitization of the permeases to regulation, and inactivation of adenylate cyclase, are slow processes which are blocked by chloramphenicol and are therefore presumably dependent on protein synthesis. Several sugar substrates of the phosphotransferase system are capable of regulating the sensitive carbohydrate transport systems. The evidence suggests that desensitization to this regulation does not result from a direct effect on the functioning of Enzyme I, a small heat-stable protein of the phosphotransferase system, HPr, or an Enzyme II of the phosphotransferase system, but specifically uncouples the permease systems from regulation.

  6. Bone Abnormalities in Mice with Protein Kinase A (PKA) Defects Reveal a Role of Cyclic AMP Signaling in Bone Stromal Cell-Dependent Tumor Development.

    Science.gov (United States)

    Liu, S; Shapiro, J M; Saloustros, E; Stratakis, C A

    2016-11-01

    Protein kinase A (PKA) is an important enzyme for all eukaryotic cells. PKA phosphorylates other proteins, thus, it is essential for the regulation of many diverse cellular functions, including cytoplasmic trafficking and signaling, organelle structure and mitochondrial oxidation, nuclear gene expression, the cell cycle, and cellular division. The PKA holoenzyme is composed of 2 regulatory and 2 catalytic subunits. Four regulatory (R1α, R1β, R2α, and R2β) and 4 catalytic subunits (Cα, Cβ, Cγ, and Prkx) have been identified, giving rise to mainly PKA-I (when the 2 regulatory subunits are either R1α or R1β), or PKA-II (when the 2 regulatory subunits are either R2α or R2β). Mutations in the PKA subunits can lead to altered total PKA activity or abnormal PKA-I to PKA-II ratio, leading to various abnormalities in both humans and mice. These effects can be tissue-specific. We studied the effect of PKA subunit defects on PKA activity and bone morphology of mice that were single or double heterozygous for null alleles of the various PKA subunit genes. Bone lesions including fibrous dysplasia, myxomas, osteo-sarcomas, -chondromas and -chondrosarcomas were found in these mice. Observational and molecular studies showed that these lesions were derived from bone stromal cells (BSCs). We conclude that haploinsufficiency for different PKA subunit genes affected bone lesion formation, new bone generation, organization, and mineralization in variable ways. This work identified a PKA subunit- and activity-dependent pathway of bone lesion formation from BSCs with important implications for understanding how cyclic AMP affects the skeleton and its tumorigenesis. © Georg Thieme Verlag KG Stuttgart · New York.

  7. The cyclic AMP receptor protein, CRP, is required for both virulence and expression of the minimal CRP regulon in Yersinia pestis biovar microtus.

    Science.gov (United States)

    Zhan, Lingjun; Han, Yanping; Yang, Lei; Geng, Jing; Li, Yingli; Gao, He; Guo, Zhaobiao; Fan, Wei; Li, Gang; Zhang, Lianfeng; Qin, Chuan; Zhou, Dongsheng; Yang, Ruifu

    2008-11-01

    The cyclic AMP receptor protein (CRP) is a bacterial regulator that controls more than 100 promoters, including those involved in catabolite repression. In the present study, a null deletion of the crp gene was constructed for Yersinia pestis bv. microtus strain 201. Microarray expression analysis disclosed that at least 6% of Y. pestis genes were affected by this mutation. Further reverse transcription-PCR and electrophoretic mobility shift assay analyses disclosed a set of 37 genes or putative operons to be the direct targets of CRP, and thus they constitute the minimal CRP regulon in Y. pestis. Subsequent primer extension and DNase I footprinting assays mapped transcriptional start sites, core promoter elements, and CRP binding sites within the DNA regions upstream of pla and pst, revealing positive and direct control of these two laterally acquired plasmid genes by CRP. The crp disruption affected both in vitro and in vivo growth of the mutant and led to a >15,000-fold loss of virulence after subcutaneous infection but a pestis and, particularly, is more important for infection by subcutaneous inoculation. It can further be concluded that the reduced in vivo growth phenotype of the crp mutant should contribute, at least partially, to its attenuation of virulence by both routes of infection. Consistent with a previous study of Y. pestis bv. medievalis, lacZ reporter fusion analysis indicated that the crp deletion resulted in the almost absolute loss of pla promoter activity. The plasminogen activator encoded by pla was previously shown to specifically promote Y. pestis dissemination from peripheral infection routes (subcutaneous infection [flea bite] or inhalation). The above evidence supports the notion that in addition to the reduced in vivo growth phenotype, the defect of pla expression in the crp mutant will greatly contribute to the huge loss of virulence of this mutant strain in subcutaneous infection.

  8. cAMP-induced activation of protein kinase A and p190B RhoGAP mediates down-regulation of TC10 activity at the plasma membrane and neurite outgrowth.

    Science.gov (United States)

    Koinuma, Shingo; Takeuchi, Kohei; Wada, Naoyuki; Nakamura, Takeshi

    2017-11-01

    Cyclic AMP plays a pivotal role in neurite growth. During outgrowth, a trafficking system supplies membrane at growth cones. However, the cAMP-induced signaling leading to the regulation of membrane trafficking remains unknown. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking. Recent studies have shown a role of TC10 in neurite growth in NGF-treated PC12 cells. Here, we investigated a mechanical linkage between cAMP and TC10 in neuritogenesis. Plasmalemmal TC10 activity decreased abruptly after cAMP addition in neuronal cells. TC10 was locally inactivated at extending neurite tips in cAMP-treated PC12 cells. TC10 depletion led to a decrease in cAMP-induced neurite outgrowth. Constitutively active TC10 could not rescue this growth reduction, supporting our model for a role of GTP hydrolysis of TC10 in neuritogenesis by accelerating vesicle fusion. The cAMP-induced TC10 inactivation was mediated by PKA. Considering cAMP-induced RhoA inactivation, we found that p190B, but not p190A, mediated inactivation of TC10 and RhoA. Upon cAMP treatment, p190B was recruited to the plasma membrane. STEF depletion and Rac1-N17 expression reduced cAMP-induced TC10 inactivation. Together, the PKA-STEF-Rac1-p190B pathway leading to inactivation of TC10 and RhoA at the plasma membrane plays an important role in cAMP-induced neurite outgrowth. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  9. cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission.

    Directory of Open Access Journals (Sweden)

    Ghania Ramdani

    2015-05-01

    Full Text Available Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites.

  10. Lack of effect of the alpha2C-adrenoceptor Del322-325 polymorphism on inhibition of cyclic AMP production in HEK293 cells.

    Science.gov (United States)

    Montgomery, M D; Bylund, D B

    2010-02-01

    The alpha(2C)-adrenoceptor has multiple functions, including inhibiting release of noradrenaline from presynaptic nerve terminals. A human alpha(2C) polymorphism, Del322-325, a potential risk factor for heart failure, has been reported to exhibit reduced signalling in CHO cells. To further understand the role of the Del322-325 polymorphism on receptor signalling, we attempted to replicate and further study the reduced signalling in HEK293 cells. Human alpha(2C) wild-type (WT) and Del322-325 adrenoceptors were stably transfected into HEK293 cells. Radioligand binding was performed to determine affinities for both receptors. In intact cells, inhibition of forskolin-stimulated cyclic AMP production by WT and Del322-325 clones with a range of receptor densities (200-2320 fmol.mg(-1) protein) was measured following agonist treatment. Noradrenaline, brimonidine and clonidine exhibited similar binding affinities for WT and Del322-325. Brimonidine and clonidine also had similar efficacies and potencies for both receptors for the inhibition of cyclic AMP production at all receptor densities tested. A linear regression analysis comparing efficacy and potency with receptor expression levels showed no differences in slopes between WT and Del322-325. The alpha(2C) WT and Del322-325 adrenoceptors exhibited similar binding properties. Additionally, inhibition of cyclic AMP production by Del322-325 was similar to that of WT over a range of receptor densities. Therefore, in intact HEK293 cells, the alpha(2C)-Del322-325 polymorphism does not exhibit reduced signalling to adenylyl cyclase and may not represent a clinically important phenotype.

  11. Electrophysiological and biochemical studies of slow responses to serotonin and dopamine of snail identified neurons. Mediating role of the cyclic AMP

    International Nuclear Information System (INIS)

    Deterre, Philippe

    1983-01-01

    In this research thesis, the electrophysiological study of slow incoming currents induced in some identified neurons of the Helix aspersa snail by serotonin and dopamine shows that they are associated with a decrease of a potassium conductance involved in the modulation of the action potential duration. By means of enzymatic tests performed on a single cell, and of electrophysiological experiments, the author shows that the cyclic AMP is an intracellular mediator involved in the genesis of these slow responses. Moreover, the obtained results show that serotonin and dopamine act by binding to specific receptors, and that these receptors activate the adenylate-cyclase through a GTP binding protein [fr

  12. Type I interferon induction by Neisseria gonorrhoeae: Dual requirement of cyclic GMP-AMP synthase and Toll-like receptor 4

    OpenAIRE

    Andrade, Warrison A.; Agarwal, Sarika; Mo, Shunyan; Shaffer, Scott A.; Dillard, Joseph P.; Schmidt, Tobias; Hornung, Veit; Fitzgerald, Katherine A.; Kurt-Jones, Evelyn A.; Golenbock, Douglas T.

    2016-01-01

    The innate immune system is the first line of defense against Neisseria gonorrhoeae (GC). Exposure of cells to GC lipooligosaccharides induces a strong immune response, leading to type I interferon (IFN) production via TLR4/MD-2. In addition to living freely in the extracellular space, GC can invade the cytoplasm to evade detection and elimination. Double-stranded DNA introduced into the cytosol binds and activates the enzyme cyclic-GMP-AMP synthase (cGAS), which produces 2′3′-cGAMP and trigg...

  13. Effect of glucagon on cyclic AMP, albumin metabolism and incorporation of 14C-leucine into proteins in isolated parenchymal rat liver cells

    DEFF Research Database (Denmark)

    Dich, J; Gluud, C N

    1976-01-01

    wet wt. This is about the rate found in the perfused liver, Glucagon (10(-8-10(-6) M) inhibited albumin secretion and the incorporation of 14C-leucine into albumin, into total proteins in the medium and into total proteins in the cell suspension. The effect of glucagon on albumin secretion...... is compatible with an effect on the rate of synthesis. A positive correlation existed between the maximal level of cyclic AMP after glucagon administration and the inhibition of both albumin secretion and the incorporation of 149leucine....

  14. Biochemical studies on the DNA binding function of the cyclic-amp reactor protein of Escherichia coli

    International Nuclear Information System (INIS)

    Angulo, J.A.

    1986-01-01

    The cAMP receptor protein (CRP) is an allosteric protein in which binding of cAMP effects a conformational change with a consequent increased affinity for DNA. Binding of double-stranded deoxyribopolynucleotides and calf thymus DNA by cAMP-CRP confers protection against attack by trypsin, subtilisin, Staph. aureus V8 protease and clostripain. Of the single-stranded deoxy- and ribopolynucleotides tested, only r(I)/sub n/ and r(A)/sub n/ gave significant protection against attack by these proteases. In the absence of cAMP, CRP is resistant to proteolysis. Incubation of CRP-DNA with trypsin results in the accumulation of two novel fragments. CRP-DNA is partially sensitive to digestion by chymotrypsin but resistant to attack by subtilisin, the Staph. aureus V8 protease and clostripain. Cleavage of CRP-DNA to fragments is accompanied by the loss of 3 H-cAMP binding activity. Modification of the arginines with phenylglyoxal or butanedione results in loss of DNA binding activity. cAMP-CRP incorporates more 14 C-phenylglyoxal than unliganded CRP. Titration of the arginines with 14 C-phenylglyoxal to where over 90% of the DNA binding activity is lost results in incorporation of one mole of reagent per mole of subunit

  15. Cyclic [G(2′,5′)pA(3′,5′)p] Is the Metazoan Second Messenger Produced by DNA-Activated Cyclic GMP-AMP Synthase

    OpenAIRE

    Gao, Pu; Ascano, Manuel; Wu, Yang; Barchet, Winfried; Gaffney, Barbara L.; Zillinger, Thomas; Serganov, Artem A.; Liu, Yizhou; Jones, Roger A.; Hartmann, Gunther; Tuschl, Thomas; Patel, Dinshaw J.

    2013-01-01

    Recent studies identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical, biochemical, and cellular assays to demonstrate that this second messenger contains G(2′,5′)pA and A(3′,5′)pG phosphodiester linkages, designated c[G(2′,5′) pA(3′,5′)p]. We show that, upon dsDNA binding, cGAS is activated through conformational transitions, res...

  16. PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling.

    Science.gov (United States)

    Monterisi, Stefania; Lobo, Miguel J; Livie, Craig; Castle, John C; Weinberger, Michael; Baillie, George; Surdo, Nicoletta C; Musheshe, Nshunge; Stangherlin, Alessandra; Gottlieb, Eyal; Maizels, Rory; Bortolozzi, Mario; Micaroni, Massimo; Zaccolo, Manuela

    2017-05-02

    cAMP/PKA signalling is compartmentalised with tight spatial and temporal control of signal propagation underpinning specificity of response. The cAMP-degrading enzymes, phosphodiesterases (PDEs), localise to specific subcellular domains within which they control local cAMP levels and are key regulators of signal compartmentalisation. Several components of the cAMP/PKA cascade are located to different mitochondrial sub-compartments, suggesting the presence of multiple cAMP/PKA signalling domains within the organelle. The function and regulation of these domains remain largely unknown. Here, we describe a novel cAMP/PKA signalling domain localised at mitochondrial membranes and regulated by PDE2A2. Using pharmacological and genetic approaches combined with real-time FRET imaging and high resolution microscopy, we demonstrate that in rat cardiac myocytes and other cell types mitochondrial PDE2A2 regulates local cAMP levels and PKA-dependent phosphorylation of Drp1. We further demonstrate that inhibition of PDE2A, by enhancing the hormone-dependent cAMP response locally, affects mitochondria dynamics and protects from apoptotic cell death.

  17. Cyclic guanosine monophosphate in the regulation of the cell function

    Directory of Open Access Journals (Sweden)

    Małgorzata Zbrojkiewicz

    2016-12-01

    Full Text Available Intracellular concentration of cGMP depends on the activity of guanylate cyclase, responsible for its synthesis, on the activity of cyclic nucleotide degrading enzymes - phosphodiesterases (PDEs. There are two forms of guanylate cyclase: the membrane-bound cyclase and the soluble form. The physiological activators of the membrane guanylate cyclase are natriuretic peptides (NPs, and of the cytosolic guanylate cyclase - nitric oxide (NO and carbon monoxide (CO. Intracellular cGMP signaling pathways arise from its direct effect on the activity of G protein kinases, phosphodiesterases and cyclic nucleotide dependent cation channels. It has been shown in recent years that cGMP can also affect other signal pathways in cell signaling activity involving Wnt proteins and sex hormones. The increased interest in the research on the role of cGMP, resulted also in the discovery of its role in the regulation of phototransduction in the eye, neurotransmission, calcium homeostasis, platelet aggregation, heartbeat, bone remodeling, lipid metabolism and the activity of the cation channels. Better understanding of the mechanisms of action of cGMP in the regulation of cell function can create new opportunities for the cGMP affecting drugs use in the pharmacotherapy.

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

    Science.gov (United States)

    Řlepokura, Katarzyna Anna

    2016-06-01

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

  19. Cyclic-AMP mediated drugs: differential or global reduction of eicosanoid synthesis in the isolated rat lung?

    Directory of Open Access Journals (Sweden)

    Mark J. Post

    1992-01-01

    Full Text Available In this study the question was addressed whether cAMP mediated drugs induce a differential reduction of branches of the arachidonic acid metabolism rather than a global reduction of eicosanoid synthesis. The isolated lungs of actively sensitized rats were employed to study prostaglandin and leukotriene release in the presence and absence of the cAMP mediated drugs theophylline, milrinone, sulmazole, isobutyl-methylxanthine and salbutamol. The release of eicosanoids as measured by RIA was predominantly basal and continuous, with a mild antigen induced stimulation only for TXB2 and the leukotrienes. All drugs reduced eicosanoid release globally. It is concluded that cAMP mediated drugs interfere with arachidonic acid metabolism at a site proximal to the branching into lipoxygenase and cyclo-oxygenase pathways.

  20. Cyclic AMP induces apoptosis in multiple myeloma cells and inhibits tumor development in a mouse myeloma model

    International Nuclear Information System (INIS)

    Follin-Arbelet, Virginie; Hofgaard, Peter O; Hauglin, Harald; Naderi, Soheil; Sundan, Anders; Blomhoff, Rune; Bogen, Bjarne; Blomhoff, Heidi K

    2011-01-01

    Multiple myeloma is an incurable disease requiring the development of effective therapies which can be used clinically. We have elucidated the potential for manipulating the cAMP signaling pathway as a target for inhibiting the growth of multiple myeloma cells. As a model system, we primarily used the murine multiple myeloma cell line MOPC315 which can be grown both in vivo and in vitro. Human multiple myeloma cell lines U266, INA-6 and the B-cell precursor acute lymphoblastic leukemia cell line Reh were used only for in vitro studies. Cell death was assessed by flow cytometry and western blot analysis after treatment with cAMP elevating agents (forskolin, prostaglandin E2 and rolipram) and cAMP analogs. We followed tumor growth in vivo after forskolin treatment by imaging DsRed-labelled MOPC315 cells transplanted subcutaneously in BALB/c nude mice. In contrast to the effect on Reh cells, 50 μM forskolin more than tripled the death of MOPC315 cells after 24 h in vitro. Forskolin induced cell death to a similar extent in the human myeloma cell lines U266 and INA-6. cAMP-mediated cell death had all the typical hallmarks of apoptosis, including changes in the mitochondrial membrane potential and cleavage of caspase 3, caspase 9 and PARP. Forskolin also inhibited the growth of multiple myeloma cells in a mouse model in vivo. Elevation of intracellular levels of cAMP kills multiple myeloma cells in vitro and inhibits development of multiple myeloma in vivo. This strongly suggests that compounds activating the cAMP signaling pathway may be useful in the field of multiple myeloma

  1. cAMP Signaling Regulates Histone H3 Phosphorylation and Mitotic Entry Through a Disruption of G2 Progression

    OpenAIRE

    Rodriguez-Collazo, Pedro; Snyder, Sara K.; Chiffer, Rebecca C.; Bressler, Erin A.; Voss, Ty C.; Anderson, Eric P.; Genieser, Hans-Gottfried; Smith, Catharine L.

    2008-01-01

    cAMP signaling is known to have significant effects on cell growth, either inhibitory or stimulatory depending on the cell type. Study of cAMP-induced growth inhibition in mammalian somatic cells has focused mainly on the combined role of protein kinase A (PKA) and mitogen-activated protein (MAP) kinases in regulation of progression through the G1 phase of the cell cycle. Here we show that cAMP signaling regulates histone H3 phosphorylation in a cell cycle-dependent fashion, increasing it in ...

  2. Regulation of cessation of respiration and killing by cyclic 3',5'-adenosine monophosphate and its receptor protein after far-ultraviolet irradiation of Escherichia coli

    International Nuclear Information System (INIS)

    Swenson, P.A.; Schenley, R.L.; Joshi, J.G.

    1978-01-01

    When Escherichia coli B/r cultures are irradiated with ultraviolet light (UV) (254 nm), those cells that are killed stop respiring by 60 min after irradiation. Post-UV treatment with cyclic adenosine 3',5'-adenosine monophosphate (cAMP) causes more cells to stop respiring and to die. We have studied these effects at a UV fluence of 52 I/m 2 in a a wild-type E. coli K 12 strain and in mutants defective in cAMP metabolism. Strain CA 8,000 has crp + and cya + genes for the cAMP receptor protein (CRP) (required for transcription of operons regulated by cAMP) and for adenylate cyclase, respectively; CA 7901 is crp - ; and CA 8306 is a cya deletion (Δ). The wild-type culture showed a small transient cessation of respiration, and addition of cAMP caused cessation to be nearly complete. The crp - culture showed no evidence of cessation of respiration, and cAMP had no effect. The Δ cya mutant also showed no cessation of respiration, but cAMP (5 mM) caused as complete inhibition as in the wild type. cAMP caused a 10-fold loss in viability of UV-irradiated wild-type and Δ cya liquid cultures but had no effect on the cpr - culture. Respiration and viability changes were also studied in a double mutant, CA8404 Δ cya crp*, which has an altered CRP that is, with respect to the lac operon, independent of cAMP. The respiration response to UV was similar to that of the wild-type culture, and both respiration and viability of cells in liquid culture were sensitive to cAMP. The survival data, obtained by plating immediately after irradiation, show the wild type, Δ cya strains, and Δ cya crp* to be equally sensitive and the crp - strain to be more resistant. We conclude that cessation of respiration and cell killing after UV irradiation are regulated by cAMP and the CRP. (orig.) [de

  3. Protein kinase A and Epac activation by cAMP regulates the expression of glial fibrillary acidic protein in glial cells

    Directory of Open Access Journals (Sweden)

    Sugimoto Naotoshi

    2016-01-01

    Full Text Available Cyclic adenosine monophosphate (cAMP controls differentiation in several types of cells during brain development. However, the molecular mechanism of cAMP-controlled differentiation is not fully understood. We investigated the role of protein kinase A (PKA and exchange protein directly activated by cAMP (Epac on cAMP-induced glial fibrillary acidic protein (GFAP, an astrocyte marker, in cultured glial cells. B92 glial cells were treated with cAMP-elevating drugs, an activator of adenylate cyclase, phosphodiesterase inhibitor and a ß adrenal receptor agonist. These cAMP-elevating agents induced dramatic morphological changes and expression of GFAP. A cAMP analog, 8-Br-cAMP, which activates Epac as well as PKA, induced GFAP expression and morphological changes, while another cAMP analog, 8-CPT-cAMP, which activates Epac with greater efficacy when compared to PKA, induced GFAP expression but very weak morphological changes. Most importantly, the treatment with a PKA inhibitor partially reduced cAMP-induced GFAP expression. Taken together, these results indicate that cAMP-elevating drugs lead to the induction of GFAP via PKA and/or Epac activation in B92 glial cells.

  4. Modulation of cyclic amp-dependent protein kinase isozyme expression associated with activation of a macrophage cell line

    International Nuclear Information System (INIS)

    Justement, L.B.; Aldrich, W.A.; Wenger, G.D.; O'Dorisio, M.S.; Zwilling, B.S.

    1986-01-01

    The RAW 264.7 macrophage (MO) cell line was used to study cAMPdPK isozymes during activation by lymphokine (LK) and lipopolysaccharide (LPS). Untreated cells were found to have two isozymes of cAMPdPK in their cytosol. PKI and PKII were differentiated based on the M/sub r/ of their regulatory subunits (RI, 45,500; and RII, 52,000, respectively) as determined by photoactivated incorporation of the cAMP analog 8-N 3 -[ 32 P]cAMP. Loss of the RI subunit of PKI occurred in association with activation of the cell line by suboptimal concentrations of LK and LPS. No modulation of the RII subunit of PKII was observed under these conditions. The addition of a suboptimal concentration of LPS after LK or a high dose of LPS alone was required for acquisition of cytolytic activity and loss of RI. The antitumor activity of the RAW 264.7 cell line was transiently expressed after activation. Cells no longer exhibited tumoricidal activity 48 hr after the removal of activating agents. It was observed that the loss of cytolytic function was accompanied by the reexpression of RI in the cytosol. This study provides evidence that modulation of cAMPdPK isozymes occurs during activation, suggesting a potential mechanism for controlling the effects of cAMP on the MO

  5. Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter.

    Science.gov (United States)

    Attey, A; Belyaeva, T; Savery, N; Hoggett, J; Fujita, N; Ishihama, A; Busby, S

    1994-10-25

    DNAase I footprinting has been used to study open complexes between Escherichia coli RNA polymerase and the galactose operon P1 promoter, both in the absence and the presence of CRP (the cyclic AMP receptor protein, a transcription activator). From the effects of deletion of the C-terminal part of the RNA polymerase alpha subunit, we deduce that alpha binds at the upstream end of both the binary RNA polymerase-galP1 and ternary RNA polymerase-CRP-galP1 complexes. Disruption of the alpha-upstream contact suppresses open complex formation at galP1 at lower temperatures. In ternary RNA polymerase-CRP-galP1 complexes, alpha appears to make direct contact with Activating Region 1 in CRP. DNAase I footprinting has been used to detect and quantify interactions between purified alpha and CRP bound at galP1.

  6. Inhibition of insulin release by cyproheptadine: Effects on 3',5'-cyclic-AMP-content and /sup 45/Ca-accumulation of incubated mouse islets

    Energy Technology Data Exchange (ETDEWEB)

    Joost, H G; Beckmann, J; Lenzen, S; Hasselblatt, A [Goettingen Univ. (F.R. Germany)

    1976-01-01

    Cyproheptadine (1, 10 and 100 ..mu..m) significantly reduced insulin release from isolated mouse islets in response to glucose. In contrast, 1 mM cyproheptadine induced a large release of insulin into the incubation medium probably due to islet cell damage, since the islets had lost a considerable amount of their protein content. 3',5'-cyclic-AMP-levels of the islets were not significantly affected by 10 ..mu..M cyproheptadine in the presence as well as in the absence of theophylline (10 mM). As the inhibitory effect of cyproheptadine on insulin release was correlated with reduced accumulation of calcium-45, the agent may inhibit insulin release by interfering with the calcium handling of the ..beta..-cell.

  7. Transporter function and cyclic AMP turnover in normal colonic mucosa from patients with and without colorectal neoplasia

    DEFF Research Database (Denmark)

    Kleberg, Karen; Jensen, Gerda Majgaard; Christensen, Dan Ploug

    2012-01-01

    The pathogenesis of colorectal neoplasia is still unresolved but has been associated with alterations in epithelial clearance of xenobiotics and metabolic waste products. The aim of this study was to functionally characterize the transport of cyclic nucleotides in colonic biopsies from patients...

  8. L-4F Inhibits Oxidized Low-density Lipoprotein-induced Inflammatory Adipokine Secretion via Cyclic AMP/Protein Kinase A-CCAAT/Enhancer Binding Protein β Signaling Pathway in 3T3-L1 Adipocytes

    Directory of Open Access Journals (Sweden)

    Xiang-Zhu Xie

    2016-01-01

    Conclusions: OxLDL induces C/EBPβ protein synthesis in a time-dependent manner and enhances MCP-1 secretion and expression in 3T3-L1 adipocytes. L-4F dose-dependently counterbalances the pro-inflammatory effect of oxLDL, and cyclic AMP/PKA-C/EBPβ signaling pathway may participate in it.

  9. Anti-cyclic regulation of the Ukrainian economy under current conditions of the international markets volatility

    Directory of Open Access Journals (Sweden)

    Volodymyr Satsyk

    2009-04-01

    Full Text Available In the article there are considered the theoretical and methodological basis of anti-cyclic regulation of the countries’ economy under conditions of the world economy globalization. It suggests the analysis of practices of implementing of anti-cyclic policy in highly developed states, its defining features and directions under current global financial and economic crisis. There has been researched a practical toolkit of economic cycles diagnostics and cyclic fluctuations of total business activity in Ukraine based on this study. There are suggested recommendations concerning the formation of the effective mechanism of anti-cyclic regulation of the Ukrainian economy.

  10. Secretion of [Met]enkephalyl-Arg6-Phe7-related peptides and catecholamines from bovine adrenal chromaffin cells: modification by changes in cyclic AMP and by treatment with reserpine.

    Science.gov (United States)

    Adams, M; Boarder, M R

    1987-07-01

    Investigations into the effects of culturing bovine adrenal chromaffin cells in the presence (72 h) of dibutyryl cyclic AMP, forskolin, and reserpine on the level and release of [Met]enkephalyl-Arg6-Phe7 immunoreactivity, noradrenaline, and adrenaline are reported. The assay for [Met]enkephalyl-Arg6-Phe7 immunoreactivity recognises both peptide B, the 31-amino acid carboxy-terminal segment of proenkephalin, and its heptapeptide fragment, [Met]enkephalyl-Arg6-Phe7. Treatments that elevate cyclic AMP increase the amount of peptide immunoreactivity in these cells; this is predominantly peptide B-like immunoreactivity in both control cells and cyclic AMP-elevated cells. Treatment with reserpine gives no change in total immunoreactivity levels, but does not result in increased accumulation of the heptapeptide [Met]enkephalyl-Arg6-Phe7 at the expense of immunoreactivity that elutes with its immediate precursor, peptide B. Cyclic AMP treatment causes either no change or a decrease in levels of accumulated noradrenaline and adrenaline. However, the release of [Met]enkephalin-Arg6-Phe7 immunoreactivity, noradrenaline, and adrenaline is increased by 72-h pretreatment with forskolin or dibutyryl cyclic AMP, whether release is stimulated by nicotine or elevated potassium. In each case the molecular form of [Met]enkephalyl-Arg6-Phe7 immunoreactivity that is released approximately reflects the cell content. Pretreatment with reserpine has no effect on the total [Met]enkephalyl-Arg6-Phe7 immunoreactivity released, but does result in an increased release of the heptapeptide and a decrease in release of peptide B-like immunoreactivity. The studies suggest that the levels of [Met]enkephalyl-Arg6-Phe7 and peptide B available for release are controlled both at the level of proenkephalin synthesis and at the level of double-basic residue proteolysis.

  11. Regulation of AMP-activated protein kinase by natural and synthetic activators

    Directory of Open Access Journals (Sweden)

    David Grahame Hardie

    2016-01-01

    Full Text Available The AMP-activated protein kinase (AMPK is a sensor of cellular energy status that is almost universally expressed in eukaryotic cells. While it appears to have evolved in single-celled eukaryotes to regulate energy balance in a cell-autonomous manner, during the evolution of multicellular animals its role has become adapted so that it also regulates energy balance at the whole body level, by responding to hormones that act primarily on the hypothalamus. AMPK monitors energy balance at the cellular level by sensing the ratios of AMP/ATP and ADP/ATP, and recent structural analyses of the AMPK heterotrimer that have provided insight into the complex mechanisms for these effects will be discussed. Given the central importance of energy balance in diseases that are major causes of morbidity or death in humans, such as type 2 diabetes, cancer and inflammatory disorders, there has been a major drive to develop pharmacological activators of AMPK. Many such activators have been described, and the various mechanisms by which these activate AMPK will be discussed. A particularly large class of AMPK activators are natural products of plants derived from traditional herbal medicines. While the mechanism by which most of these activate AMPK has not yet been addressed, I will argue that many of them may be defensive compounds produced by plants to deter infection by pathogens or grazing by insects or herbivores, and that many of them will turn out to be inhibitors of mitochondrial function.

  12. Evidence for a cyclic AMP-dependent pathway in angiotensin AT1-receptor activation of human omental arteries

    Directory of Open Access Journals (Sweden)

    Hoa Ytterberg

    2001-03-01

    Full Text Available Enhanced responses to vasoconstriction induced by neuropeptide Y and α2-adrenoceptor agonists have been seen following pharmacological activation of the adenylyl cyclase (AC system. Since preliminary studies revealed only minor responses to angiotensin II (Ang II in human omental arteries, we have investigated whether enhanced activity of AC may unravel further functional Ang II receptors. Human omental arteries were obtained in conjunction with elective gut surgery. After dissection of the vessel, the endothelium was removed by 10 sec of Triton X-100 treatment. Ring segments (1—2 mm long were mounted on a myograph and studied. Ang II produced small contractions, 27±5% relative to the response elicited by 60 mM K+. However, enhanced Ang II (105±10%, p<0.001 responses were seen during AC activation by forskolin (0.1—1 µM. This enhanced contractile response to Ang II was not inhibited by the angiotensin II type 2 (AT2-receptor antagonist PD 123319 (0.1 µM, but was blocked in an insurmountable way by the angiotensin II type 1 (AT1-receptor antagonist candesartan (1 nM and in a surmountable manner by losartan (0.1 µM and irbesartan (0.1 µM. Pertussis toxin (a Gi-protein blocker and the protein kinase C inhibitor, RO31—8220 (0.01, 0.1 and 1 µM, markedly reduced this response, while the protein kinase A inhibitor, H89 (1, 10 µM, had no effect. RT-PCR provided evidence for the presence of mRNA for both AT1- and AT2-receptors. The results suggest that both a cAMP-dependent and a cAMP-independent mechanism are involved in the contractile responses to Ang II in human omental arteries and that both responses are mediated via the AT1-receptor.

  13. Propofol reduced myocardial contraction of vertebrates partly by mediating the cyclic AMP-dependent protein kinase phosphorylation pathway

    International Nuclear Information System (INIS)

    Sun, Xiaotong; Zhang, Xinyu; Bo, Qiyu; Meng, Tao; Lei, Zhen; Li, Jingxin; Hou, Yonghao; Yu, Xiaoqian; Yu, Jingui

    2016-01-01

    Propofol inhibits myocardial contraction in a dose dependent manner. The present study is designed to examine the effect of propofol on PKA mediated myocardial contraction in the absence of adrenoreceptor agonist. The contraction of isolated rat heart was measured in the presence or absence of PKA inhibitor H89 or propofol, using a pressure transducer. The levels of cAMP and PKA kinase activity were detected by ELISA. The mRNA and total protein or phosphorylation level of PKA and downstream proteins were tested in the presence or absence of PKA inhibitor H89 or propofol, using RT-PCR, QPCR and western blotting. The phosphorylation level of PKA was examined thoroughly using immunofluorescence and PKA activity non-radioactive detection kit. Propofol induced a dose-dependent negative contractile response on the rat heart. The inhibitory effect of high concentration propofol (50 μM) with 45% decease of control could be partly reversed by the PKA inhibitor H89 (10 μM) and the depressant effect of propofol decreased from 45% to 10%. PKA kinase activity was inhibited by propofol in a dose-dependent manner. Propofol also induced a decrease in phosphorylation of PKA, which was also inhibited by H89, but did not alter the production of cAMP and the mRNA levels of PKA. The downstream proteins of PKA, PLN and RyR2 were phosphorylated to a lesser extent with propofol or H89 than control. These results demonstrated that propofol induced a negative myocardial contractile response partly by mediating the PKA phosphorylation pathway.

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

    International Nuclear Information System (INIS)

    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 μM), AZT monophosphate (150 μM), and 2',3'-dideoxycytidine (ddC; 1 μM) 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 μM) and ddC (1 μM). 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-γ activity; in the case of AZT, these observations may provide a mechanism for the observed long-term toxicity with this drug

  15. Cyclic [G(2′,5′)pA(3′,5′)p] Is the Metazoan Second Messenger Produced by DNA-Activated Cyclic GMP-AMP Synthase

    Science.gov (United States)

    Gao, Pu; Ascano, Manuel; Wu, Yang; Barchet, Winfried; Gaffney, Barbara L.; Zillinger, Thomas; Serganov, Artem A.; Liu, Yizhou; Jones, Roger A.; Hartmann, Gunther; Tuschl, Thomas; Patel, Dinshaw J.

    2015-01-01

    SUMMARY Recent studies identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical, biochemical, and cellular assays to demonstrate that this second messenger contains G(2′,5′)pA and A(3′,5′)pG phosphodiester linkages, designated c[G(2′,5′) pA(3′,5′)p]. We show that, upon dsDNA binding, cGAS is activated through conformational transitions, resulting in formation of a catalytically competent and accessible nucleotide-binding pocket for generation of c[G(2′,5′)pA(3′,5′)p]. We demonstrate that cyclization occurs in a stepwise manner through initial generation of 5′-pppG(2′,5′)pA prior to cyclization to c[G(2′,5′)pA(3′,5′)p], with the latter positioned precisely in the catalytic pocket. Mutants of cGAS dsDNA-binding or catalytic pocket residues exhibit reduced or abrogated activity. Our studies have identified c[G(2′,5′)pA(3′,5′)p] as a founding member of a family of metazoan 2′,5′-containing cyclic heterodinucleotide second messengers distinct from bacterial 3′,5′ cyclic dinucleotides. PMID:23647843

  16. The Role of Cyclic Nucleotide Signaling Pathways in Cancer: Targets for Prevention and Treatment

    Energy Technology Data Exchange (ETDEWEB)

    Fajardo, Alexandra M.; Piazza, Gary A. [Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Ave, Suite 3029, Mobile, AL 36604 (United States); Tinsley, Heather N., E-mail: htinsley@montevallo.edu [Department of Biology, Chemistry, and Mathematics, University of Montevallo, Station 6480, Montevallo, AL 35115 (United States)

    2014-02-26

    For more than four decades, the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) have been recognized as important signaling molecules within cells. Under normal physiological conditions, cyclic nucleotides regulate a myriad of biological processes such as cell growth and adhesion, energy homeostasis, neuronal signaling, and muscle relaxation. In addition, altered cyclic nucleotide signaling has been observed in a number of pathophysiological conditions, including cancer. While the distinct molecular alterations responsible for these effects vary depending on the specific cancer type, several studies have demonstrated that activation of cyclic nucleotide signaling through one of three mechanisms—induction of cyclic nucleotide synthesis, inhibition of cyclic nucleotide degradation, or activation of cyclic nucleotide receptors—is sufficient to inhibit proliferation and activate apoptosis in many types of cancer cells. These findings suggest that targeting cyclic nucleotide signaling can provide a strategy for the discovery of novel agents for the prevention and/or treatment of selected cancers.

  17. Cyclic AMP-dependent signaling system is a primary metabolic target for non-thermal effect of microwaves on heart muscle hydration.

    Science.gov (United States)

    Narinyan, Lilia; Ayrapetyan, Sinerik

    2017-01-01

    Previously, we have suggested that cell hydration is a universal and extra-sensitive sensor for the structural changes of cell aqua medium caused by the impact of weak chemical and physical factors. The aim of present work is to elucidate the nature of the metabolic messenger through which physiological solution (PS) treated by non-thermal (NT) microwaves (MW) could modulate heart muscle hydration of rats. For this purpose, the effects of NT MW-treated PS on heart muscle hydration, [ 3 H]-ouabain binding with cell membrane, 45 Ca 2+ uptake and intracellular cyclic nucleotides contents in vivo and in vitro experiments were studied. It is shown that intraperitoneal injections of both Sham-treated PS and NT MW-treated PS elevate heart muscle hydration. However, the effect of NT MW-treated PS on muscle hydration is more pronounced than the effect of Sham-treated PS. In vitro experiments NT MW-treated PS has dehydration effect on muscle, which is not changed by decreasing Na + gradients on membrane. Intraperitoneal injection of Sham- and NT MW-treated PS containing 45 Ca 2+ have similar dehydration effect on muscle, while NT MW-treated PS has activation effect on Na + /Ca 2+ exchange in reverse mode. The intraperitoneal injection of NT MW-treated PS depresses [ 3 H]-ouabain binding with its high-affinity membrane receptors, elevates intracellular cAMP and decreases cGMP contents. Based on the obtained data, it is suggested that cAMP-dependent signaling system serves as a primary metabolic target for NT MW effect on heart muscle hydration.

  18. Caffeine Modulates Vesicle Release and Recovery at Cerebellar Parallel Fibre Terminals, Independently of Calcium and Cyclic AMP Signalling

    Science.gov (United States)

    Dobson, Katharine L.; Jackson, Claire; Balakrishnan, Saju; Bellamy, Tomas C.

    2015-01-01

    Background Cerebellar parallel fibres release glutamate at both the synaptic active zone and at extrasynaptic sites—a process known as ectopic release. These sites exhibit different short-term and long-term plasticity, the basis of which is incompletely understood but depends on the efficiency of vesicle release and recycling. To investigate whether release of calcium from internal stores contributes to these differences in plasticity, we tested the effects of the ryanodine receptor agonist caffeine on both synaptic and ectopic transmission. Methods Whole cell patch clamp recordings from Purkinje neurons and Bergmann glia were carried out in transverse cerebellar slices from juvenile (P16-20) Wistar rats. Key Results Caffeine caused complex changes in transmission at both synaptic and ectopic sites. The amplitude of postsynaptic currents in Purkinje neurons and extrasynaptic currents in Bergmann glia were increased 2-fold and 4-fold respectively, but paired pulse ratio was substantially reduced, reversing the short-term facilitation observed under control conditions. Caffeine treatment also caused synaptic sites to depress during 1 Hz stimulation, consistent with inhibition of the usual mechanisms for replenishing vesicles at the active zone. Unexpectedly, pharmacological intervention at known targets for caffeine—intracellular calcium release, and cAMP signalling—had no impact on these effects. Conclusions We conclude that caffeine increases release probability and inhibits vesicle recovery at parallel fibre synapses, independently of known pharmacological targets. This complex effect would lead to potentiation of transmission at fibres firing at low frequencies, but depression of transmission at high frequency connections. PMID:25933382

  19. cAMP signalling in the vasculature: the role of Epac (exchange protein directly activated by cAMP).

    Science.gov (United States)

    Roberts, Owain Llŷr; Dart, Caroline

    2014-02-01

    The second messenger cAMP plays a central role in mediating vascular smooth muscle relaxation in response to vasoactive transmitters and in strengthening endothelial cell-cell junctions that regulate the movement of solutes, cells and macromolecules between the blood and the surrounding tissue. The vasculature expresses three cAMP effector proteins: PKA (protein kinase A), CNG (cyclic-nucleotide-gated) ion channels, and the most recently discovered Epacs (exchange proteins directly activated by cAMP). Epacs are a family of GEFs (guanine-nucleotide-exchange factors) for the small Ras-related GTPases Rap1 and Rap2, and are being increasingly implicated as important mediators of cAMP signalling, both in their own right and in parallel with the prototypical cAMP target PKA. In the present paper, we review what is currently known about the role of Epac within blood vessels, particularly with regard to the regulation of vascular tone, endothelial barrier function and inflammation.

  20. Regulation of proximal tubule vacuolar H+-ATPase by PKA and AMP-activated protein kinase

    Science.gov (United States)

    Al-bataineh, Mohammad M.; Gong, Fan; Marciszyn, Allison L.; Myerburg, Michael M.

    2014-01-01

    The vacuolar H+-ATPase (V-ATPase) mediates ATP-driven H+ transport across membranes. This pump is present at the apical membrane of kidney proximal tubule cells and intercalated cells. Defects in the V-ATPase and in proximal tubule function can cause renal tubular acidosis. We examined the role of protein kinase A (PKA) and AMP-activated protein kinase (AMPK) in the regulation of the V-ATPase in the proximal tubule as these two kinases coregulate the V-ATPase in the collecting duct. As the proximal tubule V-ATPases have different subunit compositions from other nephron segments, we postulated that V-ATPase regulation in the proximal tubule could differ from other kidney tubule segments. Immunofluorescence labeling of rat ex vivo kidney slices revealed that the V-ATPase was present in the proximal tubule both at the apical pole, colocalizing with the brush-border marker wheat germ agglutinin, and in the cytosol when slices were incubated in buffer alone. When slices were incubated with a cAMP analog and a phosphodiesterase inhibitor, the V-ATPase accumulated at the apical pole of S3 segment cells. These PKA activators also increased V-ATPase apical membrane expression as well as the rate of V-ATPase-dependent extracellular acidification in S3 cell monolayers relative to untreated cells. However, the AMPK activator AICAR decreased PKA-induced V-ATPase apical accumulation in proximal tubules of kidney slices and decreased V-ATPase activity in S3 cell monolayers. Our results suggest that in proximal tubule the V-ATPase subcellular localization and activity are acutely coregulated via PKA downstream of hormonal signals and via AMPK downstream of metabolic stress. PMID:24553431

  1. The μ opioid agonist morphine modulates potentiation of capsaicin-evoked TRPV1 responses through a cyclic AMP-dependent protein kinase A pathway

    Directory of Open Access Journals (Sweden)

    Roberts-Thomson Sarah J

    2006-07-01

    Full Text Available Abstract Background The vanilloid receptor 1 (TRPV1 is critical in the development of inflammatory hyperalgesia. Several receptors including G-protein coupled prostaglandin receptors have been reported to functionally interact with the TRPV1 through a cAMP-dependent protein kinase A (PKA pathway to potentiate TRPV1-mediated capsaicin responses. Such regulation may have significance in inflammatory pain. However, few functional receptor interactions that inhibit PKA-mediated potentiation of TRPV1 responses have been described. Results In the present studies we investigated the hypothesis that the μ opioid receptor (MOP agonist morphine can modulate forskolin-potentiated capsaicin responses through a cAMP-dependent PKA pathway. HEK293 cells were stably transfected with TRPV1 and MOP, and calcium (Ca2+ responses to injection of the TRPV1 agonist capsaicin were monitored in Fluo-3-loaded cells. Pre-treatment with morphine did not inhibit unpotentiated capsaicin-induced Ca2+ responses but significantly altered capsaicin responses potentiated by forskolin. TRPV1-mediated Ca2+ responses potentiated by the direct PKA activator 8-Br-cAMP and the PKC activator Phorbol-12-myristate-13-acetatewere not modulated by morphine. Immunohistochemical studies confirmed that the TRPV1 and MOP are co-expressed on cultured Dorsal Root Ganglion neurones, pointing towards the existence of a functional relationship between the G-protein coupled MOP and nociceptive TRPV1. Conclusion The results presented here indicate that the opioid receptor agonist morphine acts via inhibition of adenylate cyclase to inhibit PKA-potentiated TRPV1 responses. Targeting of peripheral opioid receptors may therefore have therapeutic potential as an intervention to prevent potentiation of TRPV1 responses through the PKA pathway in inflammation.

  2. Biosynthesis and regulation of cyclic lipopeptides in Pseudomonas fluorescens

    NARCIS (Netherlands)

    Bruijn, de I.

    2009-01-01

    Cyclic lipopeptides (CLPs) are surfactant and antibiotic metabolites produced by a variety of bacterial
    genera. For the genus Pseudomonas, many structurally different CLPs have been identified. CLPs play an
    important role in surface motility of Pseudomonas strains, but also in virulence

  3. Type I Interferon Induction by Neisseria gonorrhoeae: Dual Requirement of Cyclic GMP-AMP Synthase and Toll-like Receptor 4.

    Science.gov (United States)

    Andrade, Warrison A; Agarwal, Sarika; Mo, Shunyan; Shaffer, Scott A; Dillard, Joseph P; Schmidt, Tobias; Hornung, Veit; Fitzgerald, Katherine A; Kurt-Jones, Evelyn A; Golenbock, Douglas T

    2016-06-14

    The innate immune system is the first line of defense against Neisseria gonorrhoeae (GC). Exposure of cells to GC lipooligosaccharides induces a strong immune response, leading to type I interferon (IFN) production via TLR4/MD-2. In addition to living freely in the extracellular space, GC can invade the cytoplasm to evade detection and elimination. Double-stranded DNA introduced into the cytosol binds and activates the enzyme cyclic-GMP-AMP synthase (cGAS), which produces 2'3'-cGAMP and triggers STING/TBK-1/IRF3 activation, resulting in type I IFN expression. Here, we reveal a cytosolic response to GC DNA that also contributes to type I IFN induction. We demonstrate that complete IFN-β induction by live GC depends on both cGAS and TLR4. Type I IFN is detrimental to the host, and dysregulation of iron homeostasis genes may explain lower bacteria survival in cGAS(-/-) and TLR4(-/-) cells. Collectively, these observations reveal cooperation between TLRs and cGAS in immunity to GC infection. Copyright © 2016. Published by Elsevier Inc.

  4. Cyclic GMP-AMP Synthase Is the Cytosolic Sensor of Plasmodium falciparum Genomic DNA and Activates Type I IFN in Malaria.

    Science.gov (United States)

    Gallego-Marin, Carolina; Schrum, Jacob E; Andrade, Warrison A; Shaffer, Scott A; Giraldo, Lina F; Lasso, Alvaro M; Kurt-Jones, Evelyn A; Fitzgerald, Katherine A; Golenbock, Douglas T

    2018-01-15

    Innate immune receptors have a key role in the sensing of malaria and initiating immune responses. As a consequence of infection, systemic inflammation emerges and is directly related to signs and symptoms during acute disease. We have previously reported that plasmodial DNA is the primary driver of systemic inflammation in malaria, both within the phagolysosome and in the cytosol of effector cells. In this article, we demonstrate that Plasmodium falciparum genomic DNA delivered to the cytosol of human monocytes binds and activates cyclic GMP-AMP synthase (cGAS). Activated cGAS synthesizes 2'3'-cGAMP, which we subsequently can detect using liquid chromatography-tandem mass spectrometry. 2'3'-cGAMP acts as a second messenger for STING activation and triggers TBK1/IRF3 activation, resulting in type I IFN production in human cells. This induction of type I IFN was independent of IFI16. Access of DNA to the cytosolic compartment is mediated by hemozoin, because incubation of purified malaria pigment with DNase abrogated IFN-β induction. Collectively, these observations implicate cGAS as an important cytosolic sensor of P. falciparum genomic DNA and reveal the role of the cGAS/STING pathway in the induction of type I IFN in response to malaria parasites. Copyright © 2018 by The American Association of Immunologists, Inc.

  5. Type I Interferon Induction by Neisseria gonorrhoeae: Dual Requirement of Cyclic GMP-AMP Synthase and Toll-like Receptor 4

    Directory of Open Access Journals (Sweden)

    Warrison A. Andrade

    2016-06-01

    Full Text Available The innate immune system is the first line of defense against Neisseria gonorrhoeae (GC. Exposure of cells to GC lipooligosaccharides induces a strong immune response, leading to type I interferon (IFN production via TLR4/MD-2. In addition to living freely in the extracellular space, GC can invade the cytoplasm to evade detection and elimination. Double-stranded DNA introduced into the cytosol binds and activates the enzyme cyclic-GMP-AMP synthase (cGAS, which produces 2′3′-cGAMP and triggers STING/TBK-1/IRF3 activation, resulting in type I IFN expression. Here, we reveal a cytosolic response to GC DNA that also contributes to type I IFN induction. We demonstrate that complete IFN-β induction by live GC depends on both cGAS and TLR4. Type I IFN is detrimental to the host, and dysregulation of iron homeostasis genes may explain lower bacteria survival in cGAS−/− and TLR4−/− cells. Collectively, these observations reveal cooperation between TLRs and cGAS in immunity to GC infection.

  6. Binding of the cyclic AMP receptor protein of Escherichia coli and DNA bending at the P4 promoter of pBR322.

    Science.gov (United States)

    Brierley, I; Hoggett, J G

    1992-07-01

    The binding of the Escherichia coli cyclic AMP receptor protein (CRP) to its specific site on the P4 promoter of pBR322 has been studied by gel electrophoresis. Binding to the P4 site was about 40-50-fold weaker than to the principal CRP site on the lactose promoter at both low (0.01 M) and high (0.1 M) ionic strengths. CRP-induced bending at the P4 site was investigated from the mobilities of CRP bound to circularly permuted P4 fragments. The estimated bending angle, based on comparison with Zinkel & Crothers [(1990) Biopolymers 29, 29-38] A-tract bending standards, was found to be approximately 96 degrees, similar to that found for binding to the lac site. These observations suggest that there is not a simple relationship between strength of CRP binding and the extent of induced bending for different CRP sites. The apparent centre of bending in P4 is displaced about 6-8 bp away from the conserved TGTGA sequence and the P4 transcription start site.

  7. Cyclic AMP-dependent protein kinase interferes with GTP γS stimulated IP3 formation in differentiated HL-60 cell membranes

    International Nuclear Information System (INIS)

    Misaki, Naoyuki; Imaizumi, Taro; Watanabe, Yashuiro

    1989-01-01

    The effects of addition of activated cyclic AMP-dependent protein kinase (PKA) on the function of islet-activating protein (IAP)-sensitive GTP-binding (G) protein were studied in the plasma membranes of 3 H-inositol-labeled differentiated human leukemic (HL-60) cells. Pretreatment of the membranes with activated PKA in the presence of MgATP for 15 min. at 37 degree C decreased GTP γS-stimulated inositol trisphosphate (IP 3 ) formation by about 30%, but had no influence on Ca 2+ -stimulated IP 3 formation. And autoradiography in the phosphorylation experiments of solubilized HL-60 cell membranes by PKA showed some 32 P incorporated bands, and among them one of the major bands showed the migration at 40 kDa supporting that the G protein coupling with PI response was phosphorylated by PKA. These results showed that pretreatment with activated PKA inhibited the mediating function of the G protein between the fMLP receptor and phospholipase C by its phosphorylation

  8. Met-enkephalyl-Arg6-Phe7 immunoreactivity in a human neuroblastoma cell line: effect of dibutyryl 3':5'-cyclic AMP and reserpine.

    Science.gov (United States)

    Boarder, M R; Marriott, D; Adams, M

    1986-12-30

    The carboxy terminal part of the proenkephalin A sequence is the 31 amino acid peptide B, which has as its final seven amino acids the sequence of the opioid peptide Met-enkephalyl-Arg6-Phe7. Using a radioimmunoassay which recognises both these peptides we have investigated the relative amounts of peptide B and Met-enkephalyl-Arg6-Phe7 in a human neuroblastoma cell line. We show that these cells contain peptide B-like immunoreactivity but not its heptapeptide fragment. This may be due to lack of proteolytic activity cleaving Met-enkephalyl-Arg6-Phe7 from its precursor, peptide B. On treatment with dibutyryl cyclic AMP the level of immunoreactivity approximately doubles, due to increased amounts of peptide B-like immunoreactivity. Treatment with reserpine, which increases conversion of peptide B to the heptapeptide in bovine chromaffin cells in culture does not stimulate the accumulation of Met-enkephalyl-Arg6-Phe7 in the human neuroblastoma cells. The results are discussed with respect to peptide processing.

  9. Imaging alterations of cardiomyocyte cAMP microdomains in disease

    Directory of Open Access Journals (Sweden)

    Alexander eFroese

    2015-08-01

    Full Text Available 3’,5’-cyclic adenosine monophosphate (cAMP is an important second messenger which regulates heart function by acting in distinct subcellular microdomains. Recent years have provided deeper mechanistic insights into compartmentalized cAMP signaling and its link to cardiac disease. In this mini review, we summarize newest developments in this field achieved by cutting-edge biochemical and biophysical techniques. We further compile the data from different studies into a bigger picture of so far uncovered alterations in cardiomyocyte cAMP microdomains which occur in compensated cardiac hypertrophy and chronic heart failure. Finally, future research directions and translational perspectives are briefly discussed.

  10. The cAMP-PKA Signaling Pathway Regulates Pathogenicity, Hyphal Growth, Appressorial Formation, Conidiation, and Stress Tolerance in Colletotrichum higginsianum.

    Science.gov (United States)

    Zhu, Wenjun; Zhou, Man; Xiong, Zeyang; Peng, Fang; Wei, Wei

    2017-01-01

    Colletotrichum higginsianum is an economically important pathogen that causes anthracnose disease in a wide range of cruciferous crops. Understanding the mechanisms of the cruciferous plant- C. higginsianum interactions will be important in facilitating efficient control of anthracnose diseases. The cAMP-PKA signaling pathway plays important roles in diverse physiological processes of multiple pathogens. C. higginsianum contains two genes, ChPKA1 and ChPKA2 , that encode the catalytic subunits of cyclic AMP (cAMP)-dependent protein kinase A (PKA). To analyze the role of cAMP signaling pathway in pathogenicity and development in C. higginsianum , we characterized ChPKA1 and ChPKA2 genes, and adenylate cyclase ( ChAC ) gene. The ChPKA1 and ChAC deletion mutants were unable to cause disease and significantly reduced in hyphal growth, tolerance to cell wall inhibitors, conidiation, and appressorial formation with abnormal germ tubes, but they had an increased tolerance to elevated temperatures and exogenous H 2 O 2 . In contrast, the ChPKA2 mutant had no detectable alteration of phenotypes, suggesting that ChPKA1 contributes mainly to PKA activities in C. higginsianum . Moreover, we failed to generate Δ ChPKA1ChPKA2 double mutant, indicating that deletion of both PKA catalytic subunits is lethal in C. higginsianum and the two catalytic subunits possibly have overlapping functions. These results indicated that ChPKA1 is the major PKA catalytic subunit in cAMP-PKA signaling pathway and plays significant roles in hyphal growth, pathogenicity, appressorial formation, conidiation, and stress tolerance in C. higginsianum .

  11. The cAMP-PKA Signaling Pathway Regulates Pathogenicity, Hyphal Growth, Appressorial Formation, Conidiation, and Stress Tolerance in Colletotrichum higginsianum

    Directory of Open Access Journals (Sweden)

    Wenjun Zhu

    2017-07-01

    Full Text Available Colletotrichum higginsianum is an economically important pathogen that causes anthracnose disease in a wide range of cruciferous crops. Understanding the mechanisms of the cruciferous plant–C. higginsianum interactions will be important in facilitating efficient control of anthracnose diseases. The cAMP-PKA signaling pathway plays important roles in diverse physiological processes of multiple pathogens. C. higginsianum contains two genes, ChPKA1 and ChPKA2, that encode the catalytic subunits of cyclic AMP (cAMP-dependent protein kinase A (PKA. To analyze the role of cAMP signaling pathway in pathogenicity and development in C. higginsianum, we characterized ChPKA1 and ChPKA2 genes, and adenylate cyclase (ChAC gene. The ChPKA1 and ChAC deletion mutants were unable to cause disease and significantly reduced in hyphal growth, tolerance to cell wall inhibitors, conidiation, and appressorial formation with abnormal germ tubes, but they had an increased tolerance to elevated temperatures and exogenous H2O2. In contrast, the ChPKA2 mutant had no detectable alteration of phenotypes, suggesting that ChPKA1 contributes mainly to PKA activities in C. higginsianum. Moreover, we failed to generate ΔChPKA1ChPKA2 double mutant, indicating that deletion of both PKA catalytic subunits is lethal in C. higginsianum and the two catalytic subunits possibly have overlapping functions. These results indicated that ChPKA1 is the major PKA catalytic subunit in cAMP-PKA signaling pathway and plays significant roles in hyphal growth, pathogenicity, appressorial formation, conidiation, and stress tolerance in C. higginsianum.

  12. Regulation of Mg2+ Reabsorption and Transient Receptor Potential Melastatin Type 6 Activity by cAMP Signaling.

    NARCIS (Netherlands)

    Blanchard, M.G.; Kittikulsuth, W.; Nair, A.V.; Baaij, J.H.F. de; Latta, F.; Genzen, J.R.; Kohan, D.E.; Bindels, R.J.M.; Hoenderop, J.G.J.

    2016-01-01

    The transient receptor potential melastatin type 6 (TRPM6) epithelial Mg(2+) channels participate in transcellular Mg(2+) transport in the kidney and intestine. Previous reports suggested a hormonal cAMP-dependent regulation of Mg(2+) reabsorption in the kidney. The molecular details of this process

  13. AMP-activated protein kinase phosphorylates CtBP1 and down-regulates its activity

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jae-Hwan; Choi, Soo-Youn; Kang, Byung-Hee; Lee, Soon-Min [National Creative Research Center for Epigenome Reprogramming Network, Departments of Biomedical Sciences and Biochemistry and Molecular Biology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Park, Hyung Soon; Kang, Gum-Yong; Bang, Joo Young [Center for Biomedical Mass Spectrometry, Diatech Korea Co., Ltd., Seoul (Korea, Republic of); Cho, Eun-Jung [National Research Laboratory for Chromatin Dynamics, College of Pharmacy, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Youn, Hong-Duk, E-mail: hdyoun@snu.ac.kr [National Creative Research Center for Epigenome Reprogramming Network, Departments of Biomedical Sciences and Biochemistry and Molecular Biology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); WCU Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence and Technology, Seoul National University, Seoul (Korea, Republic of)

    2013-02-01

    Highlights: ► AMPK phosphorylates CtBP1 on serine 158. ► AMPK-mediated phosphorylation of CtBP1 causes the ubiquitination and nuclear export of CtBP1. ► AMPK downregulates the CtBP1-mediated repression of Bax transcription. -- Abstract: CtBP is a transcriptional repressor which plays a significant role in the regulation of cell proliferation and tumor progression. It was reported that glucose withdrawal causes induction of Bax due to the dissociation of CtBP from the Bax promoter. However, the precise mechanism involved in the regulation of CtBP still remains unclear. In this study, we found that an activated AMP-activated protein kinase (AMPK) phosphorylates CtBP1 on Ser-158 upon metabolic stresses. Moreover, AMPK-mediated phosphorylation of CtBP1 (S158) attenuates the repressive function of CtBP1. We also confirmed that triggering activation of AMPK by various factors resulted in an increase of Bax gene expression. These findings provide connections of AMPK with CtBP1-mediated regulation of Bax expression for cell death under metabolic stresses.

  14. Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

    Directory of Open Access Journals (Sweden)

    Hou Ssu-Yu

    2010-06-01

    Full Text Available Abstract Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors (statins have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin. Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2 formation, and phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP phosphorylation, and endothelial nitric oxide synthase (eNOS expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP-eNOS/NO-cyclic

  15. Cyclic AMP signalling in Dictyostelium : G-proteins activate separate Ras pathways using specific RasGEFs

    NARCIS (Netherlands)

    Kae, Helmut; Kortholt, Arjan; Rehmann, Holger; Insall, RobertH.; Van Haastert, Peter J. M.; Spiegelman, George B.; Weeks, Gerald

    In general, mammalian Ras guanine nucleotide exchange factors (RasGEFs) show little substrate specificity, although they are often thought to regulate specific pathways. Here, we provide in vitro and in vivo evidence that two RasGEFs can each act on specific Ras proteins. During Dictyostelium

  16. cAMP-CRP acts as a key regulator for the viable but non-culturable state in Escherichia coli.

    Science.gov (United States)

    Nosho, Kazuki; Fukushima, Hiroko; Asai, Takehiro; Nishio, Masahiro; Takamaru, Reiko; Kobayashi-Kirschvink, Koseki Joseph; Ogawa, Tetsuhiro; Hidaka, Makoto; Masaki, Haruhiko

    2018-03-01

    A variety of bacteria, including Escherichia coli, are known to enter the viable but non-culturable (VBNC) state under various stress conditions. During this state, cells lose colony-forming activities on conventional agar plates while retaining signs of viability. Diverse environmental stresses including starvation induce the VBNC state. However, little is known about the genetic mechanism inducing this state. Here, we aimed to reveal the genetic determinants of the VBNC state of E. coli. We hypothesized that the VBNC state is a process wherein specific gene products important for colony formation are depleted during the extended period of stress conditions. If so, higher expression of these genes would maintain colony-forming activities, thereby restraining cells from entering the VBNC state. From an E. coli plasmid-encoded ORF library, we identified genes that were responsible for maintaining high colony-forming activities after exposure to starvation condition. Among these, cpdA encoding cAMP phosphodiesterase exhibited higher performance in the maintenance of colony-forming activities. As cpdA overexpression decreases intracellular cAMP, cAMP or its complex with cAMP-receptor protein (CRP) may negatively regulate colony-forming activities under stress conditions. We confirmed this using deletion mutants lacking adenylate cyclase or CRP. These mutants fully maintained colony-forming activities even after a long period of starvation, while wild-type cells lost most of this activity. Thus, we concluded that the lack of cAMP-CRP effectively retains high colony-forming activities, indicating that cAMP-CRP acts as a positive regulator necessary for the induction of the VBNC state in E. coli.

  17. cAMP-response-element-binding protein positively regulates breast cancer metastasis and subsequent bone destruction

    Energy Technology Data Exchange (ETDEWEB)

    Son, Jieun; Lee, Jong-Ho; Kim, Ha-Neui; Ha, Hyunil, E-mail: hyunil74@hotmail.com; Lee, Zang Hee, E-mail: zang1959@snu.ac.kr

    2010-07-23

    Research highlights: {yields} CREB is highly expressed in advanced breast cancer cells. {yields} Tumor-related factors such as TGF-{beta} further elevate CREB expression. {yields} CREB upregulation stimulates metastatic potential of breast cancer cells. {yields} CREB signaling is required for breast cancer-induced bone destruction. -- Abstract: cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cells and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-{beta}. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.

  18. cAMP-dependent signaling regulates the adipogenic effect of n-6 polyunsaturated fatty acids

    DEFF Research Database (Denmark)

    Madsen, Lise; Pedersen, Lone Møller; Liaset, Bjørn

    2008-01-01

    The effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) on adipogenesis and obesity is controversial. Using in vitro cell culture models, we show that n-6 PUFAs was pro-adipogenic under conditions with base-line levels of cAMP, but anti-adipogenic when the levels of cAMP were elevated. The anti...

  19. EFFECT OF dbcAMP ON PROLIFERATION AND APOPTOSIS OF PORCINE GRANULOSA CELLS in vitro

    Directory of Open Access Journals (Sweden)

    Richard Alexa

    2013-02-01

    Full Text Available Cyclic nucleotide cAMP and its target protein kinase A (PKA dependent intracellular mechanisms can play an important role in regulation of ovarian cell function and in mediating gonadotropin action on these cells. The aim of the present study was to examine the effect of cAMP analogue, dibutyryl cyclic adenosine monophosphate (dbcAMP (0; 0.1; 1 and 10 µg/ml or FSH (0; 0,01; 1 IU/ml on proliferation and apoptosis of porcine granulosa cells in vitro. Indices of cell apoptosis (expression of apoptotic peptide bax and proliferation (expression of proliferation-associated peptide PCNA within ovarian granulosa cells were analysed by immunocytochemistry. It was observed that accumulation of PCNA was increased by dbcAMP and FSH at all doses added. The occurrence of bax was also stimulated by dbcAMP after exposition (at 0,1 and 1 µg/ml, but not at dose 10 µg/ml and by FSH (at all doses added. The stimulatory effect of both dbcAMP and FSH on both ovarian cell apoptosis and proliferation suggest, that these substances may promote ovarian follicular cell turnover. The similarity of dbcAMP and FSH effect may indicate that FSH can affect ovarian functions via cAMP-dependent intracellular mechanisms. The present data may provide new tools to regulate human and animal reproductive processes via cAMP-dependent mechanisms.

  20. Extracellular calmodulin regulates growth and cAMP-mediated chemotaxis in Dictyostelium discoideum

    International Nuclear Information System (INIS)

    O’Day, Danton H.; Huber, Robert J.; Suarez, Andres

    2012-01-01

    Highlights: ► Extracellular calmodulin is present throughout growth and development in Dictyostelium. ► Extracellular calmodulin localizes within the ECM during development. ► Extracellular calmodulin inhibits cell proliferation and increases chemotaxis. ► Extracellular calmodulin exists in eukaryotic microbes. ► Extracellular calmodulin may be functionally as important as intracellular calmodulin. -- Abstract: The existence of extracellular calmodulin (CaM) has had a long and controversial history. CaM is a ubiquitous calcium-binding protein that has been found in every eukaryotic cell system. Calcium-free apo-CaM and Ca 2+ /CaM exert their effects by binding to and regulating the activity of CaM-binding proteins (CaMBPs). Most of the research done to date on CaM and its CaMBPs has focused on their intracellular functions. The presence of extracellular CaM is well established in a number of plants where it functions in proliferation, cell wall regeneration, gene regulation and germination. While CaM has been detected extracellularly in several animal species, including frog, rat, rabbit and human, its extracellular localization and functions are less well established. In contrast the study of extracellular CaM in eukaryotic microbes remains to be done. Here we show that CaM is constitutively expressed and secreted throughout asexual development in Dictyostelium where the presence of extracellular CaM dose-dependently inhibits cell proliferation but increases cAMP mediated chemotaxis. During development, extracellular CaM localizes within the slime sheath where it coexists with at least one CaMBP, the matricellular CaM-binding protein CyrA. Coupled with previous research, this work provides direct evidence for the existence of extracellular CaM in the Dictyostelium and provides insight into its functions in this model amoebozoan.

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

    Directory of Open Access Journals (Sweden)

    Cédric eBOULARAN

    2015-10-01

    Full Text Available 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.

  2. [Physiopathology of cAMP/PKA signaling in neurons].

    Science.gov (United States)

    Castro, Liliana; Yapo, Cedric; Vincent, Pierre

    2016-01-01

    Cyclic adenosine monophosphate (cAMP) and the cyclic-AMP dependent protein kinase (PKA) regulate a plethora of cellular functions in virtually all eukaryotic cells. In neurons, the cAMP/PKA signaling cascade controls a number of biological properties such as axonal growth, synaptic transmission, regulation of excitability or long term changes in the nucleus. Genetically-encoded optical biosensors for cAMP or PKA considerably improved our understanding of these processes by providing a real-time measurement in living neurons. In this review, we describe the recent progresses made in the creation of biosensors for cAMP or PKA activity. These biosensors revealed profound differences in the amplitude of the cAMP signal evoked by neuromodulators between various neuronal preparations. These responses can be resolved at the level of individual neurons, also revealing differences related to the neuronal type. At the subcellular level, biosensors reported different signal dynamics in domains like dendrites, cell body, nucleus and axon. Combining this imaging approach with pharmacology or genetical models points at phosphodiesterases and phosphatases as critical regulatory proteins. Biosensor imaging will certainly help understand the mechanism of action of current drugs as well as help in devising novel therapeutic strategies for neuropsychiatric diseases. © Société de Biologie, 2017.

  3. Inhibitory effects of ginseng total saponin on up-regulation of cAMP pathway induced by repeated administration of morphine.

    Science.gov (United States)

    Seo, Jeong-Ju; Lee, Jae-Woong; Lee, Wan-Kyu; Hong, Jin-Tae; Lee, Chong-Kil; Lee, Myung-Koo; Oh, Ki-Wan

    2008-02-01

    We have reported that ginseng total saponin (GTS) inhibited the development of physical and psychological dependence on morphine. However, the possible molecular mechanisms of GTS are unclear. Therefore, this study was undertaken to understand the possible molecular mechanism of GTS on the inhibitory effects of morphine-induced dependence. It has been reported that the up-regulated cAMP pathway in the LC of the mouse brain after repeated administration of morphine contributes to the feature of withdrawals. GTS inhibited up-regulation of cAMP pathway in the LC after repeated administration of morphine in this experiment. GTS inhibited cAMP levels and protein expression of protein kinase A (PKA). In addition, GTS inhibited the increase of cAMP response element binding protein (CREB) phosphorylation. Therefore, we conclude that the inhibitory effects of GTS on morphine-induced dependence might be mediated by the inhibition of cAMP pathway.

  4. Exchange Protein Directly Activated by cAMP (epac) : A Multidomain cAMP Mediator in the Regulation of Diverse Biological Functions

    NARCIS (Netherlands)

    Schmidt, Martina; Dekker, Frank J.; Maarsingh, Harm

    Since the discovery nearly 60 years ago, cAMP is envisioned as one of the most universal and versatile second messengers. The tremendous feature of cAMP to tightly control highly diverse physiologic processes, including calcium homeostasis, metabolism, secretion, muscle contraction, cell fate, and

  5. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

    Science.gov (United States)

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

    The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells. PMID:27226623

  6. Neuro-endocrine regulation of LH secretion in cyclic pigs

    NARCIS (Netherlands)

    Dierx, J.

    1999-01-01

    The present thesis describes the study in the hormonal (endocrine) regulation of the fertility in the female pig. Fertility is optimal when during the reproductive cycle the concentrations of hormones change in a particular sequence. In the present thesis it has been shown that, amongst

  7. Transcutaneous electrical nerve stimulation (TENS) improves the diabetic cytopathy (DCP) via up-regulation of CGRP and cAMP.

    Science.gov (United States)

    Ding, Liucheng; Song, Tao; Yi, Chaoran; Huang, Yi; Yu, Wen; Ling, Lin; Dai, Yutian; Wei, Zhongqing

    2013-01-01

    The objective of this study was to investigate the effects and mechanism of Transcutaneous Electrical Nerve Stimulation (TENS) on the diabetic cytopathy (DCP) in the diabetic bladder. A total of 45 rats were randomly divided into diabetes mellitus (DM)/TENS group (n=15), DM group (n=15) and control group (n=15). The rats in the DM/TENS and TENS groups were electronically stimulated (stimulating parameters: intensity-31 V, frequency-31 Hz, and duration of stimulation of 15 min) for three weeks. Bladder histology, urodynamics and contractile responses to field stimulation and carbachol were determined. The expression of calcitonin gene-related peptide (CGRP) was analyzed by RT-PCR and Western blotting. The results showed that contractile responses of the DM rats were ameliorated after 3 weeks of TENS. Furthermore, TENS significantly increased bladder wet weight, volume threshold for micturition and reduced PVR, V% and cAMP content of the bladder. The mRNA and protein levels of CGRP in dorsal root ganglion (DRG) in the DM/TENS group were higher than those in the DM group. TENS also significantly up-regulated the cAMP content in the bladder body and base compared with diabetic rats. We conclude that TENS can significantly improve the urine contractility and ameliorate the feeling of bladder fullness in DM rats possibly via up-regulation of cAMP and CGRP in DRG.

  8. AMP-Activated Protein Kinase (AMPK) Regulates Energy Metabolism through Modulating Thermogenesis in Adipose Tissue

    Science.gov (United States)

    Wu, Lingyan; Zhang, Lina; Li, Bohan; Jiang, Haowen; Duan, Yanan; Xie, Zhifu; Shuai, Lin; Li, Jia; Li, Jingya

    2018-01-01

    Obesity occurs when excess energy accumulates in white adipose tissue (WAT), whereas brown adipose tissue (BAT), which is specialized in dissipating energy through thermogenesis, potently counteracts obesity. White adipocytes can be converted to thermogenic “brown-like” cells (beige cells; WAT browning) under various stimuli, such as cold exposure. AMP-activated protein kinase (AMPK) is a crucial energy sensor that regulates energy metabolism in multiple tissues. However, the role of AMPK in adipose tissue function, especially in the WAT browning process, is not fully understood. To illuminate the effect of adipocyte AMPK on energy metabolism, we generated Adiponectin-Cre-driven adipose tissue-specific AMPK α1/α2 KO mice (AKO). These AKO mice were cold intolerant and their inguinal WAT displayed impaired mitochondrial integrity and biogenesis, and reduced expression of thermogenic markers upon cold exposure. High-fat-diet (HFD)-fed AKO mice exhibited increased adiposity and exacerbated hepatic steatosis and fibrosis and impaired glucose tolerance and insulin sensitivity. Meanwhile, energy expenditure and oxygen consumption were markedly decreased in the AKO mice both in basal conditions and after stimulation with a β3-adrenergic receptor agonist, CL 316,243. In contrast, we found that in HFD-fed obese mouse model, chronic AMPK activation by A-769662 protected against obesity and related metabolic dysfunction. A-769662 alleviated HFD-induced glucose intolerance and reduced body weight gain and WAT expansion. Notably, A-769662 increased energy expenditure and cold tolerance in HFD-fed mice. A-769662 treatment also induced the browning process in the inguinal fat depot of HFD-fed mice. Likewise, A-769662 enhanced thermogenesis in differentiated inguinal stromal vascular fraction (SVF) cells via AMPK signaling pathway. In summary, a lack of adipocyte AMPKα induced thermogenic impairment and obesity in response to cold and nutrient-overload, respectively

  9. Insulin elevates leptin secretion and mRNA levels via cyclic AMP in 3T3-L1 adipocytes deprived of glucose

    Directory of Open Access Journals (Sweden)

    Tomomi Tsubai

    2016-11-01

    Conclusion: Insulin alone stimulates leptin secretion and elevates leptin mRNA levels via cAMP under the lack of glucose metabolism, while glucose is a significant and ambivalent effector on the insulin effects of leptin.

  10. CRP-dependent positive autoregulation and proteolytic degradation regulate competence activator Sxy of Escherichia coli

    DEFF Research Database (Denmark)

    Jaskólska, Milena; Gerdes, Kenn

    2015-01-01

    is positively autoregulated at the level of transcription by a mechanism that requires cAMP receptor protein (CRP), cyclic AMP (cAMP) and a CRP-S site in the sxy promoter. Similarly, we found no evidence that Sxy expression in E. coli was regulated at the translational level. However, our analysis revealed...

  11. Differential regulation by AMP and ADP of AMPK complexes containing different γ subunit isoforms

    DEFF Research Database (Denmark)

    Ross, Fiona A; Jensen, Thomas Elbenhardt; Hardie, D Grahame

    2016-01-01

    The g subunits of heterotrimeric AMPK complexes contain the binding sites for the regulatory adenine nucleotides AMP, ADP and ATP. We addressed whether complexes containing different g isoforms display different responses to adenine nucleotides by generating cells stably expressing FLAG-tagged ve...

  12. Independent AMP and NAD signaling regulates C2C12 differentiation and metabolic adaptation.

    Science.gov (United States)

    Hsu, Chia George; Burkholder, Thomas J

    2016-12-01

    The balance of ATP production and consumption is reflected in adenosine monophosphate (AMP) and nicotinamide adenine dinucleotide (NAD) content and has been associated with phenotypic plasticity in striated muscle. Some studies have suggested that AMPK-dependent plasticity may be an indirect consequence of increased NAD synthesis and SIRT1 activity. The primary goal of this study was to assess the interaction of AMP- and NAD-dependent signaling in adaptation of C2C12 myotubes. Changes in myotube developmental and metabolic gene expression were compared following incubation with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and nicotinamide mononucleotide (NMN) to activate AMPK- and NAD-related signaling. AICAR showed no effect on NAD pool or nampt expression but significantly reduced histone H3 acetylation and GLUT1, cytochrome C oxidase subunit 2 (COX2), and MYH3 expression. In contrast, NMN supplementation for 24 h increased NAD pool by 45 % but did not reduce histone H3 acetylation nor promote mitochondrial gene expression. The combination of AMP and NAD signaling did not induce further metabolic adaptation, but NMN ameliorated AICAR-induced myotube reduction. We interpret these results as indication that AMP and NAD contribute to C2C12 differentiation and metabolic adaptation independently.

  13. Microtubule Regulation of Kv7 Channels Orchestrates cAMP-Mediated Vasorelaxations in Rat Arterial Smooth Muscle

    DEFF Research Database (Denmark)

    Lindman, Johanna; Khammy, Makhala M; Lundegaard, Pia R

    2018-01-01

    Microtubules can regulate GPCR (G protein-coupled receptor) signaling in various cell types. In vascular smooth muscle, activation of the β-adrenoceptor leads to production of cAMP to mediate a vasorelaxation. Little is known about the role of microtubules in smooth muscle, and given the importance...... of renal and mesenteric arteries that the microtubule stabilizer, paclitaxel, prevented. Sharp microelectrode experiments showed that colchicine treatment caused increased hyperpolarization of mesenteric artery segments in response to isoprenaline. Application of the Kv7 channel blocker, XE991, attenuated...

  14. DREAM mediates cAMP-dependent, Ca2+-induced stimulation of GFAP gene expression and regulates cortical astrogliogenesis.

    Science.gov (United States)

    Cebolla, Beatriz; Fernández-Pérez, Antonio; Perea, Gertrudis; Araque, Alfonso; Vallejo, Mario

    2008-06-25

    In the developing mouse brain, once the generation of neurons is mostly completed during the prenatal period, precisely coordinated signals act on competent neural precursors to direct their differentiation into astrocytes, which occurs mostly after birth. Among these signals, those provided by neurotrophic cytokines and bone morphogenetic proteins appear to have a key role in triggering the neurogenic to gliogenic switch and in regulating astrocyte numbers. In addition, we have reported previously that the neurotrophic peptide pituitary adenylate cyclase-activating polypeptide (PACAP) is able to promote astrocyte differentiation of cortical precursors via activation of a cAMP-dependent pathway. Signals acting on progenitor cells of the developing cortex to generate astrocytes activate glial fibrillary acidic protein (GFAP) gene expression, but the transcriptional mechanisms that regulate this activation are unclear. Here, we identify the previously known transcriptional repressor downstream regulatory element antagonist modulator (DREAM) as an activator of GFAP gene expression. We found that DREAM occupies specific sites on the GFAP promoter before and after differentiation is initiated by exposure of cortical progenitor cells to PACAP. PACAP raises intracellular calcium concentration via a mechanism that requires cAMP, and DREAM-mediated transactivation of the GFAP gene requires the integrity of calcium-binding domains. Cortical progenitor cells from dream(-/-) mice fail to express GFAP in response to PACAP. Moreover, the neonatal cortex of dream(-/-) mice exhibits a reduced number of astrocytes and increased number of neurons. These results identify the PACAP-cAMP-Ca(2+)-DREAM cascade as a new pathway to activate GFAP gene expression during astrocyte differentiation.

  15. Polarized Th1 and Th2 cells are less responsive to negative feedback by receptors coupled to the AC/cAMP system compared to freshly isolated T cells

    NARCIS (Netherlands)

    Heijink, Irene H; Vellenga, Edo; Borger, Peter; Postma, Dirkje S; Monchy, Jan G R de; Kauffman, Henk F

    1 The adenylyl cyclase (AC)/cyclic adenosine monophosphate (cAMP) system is known to negatively regulate transcriptional activity of T cells, thereby possibly modulating T-cell-mediated responses at the sites of inflammation. Effects of cAMP have been widely studied in freshly isolated T cells and

  16. CCAAT/enhancer-binding protein delta activates insulin-like growth factor-I gene transcription in osteoblasts. Identification of a novel cyclic AMP signaling pathway in bone

    Science.gov (United States)

    Umayahara, Y.; Ji, C.; Centrella, M.; Rotwein, P.; McCarthy, T. L.

    1997-01-01

    Insulin-like growth factor-I (IGF-I) plays a key role in skeletal growth by stimulating bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteoblasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was essential for hormonal regulation of IGF-I gene expression. We now demonstrate that CCAAT/enhancer-binding protein (C/EBP) delta is a major component of a PGE2-stimulated DNA-protein complex involving HS3D and find that C/EBPdelta transactivates IGF-I promoter 1 through this site. Competition gel shift studies first indicated that a core C/EBP half-site (GCAAT) was required for binding of a labeled HS3D oligomer to osteoblast nuclear proteins. Southwestern blotting and UV-cross-linking studies showed that the HS3D probe recognized a approximately 35-kDa nuclear protein, and antibody supershift assays indicated that C/EBPdelta comprised most of the PGE2-activated gel-shifted complex. C/EBPdelta was detected by Western immunoblotting in osteoblast nuclear extracts after treatment of cells with PGE2. An HS3D oligonucleotide competed effectively with a high affinity C/EBP site from the rat albumin gene for binding to osteoblast nuclear proteins. Co-transfection of osteoblast cell cultures with a C/EBPdelta expression plasmid enhanced basal and PGE2-activated IGF-I promoter 1-luciferase activity but did not stimulate a reporter gene lacking an HS3D site. By contrast, an expression plasmid for the related protein, C/EBPbeta, did not alter basal IGF-I gene activity but did increase the response to PGE2. In osteoblasts and in COS-7 cells, C/EBPdelta, but not C/EBPbeta, transactivated a reporter gene containing four tandem copies of HS3D fused to a minimal promoter; neither transcription factor stimulated a gene with four copies of an HS3D mutant that was unable to bind osteoblast

  17. Inducible cAMP early repressor acts as a negative regulator for kindling epileptogenesis and long-term fear memory.

    Science.gov (United States)

    Kojima, Nobuhiko; Borlikova, Gilyana; Sakamoto, Toshiro; Yamada, Kazuyuki; Ikeda, Toshio; Itohara, Shigeyoshi; Niki, Hiroaki; Endo, Shogo

    2008-06-18

    Long-lasting neuronal plasticity as well as long-term memory (LTM) requires de novo synthesis of proteins through dynamic regulation of gene expression. cAMP-responsive element (CRE)-mediated gene transcription occurs in an activity-dependent manner and plays a pivotal role in neuronal plasticity and LTM in a variety of species. To study the physiological role of inducible cAMP early repressor (ICER), a CRE-mediated gene transcription repressor, in neuronal plasticity and LTM, we generated two types of ICER mutant mice: ICER-overexpressing (OE) mice and ICER-specific knock-out (KO) mice. Both ICER-OE and ICER-KO mice show no apparent abnormalities in their development and reproduction. A comprehensive battery of behavioral tests revealed no robust changes in locomotor activity, sensory and motor functions, and emotional responses in the mutant mice. However, long-term conditioned fear memory was attenuated in ICER-OE mice and enhanced in ICER-KO mice without concurrent changes in short-term fear memory. Furthermore, ICER-OE mice exhibited retardation of kindling development, whereas ICER-KO mice exhibited acceleration of kindling. These results strongly suggest that ICER negatively regulates the neuronal processes required for long-term fear memory and neuronal plasticity underlying kindling epileptogenesis, possibly through suppression of CRE-mediated gene transcription.

  18. AMP-activated protein kinase in contraction regulation of skeletal muscle metabolism: necessary and/or sufficient?

    DEFF Research Database (Denmark)

    Jensen, Thomas Elbenhardt; Wojtaszewski, Jørgen; Richter, Erik

    2009-01-01

    In skeletal muscle, the contraction-activated heterotrimeric 5'-AMP-activated protein kinase (AMPK) protein is proposed to regulate the balance between anabolic and catabolic processes by increasing substrate uptake and turnover in addition to regulating the transcription of proteins involved...... in mitochondrial biogenesis and other aspects of promoting an oxidative muscle phenotype. Here, the current knowledge on the expression of AMPK subunits in human quadriceps muscle and evidence from rodent studies suggesting distinct AMPK subunit expression pattern in different muscle types is reviewed. Then......, the intensity and time dependence of AMPK activation in human quadriceps and rodent muscle are evaluated. Subsequently, a major part of this review critically examines the evidence supporting a necessary and/or sufficient role of AMPK in a broad spectrum of skeletal muscle contraction-relevant processes...

  19. Thyrotropin regulates IL-6 expression in CD34+ fibrocytes: clear delineation of its cAMP-independent actions.

    Directory of Open Access Journals (Sweden)

    Nupur Raychaudhuri

    Full Text Available IL-6 plays diverse roles in normal and disease-associated immunity such as that associated with Graves' disease (GD. In that syndrome, the orbit undergoes remodeling during a process known as thyroid-associated ophthalmopathy (TAO. Recently, CD34(+ fibrocytes were found to infiltrate the orbit in TAO where they transition into CD34(+ orbital fibroblasts. Surprisingly, fibrocytes display high levels of functional thyrotropin receptor (TSHR, the central antigen in GD. We report here that TSH and the pathogenic anti-TSHR antibodies that drive hyperthyroidism in GD induce IL-6 expression in fibrocytes and orbital fibroblasts. Unlike TSHR signaling in thyroid epithelium, that occurring in fibrocytes is completely independent of adenylate cyclase activation and cAMP generation. Instead TSH activates PDK1 and both AKT/PKB and PKC pathways. Expression and use of PKCβII switches to that of PKCµ as fibrocytes transition to TAO orbital fibroblasts. This shift is imposed by CD34(- orbital fibroblasts but reverts when CD34(+ fibroblasts are isolated. The up-regulation of IL-6 by TSH results from coordinately enhanced IL-6 gene promoter activity and increased IL-6 mRNA stability. TSH-dependent IL-6 expression requires activity at both CREB (-213 to -208 nt and NF-κB (-78 to -62 nt binding sites. These results provide novel insights into the molecular action of TSH and signaling downstream for TSHR in non-thyroid cells. Fibrocytes neither express adenylate cyclase nor generate cAMP and thus these findings are free from any influence of cAMP-related signaling. They identify potential therapeutic targets for TAO.

  20. Cl- transport pathways regulated by Ca++, cAMP, and pH in human fibroblasts

    International Nuclear Information System (INIS)

    Lin, P.; Gruenstein, E.

    1987-01-01

    Under basal conditions Cl - efflux from human fibroblasts occurs with a rate constant of permeability of 0.08 min -1 . 50% of the basal efflux is due to Cl - /anion exchange and is DIDS inhibitable, 25% is due to Na + /K + /Cl - cotransport and is furosemide inhibitable, and 20% is due to an electrically conductive pathway. Increasing intracellular Ca ++ with A23187 stimulates Cl - efflux by 30%. This increase appears to occur entirely via an electrically conducting pathway, but unlike basal Cl - conductance, it is DIDS sensitive. Exposure of the cells to dibutyryl cAMP stimulates Cl - efflux by 15%. They do not yet know whether the cAMP stimulated pathway is electrically conductive, but the stimulation is additive with that caused by elevated Ca ++ suggesting that different pathways are activated. Elevation of intracellular pH by any of several standard methods increases Cl - efflux by as much as 700%. The pH effect appears to be mediated by a Cl - /anion exchange pathway since it is DIDS sensitive and electroneutral. Previous work from this laboratory describing a transient rapid efflux of Cl - followed by a slower efflux phase can now be explained as the result of a transient alkalinization of cells rather than as 2 subcellular Cl - compartments. This alkalinization occurs when cells are transferred from a 5% CO 2 atmosphere during 36 Cl - load to ambient CO 2 for efflux

  1. Regulation of thrombomodulin expression and release in human aortic endothelial cells by cyclic strain.

    Directory of Open Access Journals (Sweden)

    Fiona A Martin

    Full Text Available Thrombomodulin (TM, an integral membrane glycoprotein expressed on the lumenal surface of vascular endothelial cells, promotes anti-coagulant and anti-inflammatory properties. Release of functional TM from the endothelium surface into plasma has also been reported. Much is still unknown however about how endothelial TM is regulated by physiologic hemodynamic forces (and particularly cyclic strain intrinsic to endothelial-mediated vascular homeostasis.This study employed human aortic endothelial cells (HAECs to investigate the effects of equibiaxial cyclic strain (7.5%, 60 cycles/min, 24 hrs, and to a lesser extent, laminar shear stress (10 dynes/cm2, 24 hrs, on TM expression and release. Time-, dose- and frequency-dependency studies were performed.Our initial studies demonstrated that cyclic strain strongly downregulated TM expression in a p38- and receptor tyrosine kinase-dependent manner. This was in contrast to the upregulatory effect of shear stress. Moreover, both forces significantly upregulated TM release over a 48 hr period. With continuing focus on the cyclic strain-induced TM release, we noted both dose (0-7.5% and frequency (0.5-2.0 Hz dependency, with no attenuation of strain-induced TM release observed following inhibition of MAP kinases (p38, ERK-1/2, receptor tyrosine kinase, or eNOS. The concerted impact of cyclic strain and inflammatory mediators on TM release from HAECs was also investigated. In this respect, both TNFα (100 ng/ml and ox-LDL (10-50 µg/ml appeared to potentiate strain-induced TM release. Finally, inhibition of neither MMPs (GM6001 nor rhomboids (3,4-dichloroisocoumarin had any effect on strain-induced TM release. However, significantly elevated levels (2.1 fold of TM were observed in isolated microparticle fractions following 7.5% strain for 24 hrs.A preliminary in vitro investigation into the effects of cyclic strain on TM in HAECs is presented. Physiologic cyclic strain was observed to downregulate TM

  2. Increase in cyclic AMP concentration in a cerebral giant interneuron mimics part of a memory trace for conditioned taste aversion of the pond snail.

    Science.gov (United States)

    Otsuka, Emi; Matsunaga, Miho; Okada, Ryuichi; Yamagishi, Miki; Okuta, Akiko; Lukowiak, Ken; Ito, Etsuro

    2013-01-01

    Conditioned taste aversion (CTA) can be classically conditioned in the pond snail Lymnaea stagnalis and subsequently be consolidated into long-term memory (LTM). The neural trace that subserves CTA-LTM can be summarized as follows: A polysynaptic inhibitory postsynaptic potential recorded in the neuron 1 medial (N1M) cell in the conditioned snails as a result of activation of the cerebral giant cell (CGC) is larger and lasts longer than that in control snails. The N1M cell is ultimately activated by the CGC via the neuron 3 tonic (N3t) cell. That is, the inhibitory monosynaptic inputs from the N3t cell to the N1M cell are facilitated. The N1M and N3t cells are the members of feeding central pattern generator, whereas the CGC is a multimodal interneuron thought to play a key role in feeding behavior. Here we examined the involvement of a second messenger, cAMP, in the establishment of the memory trace. We injected cAMP into the CGC and monitored the potentials of the B3 motor neuron activated by the CGC. B3 activity is used as an index for the synaptic inputs from the N3t cell to the N1M cell. We found that the B3 potentials were transiently enlarged. Thus, when the cAMP concentration is increased in the CGC by taste aversion training, cAMP-induced changes may play a key role in the establishment of a memory trace in the N3t cell.

  3. Sweet taste receptor expressed in pancreatic beta-cells activates the calcium and cyclic AMP signaling systems and stimulates insulin secretion.

    Directory of Open Access Journals (Sweden)

    Yuko Nakagawa

    Full Text Available BACKGROUND: Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets. METHODOLOGY/PRINCIPAL FINDINGS: The expression of the sweet taste receptor was determined by RT-PCR and immunohistochemistry. Changes in cytoplasmic Ca(2+ ([Ca(2+](c and cAMP ([cAMP](c were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca(2+](c. The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca(2+](c response. The effect of sucralose on [Ca(2+](c was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a G(q inhibitor. Sucralose also induced sustained elevation of [cAMP](c, which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion. CONCLUSIONS: Sweet taste receptor is expressed in beta-cells, and activation of this receptor induces insulin secretion by Ca(2+ and cAMP-dependent mechanisms.

  4. The freeze-thaw stress response of the yeast Saccharomyces cerevisiae is growth phase specific and is controlled by nutritional state via the RAS-cyclic AMP signal transduction pathway.

    Science.gov (United States)

    Park, J I; Grant, C M; Attfield, P V; Dawes, I W

    1997-10-01

    The ability of cells to survive freezing and thawing is expected to depend on the physiological conditions experienced prior to freezing. We examined factors affecting yeast cell survival during freeze-thaw stress, including those associated with growth phase, requirement for mitochondrial functions, and prior stress treatment(s), and the role played by relevant signal transduction pathways. The yeast Saccharomyces cerevisiae was frozen at -20 degrees C for 2 h (cooling rate, less than 4 degrees C min-1) and thawed on ice for 40 min. Supercooling occurred without reducing cell survival and was followed by freezing. Loss of viability was proportional to the freezing duration, indicating that freezing is the main determinant of freeze-thaw damage. Regardless of the carbon source used, the wild-type strain and an isogenic petite mutant ([rho 0]) showed the same pattern of freeze-thaw tolerance throughout growth, i.e., high resistance during lag phase and low resistance during log phase, indicating that the response to freeze-thaw stress is growth phase specific and not controlled by glucose repression. In addition, respiratory ability and functional mitochondria are necessary to confer full resistance to freeze-thaw stress. Both nitrogen and carbon source starvation led to freeze-thaw tolerance. The use of strains affected in the RAS-cyclic AMP (RAS-cAMP) pathway or supplementation of an rca1 mutant (defective in the cAMP phosphodiesterase gene) with cAMP showed that the freeze-thaw response of yeast is under the control of the RAS-cAMP pathway. Yeast did not adapt to freeze-thaw stress following repeated freeze-thaw treatment with or without a recovery period between freeze-thaw cycles, nor could it adapt following pretreatment by cold shock. However, freeze-thaw tolerance of yeast cells was induced during fermentative and respiratory growth by pretreatment with H2O2, cycloheximide, mild heat shock, or NaCl, indicating that cross protection between freeze-thaw stress

  5. Regulation of cAMP Responsive Element Binding Protein 3-Like 1 (Creb3l1 Expression by Orphan Nuclear Receptor Nr4a1

    Directory of Open Access Journals (Sweden)

    Michael P. Greenwood

    2017-12-01

    Full Text Available Cyclic AMP (cAMP inducible transcription factor cAMP responsive element binding protein 3 like 1 (Creb3l1 is strongly activated in the hypothalamus in response to hyperosmotic cues such as dehydration (DH. We have recently shown that Creb3l1 expression is upregulated by cAMP pathways in vitro, however the exact mechanisms are not known. Here we show that increasing Creb3l1 transcription by raising cAMP levels in mouse pituitary AtT20 cells automatically initiates cleavage of Creb3l1, leading to a greater abundance of the transcriptionally active N-terminal portion. Inhibiting protein synthesis indicated that de novo protein synthesis of an intermediary transcription factor was required for Creb3l1 induction. Strategic mining of our microarray data from dehydrated rodent hypothalamus revealed four candidates, reduced to two by analysis of acute hyperosmotic-induced transcriptional activation profiles in the hypothalamus, and one, orphan nuclear receptor Nr4a1, by direct shRNA mediated silencing in AtT20 cells. We show that activation of Creb3l1 transcription by Nr4a1 involves interaction with a single NBRE site in the promoter region. The ability to activate Creb3l1 transcription by this pathway in vitro is dictated by the level of methylation of a CpG island within the proximal promoter/5′UTR of this gene. We thus identify a novel cAMP-Nr4a1-Creb3l1 transcriptional pathway in AtT20 cells and also, our evidence would suggest, in the hypothalamus.

  6. FRET-based binding assay between a fluorescent cAMP analogue and a cyclic nucleotide-binding domain tagged with a CFP.

    Science.gov (United States)

    Romero, Francisco; Santana-Calvo, Carmen; Sánchez-Guevara, Yoloxochitl; Nishigaki, Takuya

    2017-09-01

    The cyclic nucleotide-binding domain (CNBD) functions as a regulatory domain of many proteins involved in cyclic nucleotide signalling. We developed a straightforward and reliable binding assay based on intermolecular fluorescence resonance energy transfer (FRET) between an adenosine-3', 5'-cyclic monophosphate analogue labelled with fluorescein and a recombinant CNBD of human EPAC1 tagged with a cyan fluorescence protein (CFP). The high FRET efficiency of this method (~ 80%) allowed us to perform several types of binding experiments with nanomolar range of sample using conventional equipment. In addition, the CFP tag on the CNBD enabled us to perform a specific binding experiment using an unpurified protein. Considering these advantages, this technique is useful to study poorly characterized CNBDs. © 2017 Federation of European Biochemical Societies.

  7. A cAMP Biosensor-Based High-Throughput Screening Assay for Identification of Gs-Coupled GPCR Ligands and Phosphodiesterase Inhibitors

    DEFF Research Database (Denmark)

    Vedel, Line; Bräuner-Osborne, Hans; Mathiesen, Jesper Mosolff

    2015-01-01

    Cyclic adenosine 3',5'-monophosphate (cAMP) is an important second messenger, and quantification of intracellular cAMP levels is essential in studies of G protein-coupled receptors (GPCRs). The intracellular cAMP levels are regulated by the adenylate cyclase (AC) upon activation of either Gs- or ...... also observed for the other representative Gs-coupled GPCRs tested, GLP-1R and GlucagonR. The FRET-based cAMP biosensor assay is robust, reproducible, and inexpensive with good Z factors and is highly applicable for HTS....

  8. Cows are not mice: the role of cyclic AMP, phosphodiesterases, and adenosine monophosphate-activated protein kinase in the maintenance of meiotic arrest in bovine oocytes.

    Science.gov (United States)

    Bilodeau-Goeseels, Sylvie

    2011-01-01

    Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures. Copyright © 2011 Wiley Periodicals, Inc.

  9. A cyclic GMP signalling module that regulates gliding motility in a malaria parasite.

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    Robert W Moon

    2009-09-01

    Full Text Available The ookinete is a motile stage in the malaria life cycle which forms in the mosquito blood meal from the zygote. Ookinetes use an acto-myosin motor to glide towards and penetrate the midgut wall to establish infection in the vector. The regulation of gliding motility is poorly understood. Through genetic interaction studies we here describe a signalling module that identifies guanosine 3', 5'-cyclic monophosphate (cGMP as an important second messenger regulating ookinete differentiation and motility. In ookinetes lacking the cyclic nucleotide degrading phosphodiesterase delta (PDEdelta, unregulated signalling through cGMP results in rounding up of the normally banana-shaped cells. This phenotype is suppressed in a double mutant additionally lacking guanylyl cyclase beta (GCbeta, showing that in ookinetes GCbeta is an important source for cGMP, and that PDEdelta is the relevant cGMP degrading enzyme. Inhibition of the cGMP-dependent protein kinase, PKG, blocks gliding, whereas enhanced signalling through cGMP restores normal gliding speed in a mutant lacking calcium dependent protein kinase 3, suggesting at least a partial overlap between calcium and cGMP dependent pathways. These data demonstrate an important function for signalling through cGMP, and most likely PKG, in dynamically regulating ookinete gliding during the transmission of malaria to the mosquito.

  10. The AMP-activated protein kinase is involved in the regulation of ketone body production by astrocytes.

    Science.gov (United States)

    Blázquez, C; Woods, A; de Ceballos, M L; Carling, D; Guzmán, M

    1999-10-01

    The possible role of the AMP-activated protein kinase (AMPK), a highly conserved stress-activated kinase, in the regulation of ketone body production by astrocytes was studied. AMPK activity in rat cortical astrocytes was three times higher than in rat cortical neurons. AMPK in astrocytes was shown to be functionally active. Thus, incubation of astrocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a cell-permeable activator of AMPK, stimulated both ketogenesis from palmitate and carnitine palmitoyltransferase I. This was concomitant to a decrease of intracellular malonyl-CoA levels and an inhibition of acetyl-CoA carboxylase/fatty acid synthesis and 3-hydroxy-3-methylglutaryl-CoA reductase/cholesterol synthesis. Moreover, in microdialysis experiments AICAR was shown to stimulate brain ketogenesis markedly. The effect of chemical hypoxia on AMPK and the ketogenic pathway was studied subsequently. Incubation of astrocytes with azide led to a remarkable drop of fatty acid beta-oxidation. However, activation of AMPK during hypoxia compensated the depression of beta-oxidation, thereby sustaining ketone body production. This effect seemed to rely on the cascade hypoxia --> increase of the AMP/ATP ratio --> AMPK stimulation --> acetyl-CoA carboxylase inhibition --> decrease of malonyl-CoA concentration --> carnitine palmitoyltransferase I deinhibition --> enhanced ketogenesis. Furthermore, incubation of neurons with azide blunted lactate oxidation, but not 3-hydroxybutyrate oxidation. Results show that (a) AMPK plays an active role in the regulation of ketone body production by astrocytes, and (b) ketone bodies produced by astrocytes during hypoxia might be a substrate for neuronal oxidative metabolism.

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

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    Nicole M Warrington

    2015-07-01

    Full Text Available 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.

  12. Quantitative Proteomics Analysis of the cAMP/Protein Kinase A Signaling Pathway

    Science.gov (United States)

    2012-01-01

    To define the proteins whose expression is regulated by cAMP and protein kinase A (PKA), we used a quantitative proteomics approach in studies of wild-type (WT) and kin- (PKA-null) S49 murine T lymphoma cells. We also compared the impact of endogenous increases in the level of cAMP [by forskolin (Fsk) and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX)] or by a cAMP analogue (8-CPT-cAMP). We identified 1056 proteins in WT and kin- S49 cells and found that 8-CPT-cAMP and Fsk with IBMX produced differences in protein expression. WT S49 cells had a correlation coefficient of 0.41 between DNA microarray data and the proteomics analysis in cells incubated with 8-CPT-cAMP for 24 h and a correlation coefficient of 0.42 between the DNA microarray data obtained at 6 h and the changes in protein expression after incubation with 8-CPT-cAMP for 24 h. Glutathione reductase (Gsr) had a higher level of basal expression in kin- S49 cells than in WT cells. Consistent with this finding, kin- cells are less sensitive to cell killing and generation of malondialdehyde than are WT cells incubated with H2O2. Cyclic AMP acting via PKA thus has a broad impact on protein expression in mammalian cells, including in the regulation of Gsr and oxidative stress. PMID:23110364

  13. cAMP/PKA signaling pathway contributes to neuronal apoptosis via regulating IDE expression in a mixed model of type 2 diabetes and Alzheimer's disease.

    Science.gov (United States)

    Li, Huajie; Yang, Song; Wu, Jian; Ji, Lei; Zhu, Linfeng; Cao, Liping; Huang, Jinzhong; Jiang, Qingqing; Wei, Jiang; Liu, Meng; Mao, Keshi; Wei, Ning; Xie, Wei; Yang, Zhilong

    2018-02-01

    Type 2 diabetes (T2D) may play a relevant role in the development of Alzheimer's disease (AD), however, the underlying mechanism was not clear yet. We developed an animal model presenting both AD and T2D, morris water maze (MWM) test and recognition task were performed to trace the cognitive function. Fasting plasma glucose (FPG) and oral glucose tolerance test (OGTT) were determined to trace the metabolism evolution. TUNEL assay and apoptosis-related protein levels were analyzed for the detection of neuronal apoptosis. Cyclic adenosine monophosphate (cAMP) agonist bucladesine or protein kinase (PKA) inhibitor H-89 were used to determine the effects of cAMP/PKA signaling pathway on IDE expression and neuronal apoptosis. The results showed that T2D contributes to the AD progress by accelerating and worsening spatial memory and recognition dysfunctions. Metabolic parameters and glucose tolerance were significantly changed in the presence of the AD and T2D. The significantly induced neuronal apoptosis and increased pro-apoptotic proteins in mice with AD and T2D were also observed. We showed the decreased expression level of IDE and the activating of cAMP/PKA signaling pathway in AD and T2D mice. Further studies indicated that cAMP agonist decreased the expression level of IDE and induced the neuronal apoptosis in mice with AD and T2D; whereas PKA inhibitor H-89 treatment showed the completely opposite results. Our study indicated that, in the T2D and AD mice, cAMP/PKA signaling pathway and IDE may participate in the contribute role of T2D in accelerating the pathological process of AD via causing the accumulation of Aβ and neuronal apoptosis. © 2017 Wiley Periodicals, Inc.

  14. Enhancing E. coli tolerance towards oxidative stress via engineering its global regulator cAMP receptor protein (CRP.

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    Souvik Basak

    Full Text Available Oxidative damage to microbial hosts often occurs under stressful conditions during bioprocessing. Classical strain engineering approaches are usually both time-consuming and labor intensive. Here, we aim to improve E. coli performance under oxidative stress via engineering its global regulator cAMP receptor protein (CRP, which can directly or indirectly regulate redox-sensing regulators SoxR and OxyR, and other ~400 genes in E. coli. Error-prone PCR technique was employed to introduce modifications to CRP, and three mutants (OM1~OM3 were identified with improved tolerance via H(2O(2 enrichment selection. The best mutant OM3 could grow in 12 mM H(2O(2 with the growth rate of 0.6 h(-1, whereas the growth of wild type was completely inhibited at this H(2O(2 concentration. OM3 also elicited enhanced thermotolerance at 48°C as well as resistance against cumene hydroperoxide. The investigation about intracellular reactive oxygen species (ROS, which determines cell viability, indicated that the accumulation of ROS in OM3 was always lower than in WT with or without H(2O(2 treatment. Genome-wide DNA microarray analysis has shown not only CRP-regulated genes have demonstrated great transcriptional level changes (up to 8.9-fold, but also RpoS- and OxyR-regulated genes (up to 7.7-fold. qRT-PCR data and enzyme activity assay suggested that catalase (katE could be a major antioxidant enzyme in OM3 instead of alkyl hydroperoxide reductase or superoxide dismutase. To our knowledge, this is the first work on improving E. coli oxidative stress resistance by reframing its transcription machinery through its native global regulator. The positive outcome of this approach may suggest that engineering CRP can be successfully implemented as an efficient strain engineering alternative for E. coli.

  15. Glucose Regulates Hypothalamic Long-chain Fatty Acid Metabolism via AMP-activated Kinase (AMPK) in Neurons and Astrocytes*

    Science.gov (United States)

    Taïb, Bouchra; Bouyakdan, Khalil; Hryhorczuk, Cécile; Rodaros, Demetra; Fulton, Stephanie; Alquier, Thierry

    2013-01-01

    Hypothalamic controls of energy balance rely on the detection of circulating nutrients such as glucose and long-chain fatty acids (LCFA) by the mediobasal hypothalamus (MBH). LCFA metabolism in the MBH plays a key role in the control of food intake and glucose homeostasis, yet it is not known if glucose regulates LCFA oxidation and esterification in the MBH and, if so, which hypothalamic cell type(s) and intracellular signaling mechanisms are involved. The aim of this study was to determine the impact of glucose on LCFA metabolism, assess the role of AMP-activated Kinase (AMPK), and to establish if changes in LCFA metabolism and its regulation by glucose vary as a function of the kind of LCFA, cell type, and brain region. We show that glucose inhibits palmitate oxidation via AMPK in hypothalamic neuronal cell lines, primary hypothalamic astrocyte cultures, and MBH slices ex vivo but not in cortical astrocytes and slice preparations. In contrast, oleate oxidation was not affected by glucose or AMPK inhibition in MBH slices. In addition, our results show that glucose increases palmitate, but not oleate, esterification into neutral lipids in neurons and MBH slices but not in hypothalamic astrocytes. These findings reveal for the first time the metabolic fate of different LCFA in the MBH, demonstrate AMPK-dependent glucose regulation of LCFA oxidation in both astrocytes and neurons, and establish metabolic coupling of glucose and LCFA as a distinguishing feature of hypothalamic nuclei critical for the control of energy balance. PMID:24240094

  16. Glucose regulates hypothalamic long-chain fatty acid metabolism via AMP-activated kinase (AMPK) in neurons and astrocytes.

    Science.gov (United States)

    Taïb, Bouchra; Bouyakdan, Khalil; Hryhorczuk, Cécile; Rodaros, Demetra; Fulton, Stephanie; Alquier, Thierry

    2013-12-27

    Hypothalamic controls of energy balance rely on the detection of circulating nutrients such as glucose and long-chain fatty acids (LCFA) by the mediobasal hypothalamus (MBH). LCFA metabolism in the MBH plays a key role in the control of food intake and glucose homeostasis, yet it is not known if glucose regulates LCFA oxidation and esterification in the MBH and, if so, which hypothalamic cell type(s) and intracellular signaling mechanisms are involved. The aim of this study was to determine the impact of glucose on LCFA metabolism, assess the role of AMP-activated Kinase (AMPK), and to establish if changes in LCFA metabolism and its regulation by glucose vary as a function of the kind of LCFA, cell type, and brain region. We show that glucose inhibits palmitate oxidation via AMPK in hypothalamic neuronal cell lines, primary hypothalamic astrocyte cultures, and MBH slices ex vivo but not in cortical astrocytes and slice preparations. In contrast, oleate oxidation was not affected by glucose or AMPK inhibition in MBH slices. In addition, our results show that glucose increases palmitate, but not oleate, esterification into neutral lipids in neurons and MBH slices but not in hypothalamic astrocytes. These findings reveal for the first time the metabolic fate of different LCFA in the MBH, demonstrate AMPK-dependent glucose regulation of LCFA oxidation in both astrocytes and neurons, and establish metabolic coupling of glucose and LCFA as a distinguishing feature of hypothalamic nuclei critical for the control of energy balance.

  17. Different cAMP sources are critically involved in G protein-coupled receptor CRHR1 signaling.

    Science.gov (United States)

    Inda, Carolina; Dos Santos Claro, Paula A; Bonfiglio, Juan J; Senin, Sergio A; Maccarrone, Giuseppina; Turck, Christoph W; Silberstein, Susana

    2016-07-18

    Corticotropin-releasing hormone receptor 1 (CRHR1) activates G protein-dependent and internalization-dependent signaling mechanisms. Here, we report that the cyclic AMP (cAMP) response of CRHR1 in physiologically relevant scenarios engages separate cAMP sources, involving the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). cAMP produced by tmACs and sAC is required for the acute phase of extracellular signal regulated kinase 1/2 activation triggered by CRH-stimulated CRHR1, but only sAC activity is essential for the sustained internalization-dependent phase. Thus, different cAMP sources are involved in different signaling mechanisms. Examination of the cAMP response revealed that CRH-activated CRHR1 generates cAMP after endocytosis. Characterizing CRHR1 signaling uncovered a specific link between CRH-activated CRHR1, sAC, and endosome-based signaling. We provide evidence of sAC being involved in an endocytosis-dependent cAMP response, strengthening the emerging model of GPCR signaling in which the cAMP response does not occur exclusively at the plasma membrane and introducing the notion of sAC as an alternative source of cAMP. © 2016 Inda et al.

  18. Parathyroid hormone contributes to the down-regulation of cytochrome P450 3A through the cAMP/PI3K/PKC/PKA/NF-κB signaling pathway in secondary hyperparathyroidism.

    Science.gov (United States)

    Watanabe, Hiroshi; Sugimoto, Ryusei; Ikegami, Komei; Enoki, Yuki; Imafuku, Tadashi; Fujimura, Rui; Bi, Jing; Nishida, Kento; Sakaguchi, Yoshiaki; Murata, Michiya; Maeda, Hitoshi; Hirata, Kenshiro; Jingami, Sachiko; Ishima, Yu; Tanaka, Motoko; Matsushita, Kazutaka; Komaba, Hirotaka; Fukagawa, Masafumi; Otagiri, Masaki; Maruyama, Toru

    2017-12-01

    Chronic kidney disease (CKD), which affects, not only renal clearance, but also non-renal clearance, is accompanied by a decline in renal function. Although it has been suggested that humoral factors, such as uremic toxins that accumulate in the body under CKD conditions, could be involved in the changes associated with non-renal drug clearance, the overall process is not completely understood. In this study, we report on the role of parathyroid hormone (PTH), a middle molecule uremic toxin, on the expression of drug metabolizing or transporting proteins using rats with secondary hyperparathyroidism (SHPT) as models. In SHPT rats, hepatic and intestinal CYP3A expression was suppressed, but the changes were recovered by the administration of the calcimimetic cinacalcet, a PTH suppressor. Under the same experimental conditions, a pharmacokinetic study using orally administered midazolam, a substrate for CYP3A, showed that the AUC was increased by 5 times in SHPT rats, but that was partially recovered by a cinacalcet treatment. This was directly tested in rat primary hepatocytes and intestinal Caco-2 cells where the expression of the CYP3A protein was down-regulated by PTH (1-34). In Caco-2 cells, PTH (1-34) down-regulated the expression of CYP3A mRNA, but an inactive PTH derivative (13-34) had no effect. 8-Bromo-cyclic adenosine monophosphate, a membrane-permeable cAMP analog, reduced mRNA expression of CYP3A whereas the inhibitors of PI3K, NF-κB, PKC and PKA reversed the PTH-induced CYP3A down-regulation. These results suggest that PTH down-regulates CYP3A through multiple signaling pathways, including the PI3K/PKC/PKA/NF-κB pathway after the elevation of intracellular cAMP, and the effect of PTH can be prevented by cinacalcet treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis

    Czech Academy of Sciences Publication Activity Database

    Škopová, Karolína; Tomalová, Barbora; Kanchev, Ivan; Rossmann, Pavel; Švédová, Martina; Adkins, Irena; Bíbová, Ilona; Tomala, Jakub; Mašín, Jiří; Guiso, N.; Osička, Radim; Sedláček, Radislav; Kovář, Marek; Šebo, Peter

    2017-01-01

    Roč. 85, č. 6 (2017), s. 1-22, č. článku e00937-16. ISSN 0019-9567 R&D Projects: GA MZd(CZ) NV16-28126A; GA ČR(CZ) GA13-14547S; GA ČR GA13-12885S; GA ČR GA15-09157S; GA ČR(CZ) GAP302/12/0460; GA MŠk(CZ) LM2015064; GA MŠk(CZ) LM2015040 Institutional support: RVO:61388971 ; RVO:68378050 Keywords : Bordetella pertussis * adenylate cyclase toxin-hemolysin * cAMP intoxication Subject RIV: EE - Microbiology, Virology; EE - Microbiology, Virology (UMG-J) OBOR OECD: Microbiology; Microbiology (UMG-J) Impact factor: 3.593, year: 2016

  20. Decoding spatial and temporal features of neuronal cAMP/PKA signaling with FRET biosensors.

    Science.gov (United States)

    Castro, Liliana R V; Guiot, Elvire; Polito, Marina; Paupardin-Tritsch, Daniéle; Vincent, Pierre

    2014-02-01

    Cyclic adenosine monophosphate (cAMP) and the cyclic-AMP-dependent protein kinase (PKA) regulate a plethora of cellular functions in virtually all eukaryotic cells. In neurons, the cAMP/PKA signaling cascade controls a number of biological properties such as axonal growth, pathfinding, efficacy of synaptic transmission, regulation of excitability, or long term changes. Genetically encoded optical biosensors for cAMP or PKA are considerably improving our understanding of these processes by providing a real-time measurement in living neurons. In this review, we describe the recent progress made in the creation of biosensors for cAMP or PKA activity. These biosensors revealed profound differences in the amplitude of the cAMP signal evoked by neuromodulators between various neuronal preparations. These responses can be resolved at the level of individual neurons, also revealing differences related to the neuronal type. At the sub-cellular level, biosensors reported different signal dynamics in domains like dendrites, cell body, nucleus, and axon. Combining this imaging approach with pharmacology or genetic models points at phosphodiesterases and phosphatases as critical regulatory proteins. Biosensor imaging will certainly emerge as a forefront tool to decipher the subtle mechanics of intracellular signaling. This will certainly help us to understand the mechanism of action of current drugs and foster the development of novel molecules for neuropsychiatric diseases. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Role of cyclic AMP sensor Epac1 in masseter muscle hypertrophy and myosin heavy chain transition induced by β2-adrenoceptor stimulation.

    Science.gov (United States)

    Ohnuki, Yoshiki; Umeki, Daisuke; Mototani, Yasumasa; Jin, Huiling; Cai, Wenqian; Shiozawa, Kouichi; Suita, Kenji; Saeki, Yasutake; Fujita, Takayuki; Ishikawa, Yoshihiro; Okumura, Satoshi

    2014-12-15

    The predominant isoform of β-adrenoceptor (β-AR) in skeletal muscle is β2-AR and that in the cardiac muscle is β1-AR. We have reported that Epac1 (exchange protein directly activated by cAMP 1), a new protein kinase A-independent cAMP sensor, does not affect cardiac hypertrophy in response to pressure overload or chronic isoproterenol (isoprenaline) infusion. However, the role of Epac1 in skeletal muscle hypertrophy remains poorly understood. We thus examined the effect of disruption of Epac1, the major Epac isoform in skeletal muscle, on masseter muscle hypertrophy induced by chronic β2-AR stimulation with clenbuterol (CB) in Epac1-null mice (Epac1KO). The masseter muscle weight/tibial length ratio was similar in wild-type (WT) and Epac1KO at baseline and was significantly increased in WT after CB infusion, but this increase was suppressed in Epac1KO. CB treatment significantly increased the proportion of myosin heavy chain (MHC) IIb at the expense of that of MHC IId/x in both WT and Epac1KO, indicating that Epac1 did not mediate the CB-induced MHC isoform transition towards the faster isoform. The mechanism of suppression of CB-mediated hypertrophy in Epac1KO is considered to involve decreased activation of Akt signalling. In addition, CB-induced histone deacetylase 4 (HDAC4) phosphorylation on serine 246 mediated by calmodulin kinase II (CaMKII), which plays a role in skeletal muscle hypertrophy, was suppressed in Epac1KO. Our findings suggest that Epac1 plays a role in β2-AR-mediated masseter muscle hypertrophy, probably through activation of both Akt signalling and CaMKII/HDAC4 signalling. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

  2. Cyclic-AMP metabolism in synaptic growth, strength and precision: Neural and behavioral phenotype-specific counterbalancing effects between dnc PDE and rut AC mutations

    Science.gov (United States)

    Ueda, Atsushi; Wu, Chun-Fang

    2012-01-01

    Two classic learning mutants in Drosophila, rutabaga (rut) and dunce (dnc), are defective in cAMP synthesis and degradation, respectively, exhibiting a variety of neuronal and behavioral defects. We ask how the opposing effects of these mutations on cAMP levels modify subsets of phenotypes, and whether any specific phenotypes could be ameliorated by biochemical counter balancing effects in dnc rut double mutants. Our study at larval neuromuscular junctions (NMJs) demonstrate that dnc mutations caused severe defects in nerve terminal morphology, characterized by unusually large synaptic boutons and aberrant innervation patterns. Interestingly, a counterbalancing effect led to rescue of the aberrant innervation patterns but the enlarged boutons in dnc rut double mutant remained as extreme as those in dnc. In contrast to dnc, rut mutations strongly affect synaptic transmission. Focal loose-patch recording data accumulated over 4 years suggest that synaptic currents in rut boutons were characterized by unusually large temporal dispersion and a seasonal variation in the amount of transmitter release, with diminished synaptic currents in summer months. Experiments with different rearing temperatures revealed that high temperature (29–30 °C) decreased synaptic transmission in rut, but did not alter dnc and WT. Importantly, the large temporal dispersion and abnormal temperature dependence of synaptic transmission, characteristic of rut, still persisted in dnc rut double mutants. To interpret these results in a proper perspective, we reviewed previously documented differential effects of dnc and rut mutations and their genetic interactions in double mutants on a variety of physiological and behavioral phenotypes. The cases of rescue in double mutants are associated with gradual developmental and maintenance processes whereas many behavioral and physiological manifestations on faster time scales could not be rescued. We discuss factors that could contribute to the

  3. Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration.

    Science.gov (United States)

    Plikus, Maksim V; Mayer, Julie Ann; de la Cruz, Damon; Baker, Ruth E; Maini, Philip K; Maxson, Robert; Chuong, Cheng-Ming

    2008-01-17

    In the age of stem cell engineering it is critical to understand how stem cell activity is regulated during regeneration. Hairs are mini-organs that undergo cyclic regeneration throughout adult life, and are an important model for organ regeneration. Hair stem cells located in the follicle bulge are regulated by the surrounding microenvironment, or niche. The activation of such stem cells is cyclic, involving periodic beta-catenin activity. In the adult mouse, regeneration occurs in waves in a follicle population, implying coordination among adjacent follicles and the extrafollicular environment. Here we show that unexpected periodic expression of bone morphogenetic protein 2 (Bmp2) and Bmp4 in the dermis regulates this process. This BMP cycle is out of phase with the WNT/beta-catenin cycle, thus dividing the conventional telogen into new functional phases: one refractory and the other competent for hair regeneration, characterized by high and low BMP signalling, respectively. Overexpression of noggin, a BMP antagonist, in mouse skin resulted in a markedly shortened refractory phase and faster propagation of the regenerative wave. Transplantation of skin from this mutant onto a wild-type host showed that follicles in donor and host can affect their cycling behaviours mutually, with the outcome depending on the equilibrium of BMP activity in the dermis. Administration of BMP4 protein caused the competent region to become refractory. These results show that BMPs may be the long-sought 'chalone' inhibitors of hair growth postulated by classical experiments. Taken together, results presented in this study provide an example of hierarchical regulation of local organ stem cell homeostasis by the inter-organ macroenvironment. The expression of Bmp2 in subcutaneous adipocytes indicates physiological integration between these two thermo-regulatory organs. Our findings have practical importance for studies using mouse skin as a model for carcinogenesis, intra-cutaneous drug

  4. AMP-activated protein kinase (AMPK mediates nutrient regulation of thioredoxin-interacting protein (TXNIP in pancreatic beta-cells.

    Directory of Open Access Journals (Sweden)

    Maayan Shaked

    Full Text Available Thioredoxin-interacting protein (TXNIP regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP. Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment

  5. Dopamine receptors D3 and D5 regulate CD4(+)T-cell activation and differentiation by modulating ERK activation and cAMP production.

    Science.gov (United States)

    Franz, Dafne; Contreras, Francisco; González, Hugo; Prado, Carolina; Elgueta, Daniela; Figueroa, Claudio; Pacheco, Rodrigo

    2015-07-15

    Dopamine receptors have been described in T-cells, however their signalling pathways coupled remain unknown. Since cAMP and ERKs play key roles regulating T-cell physiology, we aim to determine whether cAMP and ERK1/2-phosphorylation are modulated by dopamine receptor 3 (D3R) and D5R, and how this modulation affects CD4(+) T-cell activation and differentiation. Our pharmacologic and genetic evidence shows that D3R-stimulation reduced cAMP levels and ERK2-phosphorylation, consequently increasing CD4(+) T-cell activation and Th1-differentiation, respectively. Moreover, D5R expression reinforced TCR-triggered ERK1/2-phosphorylation and T-cell activation. In conclusion, these findings demonstrate how D3R and D5R modulate key signalling pathways affecting CD4(+) T-cell activation and Th1-differentiation. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Acetic acid activates the AMP-activated protein kinase signaling pathway to regulate lipid metabolism in bovine hepatocytes.

    Directory of Open Access Journals (Sweden)

    Xinwei Li

    Full Text Available The effect of acetic acid on hepatic lipid metabolism in ruminants differs significantly from that in monogastric animals. Therefore, the aim of this study was to investigate the regulation mechanism of acetic acid on the hepatic lipid metabolism in dairy cows. The AMP-activated protein kinase (AMPK signaling pathway plays a key role in regulating hepatic lipid metabolism. In vitro, bovine hepatocytes were cultured and treated with different concentrations of sodium acetate (neutralized acetic acid and BML-275 (an AMPKα inhibitor. Acetic acid consumed a large amount of ATP, resulting in an increase in AMPKα phosphorylation. The increase in AMPKα phosphorylation increased the expression and transcriptional activity of peroxisome proliferator-activated receptor α, which upregulated the expression of lipid oxidation genes, thereby increasing lipid oxidation in bovine hepatocytes. Furthermore, elevated AMPKα phosphorylation reduced the expression and transcriptional activity of the sterol regulatory element-binding protein 1c and the carbohydrate responsive element-binding protein, which reduced the expression of lipogenic genes, thereby decreasing lipid biosynthesis in bovine hepatocytes. In addition, activated AMPKα inhibited the activity of acetyl-CoA carboxylase. Consequently, the triglyceride content in the acetate-treated hepatocytes was significantly decreased. These results indicate that acetic acid activates the AMPKα signaling pathway to increase lipid oxidation and decrease lipid synthesis in bovine hepatocytes, thereby reducing liver fat accumulation in dairy cows.

  7. Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition

    Science.gov (United States)

    Musante, Veronica; Li, Lu; Kanyo, Jean; Lam, Tukiet T; Colangelo, Christopher M; Cheng, Shuk Kei; Brody, A Harrison; Greengard, Paul; Le Novère, Nicolas; Nairn, Angus C

    2017-01-01

    ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition. DOI: http://dx.doi.org/10.7554/eLife.24998.001 PMID:28613156

  8. Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition.

    Science.gov (United States)

    Musante, Veronica; Li, Lu; Kanyo, Jean; Lam, Tukiet T; Colangelo, Christopher M; Cheng, Shuk Kei; Brody, A Harrison; Greengard, Paul; Le Novère, Nicolas; Nairn, Angus C

    2017-06-14

    ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition.

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

    Directory of Open Access Journals (Sweden)

    Yosuke Osawa

    2015-11-01

    Full Text Available 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 Ly6Clow 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.

  10. Nitric oxide increases cyclic GMP levels, AMP-activated protein kinase (AMPK)alpha1-specific activity and glucose transport in human skeletal muscle

    DEFF Research Database (Denmark)

    Deshmukh, A S; Long, Y C; de Castro Barbosa, T

    2010-01-01

    -nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) would increase intracellular cyclic GMP (cGMP) levels and promote glucose transport. METHODS: Skeletal muscle strips were prepared from vastus lateralis muscle biopsies obtained from seven healthy men. Muscle strips were incubated in the absence or presence...... of 5 mmol/l spermine NONOate or 120 nmol/l insulin. The L6 muscle cells were treated with spermine NONOate (20 micromol/l) and incubated in the absence or presence of insulin (120 nmol/l). The direct effect of spermine NONOate and insulin on glucose transport, cGMP levels and signal transduction...... was determined. RESULTS: In human skeletal muscle, spermine NONOate increased glucose transport 2.4-fold (p GMP levels (80-fold, p

  11. AMP-Activated Kinase Regulates Lipid Droplet Localization and Stability of Adipose Triglyceride Lipase in C. elegans Dauer Larvae.

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    Meng Xie

    Full Text Available Animals have developed diverse mechanisms to adapt to their changing environment. Like many organisms the free-living nematode C. elegans can alternate between a reproductive mode or a diapause-like "dauer" stage during larval development to circumvent harsh environmental conditions. The master metabolic regulator AMP-activated protein kinase (AMPK is critical for survival during the dauer stage, where it phosphorylates adipose triglyceride lipase (ATGL-1 at multiple sites to block lipid hydrolysis and ultimately protect the cellular triglyceride-based energy depot from rapid depletion. However, how the AMPK-mediated phosphorylation affects the function of ATGL-1 has not been characterised at the molecular level. Here we show that AMPK phosphorylation leads to the generation of 14-3-3 binding sites on ATGL-1, which are recognized by the C. elegans 14-3-3 protein orthologue PAR-5. Physical interaction of ATGL-1 with PAR-5 results in sequestration of ATGL-1 away from the lipid droplets and eventual proteasome-mediated degradation. In addition, we also show that the major AMPK phosphorylation site on ATGL-1, Ser 303, is required for both modification of its lipid droplet localization and its degradation. Our data provide mechanistic insight as to how AMPK functions to enhance survival through its ability to protect the accumulated triglyceride deposits from rapid hydrolysis to preserve the energy stores during periods of extended environmental duress.

  12. cAMP and forskolin decrease γ-aminobutyric acid-gated chloride flux in rat brain synaptoneurosomes

    International Nuclear Information System (INIS)

    Heuschneider, G.; Schwartz, R.D.

    1989-01-01

    The effects of the cyclic nucleotide cAMP on γ-aminobutyric acid-gated chloride channel function were investigated. The membrane-permeant cAMP analog N 6 , O 2' -dibutyryladenosine 3',5'-cyclic monophosphate inhibited muscimol-induced 36 Cl - uptake into rat cerebral cortical synaptoneurosomes in a concentration-dependent manner. The inhibition was due to a decrease in the maximal effect of muscimol, with no change in potency. Similar effects were observed with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, 8-bromoadenosine 3',5'-cyclic monophosphate, and the phosphodiesterase inhibitor isobutylmethylxanthine. The effect of endogenous cAMP accumulation on the γ-aminobutyric acid-gated Cl - channel was studied with forskolin, an activator of adenylate cyclase. Under identical conditions, in the intact synaptoneurosomes, forskolin inhibited muscimol-induced 36 Cl - uptake and generated cAMP with similar potencies. Surprisingly, 1,9-dideoxyforskolin, which does not activate adenylate cyclase, also inhibited the muscimol response, suggesting that forskolin and its lipophilic derivatives may interact with the Cl - channel directly. The data suggest that γ-aminobutyric acid (GABA A ) receptor function in brain can be regulated by cAMP-dependent phosphorylation

  13. Down-Regulation of the Na+-Coupled Phosphate Transporter NaPi-IIa by AMP-Activated Protein Kinase

    Directory of Open Access Journals (Sweden)

    Miribane Dërmaku-Sopjani

    2013-11-01

    Full Text Available Background/Aims: The Na+-coupled phosphate transporter NaPi-IIa is the main carrier accomplishing renal tubular phosphate reabsorption. It is driven by the electrochemical Na+ gradient across the apical cell membrane, which is maintained by Na+ extrusion across the basolateral cell membrane through the Na+/K+ ATPase. The operation of NaPi-IIa thus requires energy in order to avoid cellular Na+ accumulation and K+ loss with eventual decrease of cell membrane potential, Cl- entry and cell swelling. Upon energy depletion, early inhibition of Na+-coupled transport processes may delay cell swelling and thus foster cell survival. Energy depletion is sensed by the AMP-activated protein kinase (AMPK, a serine/threonine kinase stimulating several cellular mechanisms increasing energy production and limiting energy utilization. The present study explored whether AMPK influences the activity of NAPi-IIa. Methods: cRNA encoding NAPi-IIa was injected into Xenopus oocytes with or without additional expression of wild-type AMPK (AMPKα1-HA+AMPKβ1-Flag+AMPKγ1-HA, of inactive AMPKαK45R (AMPKα1K45R+AMPKβ1-Flag+AMPKγ1-HA or of constitutively active AMPKγR70Q (AMPKα1-HA+AMPKβ1-Flag+AMPKγ1R70Q. NaPi-IIa activity was estimated from phosphate-induced current in dual electrode voltage clamp experiments. Results: In NaPi-IIa-expressing, but not in water-injected Xenopus oocytes, the addition of phosphate (1 mM to the extracellular bath solution generated a current (Ip, which was significantly decreased by coexpression of wild-type AMPK and of AMPKγR70Q but not of AMPKαK45R. The phosphate-induced current in NaPi-IIa- and AMPK-expressing Xenopus ooocytes was significantly increased by AMPK inhibitor Compound C (20 µM. Kinetic analysis revealed that AMPK significantly decreased the maximal transport rate. Conclusion: The AMP-activated protein kinase AMPK is a powerful regulator of NaPi-IIa and thus of renal tubular phosphate transport.

  14. Regulation of autophagy by AMP-activated protein kinase/ sirtuin 1 pathway reduces spinal cord neurons damage

    Directory of Open Access Journals (Sweden)

    Peng Yan

    2017-09-01

    Full Text Available Objective(s: AMP-activated protein kinase/sirtuin 1 (AMPK/SIRT1 signaling pathway has been proved to be involved in the regulation of autophagy in various models. The aim of this study was to evaluate the effect of AMPK/SIRT1 pathway on autophagy after spinal cord injury (SCI. Materials and Methods:The SCI model was established in rats in vivo and the primary spinal cord neurons were subjected to mechanical injury (MI in vitro. The apoptosis in spinal cord tissue and neurons was assessed by TUNEL staining and Hoechst 33342 staining, respectively. The autophagy-related proteins levels were detected by Western blot. The activation of AMPK/SIRT1 pathway was determined by Western blot and immunohistochemical staining. Results: We found that the apoptosis of spinal cord tissue and cell damage of spinal cord neurons was obvious after the trauma. The ratio of LC3II/LC3I and level of p62 were first increased significantly and then decreased after the trauma in vivo and in vitro, indicating the defect in autophagy. The levels of p-AMPK and SIRT1 were increased obviously after the trauma in vivo and in vitro. Further activation of the AMPK/SIRT1 pathway by pretreatment with resveratrol, a confirmed activator of the AMPK/SIRT1 pathway, alleviated the cell damage and promoted the autophagy flux via downregulation of p62 in spinal cord neurons at 24 hr after MI. Conclusion: Our results demonstrate that regulation of autophagy by AMPK/SIRT1 pathway can restrain spinal cord neurons damage, which may be a potential intervention of SCI.

  15. Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE.

    Science.gov (United States)

    Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A; Moraes, Carlos T; Sanderson, Thomas H; Stemmler, Timothy L; Grossman, Lawrence I; Kagan, Valerian E; Brunzelle, Joseph S; Salomon, Arthur R; Edwards, Brian F P; Hüttemann, Maik

    2017-01-06

    Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr 28 , leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨ m ), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr 28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr 28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr 28 in vivo We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ΔΨ m hyperpolarization, a known cause of ROS and trigger of apoptosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility

    Directory of Open Access Journals (Sweden)

    Violeta Calle-Guisado

    2017-01-01

    Full Text Available AMP-activated kinase (AMPK, a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work′s aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%-80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS in the presence or absence of the AMPK inhibitor compound C (CC. AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied.

  17. Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE

    Energy Technology Data Exchange (ETDEWEB)

    Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A.; Moraes, Carlos T.; Sanderson, Thomas H.; Stemmler, Timothy L.; Grossman, Lawrence I.; Kagan, Valerian E.; Brunzelle, Joseph S.; Salomon, Arthur R.; Edwards, Brian F. P.; Hüttemann, Maik

    2016-10-07

    Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

  18. AMP-activated kinase in human spermatozoa: identification, intracellular localization, and key function in the regulation of sperm motility

    Science.gov (United States)

    Calle-Guisado, Violeta; de Llera, Ana Hurtado; Martin-Hidalgo, David; Mijares, Jose; Gil, Maria C; Alvarez, Ignacio S; Bragado, Maria J; Garcia-Marin, Luis J

    2017-01-01

    AMP-activated kinase (AMPK), a protein that regulates energy balance and metabolism, has recently been identified in boar spermatozoa where regulates key functional sperm processes essential for fertilization. This work's aims are AMPK identification, intracellular localization, and their role in human spermatozoa function. Semen was obtained from healthy human donors. Sperm AMPK and phospho-Thr172-AMPK were analyzed by Western blotting and indirect immunofluorescence. High- and low-quality sperm populations were separated by a 40%–80% density gradient. Human spermatozoa motility was evaluated by an Integrated Semen Analysis System (ISAS) in the presence or absence of the AMPK inhibitor compound C (CC). AMPK is localized along the human spermatozoa, at the entire acrosome, midpiece and tail with variable intensity, whereas its active form, phospho-Thr172-AMPK, shows a prominent staining at the acrosome and sperm tail with a weaker staining in the midpiece and the postacrosomal region. Interestingly, spermatozoa bearing an excess residual cytoplasm show strong AMPK staining in this subcellular compartment. Both AMPK and phospho-Thr172-AMPK human spermatozoa contents exhibit important individual variations. Moreover, active AMPK is predominant in the high motility sperm population, where shows a stronger intensity compared with the low motility sperm population. Inhibition of AMPK activity in human spermatozoa by CC treatment leads to a significant reduction in any sperm motility parameter analyzed: percent of motile sperm, sperm velocities, progressivity, and other motility coefficients. This work identifies and points out AMPK as a new molecular mechanism involved in human spermatozoa motility. Further AMPK implications in the clinical efficiency of assisted reproduction and in other reproductive areas need to be studied. PMID:27678462

  19. Regulation of autophagy by AMP-activated protein kinase/sirtuin 1 pathway reduces spinal cord neurons damage.

    Science.gov (United States)

    Yan, Peng; Bai, Liangjie; Lu, Wei; Gao, Yuzhong; Bi, Yunlong; Lv, Gang

    2017-09-01

    AMP-activated protein kinase/sirtuin 1 (AMPK/SIRT1) signaling pathway has been proved to be involved in the regulation of autophagy in various models. The aim of this study was to evaluate the effect of AMPK/SIRT1 pathway on autophagy after spinal cord injury (SCI). The SCI model was established in rats in vivo and the primary spinal cord neurons were subjected to mechanical injury (MI) in vitro . The apoptosis in spinal cord tissue and neurons was assessed by TUNEL staining and Hoechst 33342 staining, respectively. The autophagy-related proteins levels were detected by Western blot. The activation of AMPK/SIRT1 pathway was determined by Western blot and immunohistochemical staining. We found that the apoptosis of spinal cord tissue and cell damage of spinal cord neurons was obvious after the trauma. The ratio of LC3II/LC3I and level of p62 were first increased significantly and then decreased after the trauma in vivo and in vitro , indicating the defect in autophagy. The levels of p-AMPK and SIRT1 were increased obviously after the trauma in vivo and in vitro . Further activation of the AMPK/SIRT1 pathway by pretreatment with resveratrol, a confirmed activator of the AMPK/SIRT1 pathway, alleviated the cell damage and promoted the autophagy flux via downregulation of p62 in spinal cord neurons at 24 hr after MI. Our results demonstrate that regulation of autophagy by AMPK/SIRT1 pathway can restrain spinal cord neurons damage, which may be a potential intervention of SCI.

  20. Ca2+-regulated-cAMP/PKA signaling in cardiac pacemaker cells links ATP supply to demand.

    Science.gov (United States)

    Yaniv, Yael; Juhaszova, Magdalena; Lyashkov, Alexey E; Spurgeon, Harold A; Sollott, Steven J; Lakatta, Edward G

    2011-11-01

    In sinoatrial node cells (SANC), Ca(2+) activates adenylate cyclase (AC) to generate a high basal level of cAMP-mediated/protein kinase A (PKA)-dependent phosphorylation of Ca(2+) cycling proteins. These result in spontaneous sarcoplasmic-reticulum (SR) generated rhythmic Ca(2+) oscillations during diastolic depolarization, that not only trigger the surface membrane to generate rhythmic action potentials (APs), but, in a feed-forward manner, also activate AC/PKA signaling. ATP is consumed to pump Ca(2+) to the SR, to produce cAMP, to support contraction and to maintain cell ionic homeostasis. Since feedback mechanisms link ATP-demand to ATP production, we hypothesized that (1) both basal ATP supply and demand in SANC would be Ca(2+)-cAMP/PKA dependent; and (2) due to its feed-forward nature, a decrease in flux through the Ca(2+)-cAMP/PKA signaling axis will reduce the basal ATP production rate. O(2) consumption in spontaneous beating SANC was comparable to ventricular myocytes (VM) stimulated at 3 Hz. Graded reduction of basal Ca(2+)-cAMP/PKA signaling to reduce ATP demand in rabbit SANC produced graded ATP depletion (r(2)=0.96), and reduced O(2) consumption and flavoprotein fluorescence. Neither inhibition of glycolysis, selectively blocking contraction nor specific inhibition of mitochondrial Ca(2+) flux reduced the ATP level. Feed-forward basal Ca(2+)-cAMP/PKA signaling both consumes ATP to drive spontaneous APs in SANC and is tightly linked to mitochondrial ATP production. Interfering with Ca(2+)-cAMP/PKA signaling not only slows the firing rate and reduces ATP consumption, but also appears to reduce ATP production so that ATP levels fall. This distinctly differs from VM, which lack this feed-forward basal cAMP/PKA signaling, and in which ATP level remains constant when the demand changes. Published by Elsevier Ltd.

  1. Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

    Science.gov (United States)

    Sato, Shogo; Jung, Hunmin; Nakagawa, Tsutomu; Pawlosky, Robert; Takeshima, Tomomi; Lee, Wan-Ru; Sakiyama, Haruhiko; Laxman, Sunil; Wynn, R Max; Tu, Benjamin P; MacMillan, John B; De Brabander, Jef K; Veech, Richard L; Uyeda, Kosaku

    2016-05-13

    The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

    International Nuclear Information System (INIS)

    Bhattacharjee, Rajesh; Xiang, Wenpei; Wang, Yinna; Zhang, Xiaoying; Billiar, Timothy R.

    2012-01-01

    Highlights: ► cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. ► cAMP blocks NF-κB activation induced by TNF and actinomycin D. ► cAMP blocks DISC formation following TNF and actinomycin D exposure. ► cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor α (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found that cAMP exerts its affect at the proximal level of TNF signaling by inhibiting the formation of the DISC

  3. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, Rajesh [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Xiang, Wenpei [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People' s Republic of China (China); Wang, Yinna [Vascular Medicine Institute, University of Pittsburgh School of Medicine, 10051-5A BST 3, 3501 Fifth Avenue, Pittsburgh, PA 15261 (United States); Zhang, Xiaoying [Department of Medicine/Endocrinology Division, University of Pittsburgh Medical Center, 200 Lothrop St., Pittsburgh, PA 15213 (United States); Billiar, Timothy R., E-mail: billiartr@upmc.edu [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. Black-Right-Pointing-Pointer cAMP blocks NF-{kappa}B activation induced by TNF and actinomycin D. Black-Right-Pointing-Pointer cAMP blocks DISC formation following TNF and actinomycin D exposure. Black-Right-Pointing-Pointer cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor {alpha} (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found

  4. cAMP and EPAC are key players in the regulation of the signal transduction pathway involved in the α-hemolysin autophagic response.

    Directory of Open Access Journals (Sweden)

    María Belén Mestre

    Full Text Available Staphylococcus aureus is a microorganism that causes serious diseases in the human being. This microorganism is able to escape the phagolysosomal pathway, increasing intracellular bacterial survival and killing the eukaryotic host cell to spread the infection. One of the key features of S. aureus infection is the production of a series of virulence factors, including secreted enzymes and toxins. We have shown that the pore-forming toxin α-hemolysin (Hla is the S. aureus-secreted factor responsible for the activation of the autophagic pathway and that this response occurs through a PI3K/Beclin1-independent form. In the present report we demonstrate that cAMP has a key role in the regulation of this autophagic response. Our results indicate that cAMP is able to inhibit the autophagy induced by Hla and that PKA, the classical cAMP effector, does not participate in this regulation. We present evidence that EPAC and Rap2b, through calpain activation, are the proteins involved in the regulation of Hla-induced autophagy. Similar results were obtained in cells infected with different S. aureus strains. Interestingly, in this report we show, for the first time to our knowledge, that both EPAC and Rap2b are recruited to the S. aureus-containing phagosome. We believe that our findings have important implications in understanding innate immune processes involved in intracellular pathogen invasion of the host cell.

  5. Biophysical Techniques for Detection of cAMP and cGMP in Living Cells

    Directory of Open Access Journals (Sweden)

    Viacheslav O. Nikolaev

    2013-04-01

    Full Text Available Cyclic nucleotides cAMP and cGMP are ubiquitous second messengers which regulate myriads of functions in virtually all eukaryotic cells. Their intracellular effects are often mediated via discrete subcellular signaling microdomains. In this review, we will discuss state-of-the-art techniques to measure cAMP and cGMP in biological samples with a particular focus on live cell imaging approaches, which allow their detection with high temporal and spatial resolution in living cells and tissues. Finally, we will describe how these techniques can be applied to the analysis of second messenger dynamics in subcellular signaling microdomains.

  6. The effect of ultraviolet light on the cyclic nucleotide system of human fibroblasts

    International Nuclear Information System (INIS)

    Fertel, R.H.; Tejwani, G.A.; Albrightson, C.R.; Hart, R.W.

    1981-01-01

    The concentrations of cyclic AMP and cyclic GMP in in human skin fibroblasts in culture were determined after exposing the cells to varying fluences of UV (254 nm) light. The cyclic nucleotide concentrations of cells irradiated in the log phase of growth were unchanged relative to controls. In contrast, there was a rise in the concentration of cyclic AMP in cells irradiated after they reached confluency. The increase in concentration was observed as early as 30 min after irradiation, reached a maximum of about 200% of control at 4 to 6 h after exposure, and returned to control values by 24 h after irradiation. The effect was proportional to a UV fluence from 5 to 20 J/m 2 , and was blocked by the addition of the UV absorbing agent para-aminobenzoic acid. In contrast, the results indicated that UV light had no effect on the concentration of cyclic GMP in human fibroblast cell cultures. Because of the importance of cyclic nucleotides in the regulation of cellular function, it is reasonable to hypothesize that changes in cyclic AMP induced by UV light may effect the extranuclear functions of irradiated cells. (author)

  7. Cyclic AMP-Responsive Element-Binding Protein (CREB is Critical in Autoimmunity by Promoting Th17 but Inhibiting Treg Cell Differentiation

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    Xiaohu Wang

    2017-11-01

    Full Text Available The molecular mechanisms that govern differential T cell development into pro-inflammatory Th17 vs. regulatory T (Treg cells remain unclear. Here, we show that selective deletion of CREB in T cells or Th17 cells impaired Th17 cell differentiation in vitro and in vivo, and led to resistance to autoimmune diseases. Mechanistically, CREB, activated by CD3-PKC-ϴ signaling, plays a key role in regulating Th17 cell differentiation, at least in part through directly binding to the Il17-Il17f gene locus. Unexpectedly, although dispensable for FOXP3 expression and for the homeostasis and suppressive function of thymus-derived Treg cells, CREB negatively regulates the survival of TGF-β-induced Treg cells, and deletion of CREB resulted in increased FOXP3+ Treg cells in the intestine and protection in a colitis model. Thus, CREB is critical in autoimmune diseases by promoting Th17 cell and inhibiting de novo Treg cell generation.

  8. Synthesis and degradation of cyclic nucleotides in brain after a high dose of ionizing radiation

    International Nuclear Information System (INIS)

    Hunt, W.A.; Dalton, T.K.

    1981-01-01

    Previous data from our laboratory have indicated that a high dose of ionizing radiation can deplete the cyclic nucleotides guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) on several areas of the rat brain. cGMP is more sensitive to radiation than cAMP and does not recover for at least 24 h after irradiation. The response of cAMP is transient and recovery occurs within 4 h. The purpose of the present paper is to determine whether alternations in the activity of the synthetic and degradative enzymes that regulate cyclic nucleotide levels could account for the observed effects. Guanylate and adenylate cyclase and cGMP and cAMP phosphodiesterase activities were determined 10 min after irradiation with 10,000 rad of high-energy electrons. No alteration was detected under these experimental conditions. The data suggest that the reduction in cyclic nucleotides is not a direct effect on their metabolic enzymes and is probably secondary to some as yet-undefined action of radiation on the brain

  9. AMP-regulated protein kinase activity in the hearts of mice treated with low- or high-fat diet measured using novel LC-MS method.

    Science.gov (United States)

    Rybakowska, I M; Slominska, E M; Romaszko, P; Olkowicz, M; Kaletha, K; Smolenski, R T

    2015-06-01

    AMP-regulated protein kinase (AMPK) is involved in regulation of energy-generating pathways in response to the metabolic needs in different organs including the heart. The activity of AMPK is mainly controlled by AMP concentration that in turn could be affected by nucleotide metabolic pathways. This study aimed to develop a procedure for measurement of AMPK activity together with nucleotide metabolic enzymes and its application for studies of mice treated with high-fat diet. The method developed was based on analysis of conversion of AMARA peptide to pAMARA by partially purified heart homogenate by liquid chromatography/mass spectrometry (LC/MS). Activities of the enzymes of nucleotide metabolism were evaluated by analysis of conversion of substrates into products by HPLC. The method was applied for analysis of hearts of mice fed 12 weeks with low- (LFD) or high-fat diet (HFD). The optimized method for AMPK activity analysis (measured in presence of AMP) revealed change of activity from 0.089 ± 0.035 pmol/min/mg protein in LFD to 0.024 ± 0.002 in HFD. This coincided with increase of adenosine deaminase (ADA) activity from 0.11 ± 0.02 to 0.19 ± 0.06 nmol/mg tissue/min and decrease of AMP-deaminase (AMPD) activity from 1.26 ± 0.35 to 0.56 ± 0.15 nmol/mg tissue/min for LFD and HFD, respectively. We have proven quality of our LC/MS method for analysis of AMPK activity. We observed decrease in AMPK activity in the heart of mice treated with high-fat diet. However, physiological consequences of this change could be modulated by decrease in AMPD activity.

  10. SIMULTANEOUS EXPRESSION AND REGULATION OF G-CSF AND IL-6 MESSENGER-RNA IN ADHERENT HUMAN MONOCYTES AND FIBROBLASTS

    NARCIS (Netherlands)

    VELLENGA, E; VANDERVINNE, B; DEWOLF, JTM; HALIE, MR

    The regulation of granulocyte-colony stimulating factor (G-CSF) and interleukin-6 (IL-6) mRNA was studied in human adherent monocytes in response to the protein kinase C activator, oleolyl-acetylglycerol (OAG), the calcium-ionophore A23187 and the cyclic AMP elevating agents, dibutyryl c-AMP

  11. Proteomic signatures implicate cAMP in light and temperature responses in Arabidopsis thaliana

    KAUST Repository

    Thomas, Ludivine; Marondedze, Claudius; Ederli, Luisa; Pasqualini, Stefania; Gehring, Christoph A

    2013-01-01

    The second messenger 3'-5'-cyclic adenosine monophosphate (cAMP) and adenylyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, are increasingly recognized as important signaling molecules in a number of physiological responses

  12. Angiopoietin-like protein 4 is an exercise-induced hepatokine in humans, regulated by glucagon and cAMP

    Directory of Open Access Journals (Sweden)

    Bodil Ingerslev

    2017-10-01

    Conclusions: The data suggest that exercise-induced ANGPTL4 is secreted from the liver and driven by a glucagon-cAMP-PKA pathway in humans. These findings link the liver, insulin/glucagon, and lipid metabolism together, which could implicate a role of ANGPTL4 in metabolic diseases.

  13. Cyclic Mechanical Stretch Up-regulates Hepatoma-Derived Growth Factor Expression in Cultured Rat Aortic Smooth Muscle Cells.

    Science.gov (United States)

    Kao, Ying-Hsien; Chen, Po-Han; Sun, Cheuk-Kwan; Chang, Yo-Chen; Lin, Yu-Chun; Tsai, Ming-Shian; Lee, Po-Huang; Cheng, Cheng-I

    2018-02-21

    Hepatoma-derived growth factor (HDGF) is a potent mitogen for vascular smooth muscle cells (SMCs) during embryogenesis and injury repair of vessel walls. Whether mechanical stimuli modulate HDGF expression remains unknown. This study aimed at investigating whether cyclic mechanical stretch plays a regulatory role in HDGF expression and regenerative cytokine production in aortic SMCs. A SMC cell line was grown on a silicone-based elastomer chamber with extracellular matrix coatings (either type I collagen or fibronectin) and received cyclic and uni-axial mechanical stretches with 10% deformation at frequency 1 Hz. Morphological observation showed that fibronectin coating provided better cell adhesion and spreading and that consecutive 6 hours of cyclic mechanical stretch remarkably induced reorientation and realignment of SMCs. Western blotting detection demonstrated that continuous mechanical stimuli elicited up-regulation of HDGF and PCNA, a cell proliferative marker. Signal kinetic profiling study indicated that cyclic mechanical stretch induced signaling activity in RhoA/ROCK and PI3K/Akt cascades. Kinase inhibition study further showed that blockade of PI3K activity suppressed the stretch-induced TNF-a, whereas RhoA/ROCK inhibition significantly blunted the IL-6 production and HDGF over-expression. Moreover, siRNA-mediated HDGF gene silencing significantly suppressed constitutive expression of IL-6, but not TNF-α, in SMCs. These findings support the role of HDGF in maintaining vascular expression of IL-6, which has been regarded a crucial regenerative factor for acute vascular injury. In conclusion, cyclic mechanical stretch may maintain constitutive expression of HDGF in vascular walls and be regarded an important biophysical regulator in vascular regeneration. ©2018 The Author(s).

  14. Regulation of basal and reserve cardiac pacemaker function by interactions of cAMP mediated PKA-dependent Ca2+ cycling with surface membrane channels

    Science.gov (United States)

    Vinogradova, Tatiana M.; Lakatta, Edward G.

    2009-01-01

    Decades of intensive research of primary cardiac pacemaker, the sinoatrial node, have established potential roles of specific membrane channels in the generation of the diastolic depolarization, the major mechanism allowing sinoatrial node cells generate spontaneous beating. During the last three decades, multiple studies made either in the isolated sinoatrial node or sinoatrial node cells have demonstrated a pivotal role of Ca2+ and, specifically Ca2+-release from sarcoplasmic reticulum, for spontaneous beating of cardiac pacemaker. Recently, spontaneous, rhythmic local subsarcolemmal Ca2+ releases from ryanodine receptors during late half of the diastolic depolarization have been implicated as a vital factor in the generation of sinoatrial node cells spontaneous firing. Local Ca2+ releases are driven by a unique combination of high basal cAMP production by adenylyl cyclases, high basal cAMP degradation by phosphodiesterases and a high level of cAMP-mediated PKA-dependent phosphorylation. These local Ca2+ releases activate an inward Na+-Ca2+ exchange current which accelerates the terminal diastolic depolarization rate and, thus, controls the spontaneous pacemaker firing. Both the basal primary pacemaker beating rate and its modulation via β-adrenergic receptor stimulation appear to be critically dependent upon intact RyR function and local subsarcolemmal sarcoplasmic reticulum generated Ca2+ releases. This review aspires to integrate the traditional viewpoint that has emphasized the supremacy of the ensemble of surface membrane ion channels in spontaneous firing of the primary cardiac pacemaker, and these novel perspectives of cAMP-mediated PKA-dependent Ca2+ cycling in regulation of the heart pacemaker clock, both in the basal state and during β-adrenergic receptor stimulation. PMID:19573534

  15. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

    Directory of Open Access Journals (Sweden)

    Lee A Borthwick

    Full Text Available Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR, a cAMP-dependent protein kinase A (PKA and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2 forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A. Overlay (Far-Western and Surface Plasmon Resonance (SPR analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727. Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.

  16. Mycobacterium tuberculosis cAMP Receptor Protein (Rv3676) Differs from the Escherichia coli Paradigm in Its cAMP Binding and DNA Binding Properties and Transcription Activation Properties*

    Science.gov (United States)

    Stapleton, Melanie; Haq, Ihtshamul; Hunt, Debbie M.; Arnvig, Kristine B.; Artymiuk, Peter J.; Buxton, Roger S.; Green, Jeffrey

    2010-01-01

    The pathogen Mycobacterium tuberculosis produces a burst of cAMP upon infection of macrophages. Bacterial cyclic AMP receptor proteins (CRP) are transcription factors that respond to cAMP by binding at target promoters when cAMP concentrations increase. Rv3676 (CRPMt) is a CRP family protein that regulates expression of genes (rpfA and whiB1) that are potentially involved in M. tuberculosis persistence and/or emergence from the dormant state. Here, the CRPMt homodimer is shown to bind two molecules of cAMP (one per protomer) at noninteracting sites. Furthermore, cAMP binding by CRPMt was relatively weak, entropy driven, and resulted in a relatively small enhancement in DNA binding. Tandem CRPMt-binding sites (CRP1 at −58.5 and CRP2 at −37.5) were identified at the whiB1 promoter (PwhiB1). In vitro transcription reactions showed that CRP1 is an activating site and that CRP2, which was only occupied in the presence of cAMP or at high CRPMt concentrations in the absence of cAMP, is a repressing site. Binding of CRPMt to CRP1 was not essential for open complex formation but was required for transcription activation. Thus, these data suggest that binding of CRPMt to the PwhiB1 CRP1 site activates transcription at a step after open complex formation. In contrast, high cAMP concentrations allowed occupation of both CRP1 and CRP2 sites, resulting in inhibition of open complex formation. Thus, M. tuberculosis CRP has evolved several distinct characteristics, compared with the Escherichia coli CRP paradigm, to allow it to regulate gene expression against a background of high concentrations of cAMP. PMID:20028978

  17. Effect of ionizing radiation on calcium and cyclic nucleotides metabolism in rats of different age

    International Nuclear Information System (INIS)

    Efimova, N.I.; Libenson, S.V.

    1982-01-01

    Some features of mechanism of calcium homeostasis and cyclic nucleotide exchange breakage in case of acute radiation injury of rats of various age were studied. It is established that calcium level in blood in nonpuberal animals, calcium and cAMP excretion with urine are minimal and reach maximum at puberal age. cGMP excretion with urine and concentrational levels of cAMP and cGMP in blood do not change with age. It is shown that calcium excretion with urine decreases adaptively in conditions of acute radiation injury in rats of all age groups. Maximal shifts in cAMP/cGMP ratio were noted in nonpuberal animals, whereas maximal adaptive-compensatory abilities in the regulation system of calcium homeostasis and cyclic nucleotides are typical to adolescent puberal animals

  18. cAMP-dependent protein kinase A (PKA) regulates angiogenesis by modulating tip cell behavior in a Notch-independent manner.

    Science.gov (United States)

    Nedvetsky, Pavel I; Zhao, Xiaocheng; Mathivet, Thomas; Aspalter, Irene M; Stanchi, Fabio; Metzger, Ross J; Mostov, Keith E; Gerhardt, Holger

    2016-10-01

    cAMP-dependent protein kinase A (PKA) is a ubiquitously expressed serine/threonine kinase that regulates a variety of cellular functions. Here, we demonstrate that endothelial PKA activity is essential for vascular development, specifically regulating the transition from sprouting to stabilization of nascent vessels. Inhibition of endothelial PKA by endothelial cell-specific expression of dominant-negative PKA in mice led to perturbed vascular development, hemorrhage and embryonic lethality at mid-gestation. During perinatal retinal angiogenesis, inhibition of PKA resulted in hypersprouting as a result of increased numbers of tip cells. In zebrafish, cell autonomous PKA inhibition also increased and sustained endothelial cell motility, driving cells to become tip cells. Although these effects of PKA inhibition were highly reminiscent of Notch inhibition effects, our data demonstrate that PKA and Notch independently regulate tip and stalk cell formation and behavior. © 2016. Published by The Company of Biologists Ltd.

  19. A biphasic and brain-region selective down-regulation of cyclic adenosine monophosphate concentrations supports object recognition in the rat.

    Directory of Open Access Journals (Sweden)

    Maïte Hotte

    Full Text Available BACKGROUND: We aimed to further understand the relationship between cAMP concentration and mnesic performance. METHODS AND FINDINGS: Rats were injected with milrinone (PDE3 inhibitor, 0.3 mg/kg, i.p., rolipram (PDE4 inhibitor, 0.3 mg/kg, i.p. and/or the selective 5-HT4R agonist RS 67333 (1 mg/kg, i.p. before testing in the object recognition paradigm. Cyclic AMP concentrations were measured in brain structures linked to episodic-like memory (i.e. hippocampus, prefrontal and perirhinal cortices before or after either the sample or the testing phase. Except in the hippocampus of rolipram treated-rats, all treatment increased cAMP levels in each brain sub-region studied before the sample phase. After the sample phase, cAMP levels were significantly increased in hippocampus (1.8 fold, prefrontal (1.3 fold and perirhinal (1.3 fold cortices from controls rat while decreased in prefrontal cortex (∼0.83 to 0.62 fold from drug-treated rats (except for milrinone+RS 67333 treatment. After the testing phase, cAMP concentrations were still increased in both the hippocampus (2.76 fold and the perirhinal cortex (2.1 fold from controls animals. Minor increase were reported in hippocampus and perirhinal cortex from both rolipram (respectively, 1.44 fold and 1.70 fold and milrinone (respectively 1.46 fold and 1.56 fold-treated rat. Following the paradigm, cAMP levels were significantly lower in the hippocampus, prefrontal and perirhinal cortices from drug-treated rat when compared to controls animals, however, only drug-treated rats spent longer time exploring the novel object during the testing phase (inter-phase interval of 4 h. CONCLUSIONS: Our results strongly suggest that a "pre-sample" early increase in cAMP levels followed by a specific lowering of cAMP concentrations in each brain sub-region linked to the object recognition paradigm support learning efficacy after a middle-term delay.

  20. Noninvasive microelectrode ion flux estimation technique (MIFE) for the study of the regulation of root membrane transport by cyclic nucleotides

    KAUST Repository

    Ordoñ ez, Natalia Maria; Shabala, Lana; Gehring, Christoph A; Shabala, Sergey Nikolayevich

    2013-01-01

    Changes in ion permeability and subsequently intracellular ion concentrations play a crucial role in intracellular and intercellular communication and, as such, confer a broad array of developmental and adaptive responses in plants. These changes are mediated by the activity of plasma-membrane based transport proteins many of which are controlled by cyclic nucleotides and/or other signaling molecules. The MIFE technique for noninvasive microelectrode ion flux measuring allows concurrent quantification of net fluxes of several ions with high spatial (μm range) and temporal (ca. 5 s) resolution, making it a powerful tool to study various aspects of downstream signaling events in plant cells. This chapter details basic protocols enabling the application of the MIFE technique to study regulation of root membrane transport in general and cyclic nucleotide mediated transport in particular. © Springer Science+Business Media New York 2013.

  1. Noninvasive microelectrode ion flux estimation technique (MIFE) for the study of the regulation of root membrane transport by cyclic nucleotides

    KAUST Repository

    Ordoñez, Natalia Maria

    2013-09-03

    Changes in ion permeability and subsequently intracellular ion concentrations play a crucial role in intracellular and intercellular communication and, as such, confer a broad array of developmental and adaptive responses in plants. These changes are mediated by the activity of plasma-membrane based transport proteins many of which are controlled by cyclic nucleotides and/or other signaling molecules. The MIFE technique for noninvasive microelectrode ion flux measuring allows concurrent quantification of net fluxes of several ions with high spatial (μm range) and temporal (ca. 5 s) resolution, making it a powerful tool to study various aspects of downstream signaling events in plant cells. This chapter details basic protocols enabling the application of the MIFE technique to study regulation of root membrane transport in general and cyclic nucleotide mediated transport in particular. © Springer Science+Business Media New York 2013.

  2. Biosynthesis of the antimicrobial cyclic lipopeptides nunamycin and nunapeptin by Pseudomonas fluorescens strain In5 is regulated by the LuxR‐type transcriptional regulator NunF

    OpenAIRE

    Hennessy, Rosanna C.; Phippen, Christopher B. W.; Nielsen, Kristian F.; Olsson, Stefan; Stougaard, Peter

    2017-01-01

    Abstract Nunamycin and nunapeptin are two antimicrobial cyclic lipopeptides (CLPs) produced by Pseudomonas fluorescens In5 and synthesized by nonribosomal synthetases (NRPS) located on two gene clusters designated the nun–nup regulon. Organization of the regulon is similar to clusters found in other CLP‐producing pseudomonads except for the border regions where putative LuxR‐type regulators are located. This study focuses on understanding the regulatory role of the LuxR‐type‐encoding gene nun...

  3. TmCactin plays an important role in Gram-negative and -positive bacterial infection by regulating expression of 7 AMP genes in Tenebrio molitor

    Science.gov (United States)

    Jo, Yong Hun; Jung Kim, Yu; Beom Park, Ki; Hwan Seong, Jeong; Gon Kim, Soo; Park, Soyi; Young Noh, Mi; Seok Lee, Yong; Soo Han, Yeon

    2017-01-01

    Cactin was originally identified as an interactor of the Drosophila IκB factor Cactus and shown to play a role in controlling embryonic polarity and regulating the NF-κB signaling pathway. While subsequent studies have identified the roles for Cactin in the mammalian immune response, the immune function of Cactin in insects has not been described yet. Here, we identified a Cactin gene from the mealworm beetle, Tenebrio molitor (TmCactin) and characterized its functional role in innate immunity. TmCactin was highly expressed in prepupa to last instar stages, and its expression was high in the integument and Malpighian tubules of last instar larvae and adults. TmCactin was induced in larvae after infection with different pathogens and detectable within 3 hours of infection. The highest levels of TmCactin expression were detected at 9 hours post infection. TmCactin RNAi significantly decreased the survival rates of larvae after challenge with Escherichia coli and Staphylococcus aureus, but had no significant effect after challenge with Candida albicans. Furthermore, TmCactin RNAi significantly reduced the expression of seven antimicrobial peptide genes (AMPs) after bacterial challenge. Our results suggest that TmCactin may serve as an important regulator of innate immunity, mediating AMP responses against both Gram-positive and Gram-negative bacteria in T. molitor. PMID:28418029

  4. Human mesenchymal stem cell proliferation is regulated by PGE2 through differential activation of cAMP-dependent protein kinase isoforms

    International Nuclear Information System (INIS)

    Kleiveland, Charlotte Ramstad; Kassem, Moustapha; Lea, Tor

    2008-01-01

    The conditions used for in vitro differentiation of hMSCs contain substances that affect the activity and expression of cyclooxygenase enzymes (COX1/COX2) and thereby the synthesis of prostanoids. hMSC constitutively produce PGE2 when cultivated in vitro. In this study we have investigated effects of PGE2 on proliferation of hMSC. We here demonstrate that one of the main control molecules in the Wnt pathway, GSK-3β, is phosphorylated at the negative regulatory site ser-9 after treating the cells with PGE2. This phosphorylation is mediated by elevation of cAMP and subsequent activation of PKA. Furthermore, PGE2 treatment leads to enhanced nuclear translocation of β-catenin, thus influencing cell proliferation. The presence of two PKA isoforms, types I and II, prompted us to investigate their individual contribution in PGE2-mediated regulation of proliferation. Specific activation of PKA type II with synthetic cAMP analogues, resulted in enhancement of proliferation. On the other side, we found that treatment of hMSC with high concentrations of PGE2 inhibited cell proliferation by arresting the cells in G 0 /G 1 phase, an effect we found to be mediated by PKA I. Hence, the two different PKA isoforms seem to have opposing functions in the regulation of proliferation and differentiation in these cells

  5. Cyclic nucleotides and radioresistnace

    International Nuclear Information System (INIS)

    Kulinskij, V.I.; Mikheeva, G.A.; Zel'manovich, B.M.

    1982-01-01

    The addition of glucose to meat-peptone broth does not change the radiosensitizing effect (RSE) of cAMP at the logarithmic phase (LP) and the radioprotective effect (RPE) at the stationary phase (SP), but sensitization, characteristic of cGMP, disappears in SP and turns into RPE in LP. Introduction of glucose into the broth for 20 min eliminates all the effects of both cyclic nucleotides in the cya + strain while cya - mutant exhibits RSE. RSE of both cyclic nucleotides is only manifested on minimal media. These data brought confirmation of the dependence of the influence of cyclic media. These data brought confirmation of the dependence of the influence of cyclic nucleotides on radioresistance upon the metabolic status of the cell [ru

  6. Rac1 controls Schwann cell myelination through cAMP and NF2/merlin

    Science.gov (United States)

    Guo, Li; Moon, Chandra; Niehaus, Karen; Zheng, Yi; Ratner, Nancy

    2013-01-01

    During peripheral nervous system development, Schwann cells (SCs) surrounding single large axons differentiate into myelinating SCs. Previous studies implicate RhoGTPases in SC myelination, but the mechanisms involved in RhoGTPase regulation of SC myelination are unknown. Here, we show that SC myelination is arrested in Rac1 conditional knockout (Rac1-CKO) mice. Rac1 knockout abrogated phosphorylation of the effector p21-activated kinase (PAK) and decreased NF2/merlin phosphorylation. Mutation of NF2/merlin rescued the myelin deficit in Rac1-CKO mice in vivo, and the shortened processes in cultured Rac1-CKO SCs in vitro. Mechanistically, cyclic adenosine monophosphate (cAMP) levels and E-cadherin expression were decreased in the absence of Rac1, and both were restored by mutation of NF2/merlin. Reduced cAMP is a cause of the myelin deficiency in Rac1-CKO mice, as elevation of cAMP by rolipram in Rac1-CKO mice in vivo allowed myelin formation. Thus NF2/merlin and cAMP function downstream of Rac1 signaling in SC myelination, and cAMP levels control Rac1-regulated SC myelination. PMID:23197717

  7. Cyclic mechanical stretch down-regulates cathelicidin antimicrobial peptide expression and activates a pro-inflammatory response in human bronchial epithelial cells

    Directory of Open Access Journals (Sweden)

    Harpa Karadottir

    2015-12-01

    Full Text Available Mechanical ventilation (MV of patients can cause damage to bronchoalveolar epithelium, leading to a sterile inflammatory response, infection and in severe cases sepsis. Limited knowledge is available on the effects of MV on the innate immune defense system in the human lung. In this study, we demonstrate that cyclic stretch of the human bronchial epithelial cell lines VA10 and BCi NS 1.1 leads to down-regulation of cathelicidin antimicrobial peptide (CAMP gene expression. We show that treatment of VA10 cells with vitamin D3 and/or 4-phenyl butyric acid counteracted cyclic stretch mediated down-regulation of CAMP mRNA and protein expression (LL-37. Further, we observed an increase in pro-inflammatory responses in the VA10 cell line subjected to cyclic stretch. The mRNA expression of the genes encoding pro-inflammatory cytokines IL-8 and IL-1β was increased after cyclic stretching, where as a decrease in gene expression of chemokines IP-10 and RANTES was observed. Cyclic stretch enhanced oxidative stress in the VA10 cells. The mRNA expression of toll-like receptor (TLR 3, TLR5 and TLR8 was reduced, while the gene expression of TLR2 was increased in VA10 cells after cyclic stretch. In conclusion, our in vitro results indicate that cyclic stretch may differentially modulate innate immunity by down-regulation of antimicrobial peptide expression and increase in pro-inflammatory responses.

  8. Integration of the tricarboxylic acid (TCA cycle with cAMP signaling and Sfl2 pathways in the regulation of CO2 sensing and hyphal development in Candida albicans.

    Directory of Open Access Journals (Sweden)

    Li Tao

    2017-08-01

    Full Text Available Morphological transitions and metabolic regulation are critical for the human fungal pathogen Candida albicans to adapt to the changing host environment. In this study, we generated a library of central metabolic pathway mutants in the tricarboxylic acid (TCA cycle, and investigated the functional consequences of these gene deletions on C. albicans biology. Inactivation of the TCA cycle impairs the ability of C. albicans to utilize non-fermentable carbon sources and dramatically attenuates cell growth rates under several culture conditions. By integrating the Ras1-cAMP signaling pathway and the heat shock factor-type transcription regulator Sfl2, we found that the TCA cycle plays fundamental roles in the regulation of CO2 sensing and hyphal development. The TCA cycle and cAMP signaling pathways coordinately regulate hyphal growth through the molecular linkers ATP and CO2. Inactivation of the TCA cycle leads to lowered intracellular ATP and cAMP levels and thus affects the activation of the Ras1-regulated cAMP signaling pathway. In turn, the Ras1-cAMP signaling pathway controls the TCA cycle through both Efg1- and Sfl2-mediated transcriptional regulation in response to elevated CO2 levels. The protein kinase A (PKA catalytic subunit Tpk1, but not Tpk2, may play a major role in this regulation. Sfl2 specifically binds to several TCA cycle and hypha-associated genes under high CO2 conditions. Global transcriptional profiling experiments indicate that Sfl2 is indeed required for the gene expression changes occurring in response to these elevated CO2 levels. Our study reveals the regulatory role of the TCA cycle in CO2 sensing and hyphal development and establishes a novel link between the TCA cycle and Ras1-cAMP signaling pathways.

  9. Integration of the tricarboxylic acid (TCA) cycle with cAMP signaling and Sfl2 pathways in the regulation of CO2 sensing and hyphal development in Candida albicans.

    Science.gov (United States)

    Tao, Li; Zhang, Yulong; Fan, Shuru; Nobile, Clarissa J; Guan, Guobo; Huang, Guanghua

    2017-08-01

    Morphological transitions and metabolic regulation are critical for the human fungal pathogen Candida albicans to adapt to the changing host environment. In this study, we generated a library of central metabolic pathway mutants in the tricarboxylic acid (TCA) cycle, and investigated the functional consequences of these gene deletions on C. albicans biology. Inactivation of the TCA cycle impairs the ability of C. albicans to utilize non-fermentable carbon sources and dramatically attenuates cell growth rates under several culture conditions. By integrating the Ras1-cAMP signaling pathway and the heat shock factor-type transcription regulator Sfl2, we found that the TCA cycle plays fundamental roles in the regulation of CO2 sensing and hyphal development. The TCA cycle and cAMP signaling pathways coordinately regulate hyphal growth through the molecular linkers ATP and CO2. Inactivation of the TCA cycle leads to lowered intracellular ATP and cAMP levels and thus affects the activation of the Ras1-regulated cAMP signaling pathway. In turn, the Ras1-cAMP signaling pathway controls the TCA cycle through both Efg1- and Sfl2-mediated transcriptional regulation in response to elevated CO2 levels. The protein kinase A (PKA) catalytic subunit Tpk1, but not Tpk2, may play a major role in this regulation. Sfl2 specifically binds to several TCA cycle and hypha-associated genes under high CO2 conditions. Global transcriptional profiling experiments indicate that Sfl2 is indeed required for the gene expression changes occurring in response to these elevated CO2 levels. Our study reveals the regulatory role of the TCA cycle in CO2 sensing and hyphal development and establishes a novel link between the TCA cycle and Ras1-cAMP signaling pathways.

  10. A Serratia marcescens PigP homolog controls prodigiosin biosynthesis, swarming motility and hemolysis and is regulated by cAMP-CRP and HexS.

    Directory of Open Access Journals (Sweden)

    Robert M Q Shanks

    Full Text Available Swarming motility and hemolysis are virulence-associated determinants for a wide array of pathogenic bacteria. The broad host-range opportunistic pathogen Serratia marcescens produces serratamolide, a small cyclic amino-lipid, that promotes swarming motility and hemolysis. Serratamolide is negatively regulated by the transcription factors HexS and CRP. Positive regulators of serratamolide production are unknown. Similar to serratamolide, the antibiotic pigment, prodigiosin, is regulated by temperature, growth phase, HexS, and CRP. Because of this co-regulation, we tested the hypothesis that a homolog of the PigP transcription factor of the atypical Serratia species ATCC 39006, which positively regulates prodigiosin biosynthesis, is also a positive regulator of serratamolide production in S. marcescens. Mutation of pigP in clinical, environmental, and laboratory strains of S. marcescens conferred pleiotropic phenotypes including the loss of swarming motility, hemolysis, and severely reduced prodigiosin and serratamolide synthesis. Transcriptional analysis and electrophoretic mobility shift assays place PigP in a regulatory pathway with upstream regulators CRP and HexS. The data from this study identifies a positive regulator of serratamolide production, describes novel roles for the PigP transcription factor, shows for the first time that PigP directly regulates the pigment biosynthetic operon, and identifies upstream regulators of pigP. This study suggests that PigP is important for the ability of S. marcescens to compete in the environment.

  11. The Arabidopsis thaliana Cyclic-Nucleotide-Dependent Response – a Quantitative Proteomic and Phosphoproteomic Analysis

    KAUST Repository

    Alqurashi, May M.

    2013-11-01

    Protein phosphorylation governs many regulatory pathways and an increasing number of kinases, proteins that transfer phosphate groups, are in turn activated by cyclic nucleotides. One of the cyclic nucleotides, cyclic adenosine monophosphate (cAMP), has been shown to be a second messenger in abiotic and biotic stress responses. However, little is known about the precise role of cAMP in plants and in the down-stream activation of kinases, and hence cAMP-dependent phosphorylation. To increase our understanding of the role of cAMP, proteomic and phosphoproteomic profiles of Arabidopsis thaliana suspension culture cells were analyzed before and after treatment of cells with two different concentrations of 8-Bromo-cAMP (1 µM and 100 nM) and over a time-course of one hour. A comparative quantitative analysis was undertaken using two- dimensional gel electrophoresis and the Delta 2D software (DECODON) followed by protein spot identification by tandem mass spectrometry combined with Mascot and Scaffold. Differentially expressed proteins and regulated phosphoproteins were categorized according to their biological function using bioinformatics tools. The results revealed that the treatment with 1 µM and 100 nM 8-Bromo-cAMP was sufficient to induce specific concentration- and time-dependent changes at the proteome and phosphoproteome levels. In particular, different phosphorylation patterns were observed overtime preferentially affecting proteins in a number of functional categories, notably phosphatases, proteins that remove phosphate groups. This suggests that cAMP both transiently activates and deactivates proteins through specific phosphorylation events and provides new insight into biological mechanisms and functions at the systems level.

  12. cAMP/PKA regulates osteogenesis, adipogenesis and ratio of RANKL/OPG mRNA expression in mesenchymal stem cells by suppressing leptin.

    Directory of Open Access Journals (Sweden)

    Der-Chih Yang

    Full Text Available BACKGROUND: Mesenchymal stem cells (MSCs are a pluripotent cell type that can differentiate into adipocytes, osteoblasts and other cells. The reciprocal relationship between adipogenesis and osteogenesis was previously demonstrated; however, the mechanisms remain largely unknown. METHODS AND FINDINGS: We report that activation of PKA by 3-isobutyl-1 methyl xanthine (IBMX and forskolin enhances adipogenesis, the gene expression of PPARgamma2 and LPL, and downregulates the gene expression of Runx2 and osteopontin, markers of osteogenesis. PKA activation also decreases the ratio of Receptor Activator of the NF-kappaB Ligand to Osteoprotegerin (RANKL/OPG gene expression - the key factors of osteoclastogenesis. All these effects are mediated by the cAMP/PKA/CREB pathway by suppressing leptin, and may contribute to PKA stimulators-induced in vivo bone loss in developing zebrafish. CONCLUSIONS: Using MSCs, the center of a newly proposed bone metabolic unit, we identified cAMP/PKA signaling, one of the many signaling pathways that regulate bone homeostasis via controlling cyto-differentiation of MSCs and altering RANKL/OPG gene expression.

  13. Autonomous and nonautonomous regulation of axis formation by antagonistic signaling via 7-span cAMP receptors and GSK3 in Dictyostelium.

    Science.gov (United States)

    Ginsburg, G T; Kimmel, A R

    1997-08-15

    Early during Dictyostelium development a fundamental cell-fate decision establishes the anteroposterior (prestalk/prespore) axis. Signaling via the 7-transmembrane cAMP receptor CAR4 is essential for creating and maintaining a normal pattern; car4-null alleles have decreased levels of prestalk-specific mRNAs but enhanced expression of prespore genes. car4- cells produce all of the signals required for prestalk differentiation but lack an extracellular factor necessary for prespore differentiation of wild-type cells. This secreted factor decreases the sensitivity of prespore cells to inhibition by the prestalk morphogen DIF-1. At the cell autonomous level, CAR4 is linked to intracellular circuits that activate prestalk but inhibit prespore differentiation. The autonomous action of CAR4 is antagonistic to the positive intracellular signals mediated by another cAMP receptor, CAR1 and/or CAR3. Additional data indicate that these CAR-mediated pathways converge at the serine/threonine protein kinase GSK3, suggesting that the anterior (prestalk)/posterior (prespore) axis of Dictyostelium is regulated by an ancient mechanism that is shared by the Wnt/Fz circuits for dorsoventral patterning during early Xenopus development and establishing Drosophila segment polarity.

  14. Plasmids encoding PKI(1-31), a specific inhibitor of cAMP-stimulated gene expression, inhibit the basal transcriptional activity of some but not all cAMP-regulated DNA response elements in JEG-3 cells.

    Science.gov (United States)

    Grove, J R; Deutsch, P J; Price, D J; Habener, J F; Avruch, J

    1989-11-25

    Plasmids that encode a bioactive amino-terminal fragment of the heat-stable inhibitor of the cAMP-dependent protein kinase, PKI(1-31), were employed to characterize the role of this protein kinase in the control of transcriptional activity mediated by three DNA regulatory elements in the JEG-3 human placental cell line. The 5'-flanking sequence of the human collagenase gene contains the heptameric sequence, 5'-TGAGTCA-3', previously identified as a "phorbol ester" response element. Reporter genes containing either the intact 1.2-kilobase 5'-flanking sequence from the human collagenase gene or just the 7-base pair (bp) response element, when coupled to an enhancerless promoter, each exhibit both cAMP and phorbol ester-stimulated expression in JEG-3 cells. Cotransfection of either construct with plasmids encoding PKI(1-31) inhibits cAMP-stimulated but not basal- or phorbol ester-stimulated expression. Pretreatment of cells with phorbol ester for 1 or 2 days abrogates completely the response to rechallenge with phorbol ester but does not alter the basal expression of either construct; cAMP-stimulated expression, while modestly inhibited, remains vigorous. The 5'-flanking sequence of the human chorionic gonadotropin-alpha subunit (HCG alpha) gene has two copies of the sequence, 5'-TGACGTCA-3', contained in directly adjacent identical 18-bp segments, previously identified as a cAMP-response element. Reporter genes containing either the intact 1.5 kilobase of 5'-flanking sequence from the HCG alpha gene, or just the 36-bp tandem repeat cAMP response element, when coupled to an enhancerless promoter, both exhibit a vigorous cAMP stimulation of expression but no response to phorbol ester in JEG-3 cells. Cotransfection with plasmids encoding PKI(1-31) inhibits both basal and cAMP-stimulated expression in a parallel fashion. The 5'-flanking sequence of the human enkephalin gene mediates cAMP-stimulated expression of reporter genes in both JEG-3 and CV-1 cells. Plasmids

  15. Complex Interplay between FleQ, Cyclic Diguanylate and Multiple σ Factors Coordinately Regulates Flagellar Motility and Biofilm Development in Pseudomonas putida.

    Directory of Open Access Journals (Sweden)

    Alicia Jiménez-Fernández

    Full Text Available Most bacteria alternate between a free living planktonic lifestyle and the formation of structured surface-associated communities named biofilms. The transition between these two lifestyles requires a precise and timely regulation of the factors involved in each of the stages that has been likened to a developmental process. Here we characterize the involvement of the transcriptional regulator FleQ and the second messenger cyclic diguanylate in the coordinate regulation of multiple functions related to motility and surface colonization in Pseudomonas putida. Disruption of fleQ caused strong defects in flagellar motility, biofilm formation and surface attachment, and the ability of this mutation to suppress multiple biofilm-related phenotypes associated to cyclic diguanylate overproduction suggests that FleQ mediates cyclic diguanylate signaling critical to biofilm growth. We have constructed a library containing 94 promoters potentially involved in motility and biofilm development fused to gfp and lacZ, screened this library for FleQ and cyclic diguanylate regulation, and assessed the involvement of alternative σ factors σN and FliA in the transcription of FleQ-regulated promoters. Our results suggest a dual mode of action for FleQ. Low cyclic diguanylate levels favor FleQ interaction with σN-dependent promoters to activate the flagellar cascade, encompassing the flagellar cluster and additional genes involved in cyclic diguanylate metabolism, signal transduction and gene regulation. On the other hand, characterization of the FleQ-regulated σN- and FliA-independent PlapA and PbcsD promoters revealed two disparate regulatory mechanisms leading to a similar outcome: the synthesis of biofilm matrix components in response to increased cyclic diguanylate levels.

  16. Detection of Cyclic Dinucleotides by STING.

    Science.gov (United States)

    Du, Xiao-Xia; Su, Xiao-Dong

    2017-01-01

    STING (stimulator of interferon genes) is an essential signaling adaptor protein mediating cytosolic DNA-induced innate immunity for both microbial invasion and self-DNA leakage. STING is also a direct receptor for cytosolic cyclic dinucleotides (CDNs), including the microbial secondary messengers c-di-GMP (3',3'-cyclic di-GMP), 3',3'cGAMP (3',3'-cyclic GMP-AMP), and mammalian endogenous 2',3'cGAMP (2',3'-cyclic GMP-AMP) synthesized by cGAS (cyclic GMP-AMP synthase). Upon CDN binding, STING undergoes a conformational change to enable signal transduction by phosphorylation and finally to active IRF3 (Interferon regulatory factor 3) for type I interferon production. Here, we describe some experimental procedures such as Isothermal Titration Calorimetry and luciferase reporter assays to study the CDNs binding and activity by STING proteins.

  17. Regulation of ATP-sensitive K+ channels in insulinoma cells: Activation by somatostatin and protein kinase C and the role of cAMP

    International Nuclear Information System (INIS)

    De Weille, J.R.; Schmid-Antomarchi, H.; Fosset, M.; Lazdunski, M.

    1989-01-01

    The actions of somatostatin and of the phorbol ester 4β-phorbol 12-myristate 13-acetate (PMA) were studied in rat insulinoma (RINm5F) cells by electrophysiological and 86 Rb + flux techniques. Both PMA and somatostatin hyperpolarize insulinoma cells by activating ATP-sensitive K + channels. The presence of intracellular GTP is required for the somatostatin effects. PMA- and somatostatin-induced hyperpolarization and channel activity are inhibited by the sulfonylurea glibenclamide. Glibenclamide-sensitive 86 Rb + efflux from insulinoma cells is stimulated by somatostatin in a dose-dependent manner (half maximal effect at 0.7 nM) and abolished by pertussis toxin pretreatment. Mutual roles of a GTP-binding protein, of protein kinase C, and of cAMP in the regulation of ATP-sensitive K + channels are discussed

  18. Aspirin-triggered resolvin D1 attenuates PDGF-induced vascular smooth muscle cell migration via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway.

    Science.gov (United States)

    Mottola, Giorgio; Chatterjee, Anuran; Wu, Bian; Chen, Mian; Conte, Michael S

    2017-01-01

    Resolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been previously shown to attenuate vascular smooth muscle cell (VSMC) migration, a key process in the development of intimal hyperplasia. We sought to investigate the role of the cAMP/PKA pathway in mediating the effects of the aspirin-triggered epimer 17R-RvD1 (AT-RvD1) on VSMC migration. VSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity. Treatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor. Our results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects of AT-RvD1 on VSMC migration.

  19. Proteomic signatures implicate cAMP in light and temperature responses in Arabidopsis thaliana

    KAUST Repository

    Thomas, Ludivine

    2013-05-01

    The second messenger 3\\'-5\\'-cyclic adenosine monophosphate (cAMP) and adenylyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, are increasingly recognized as important signaling molecules in a number of physiological responses in higher plants. Here we used proteomics to identify cAMP-dependent protein signatures in Arabidopsis thaliana and identify a number of differentially expressed proteins with a role in light- and temperature-dependent responses, notably photosystem II subunit P-1, plasma membrane associated cation-binding protein and chaperonin 60 β. Based on these proteomics results we conclude that, much like in cyanobacteria, algae and fungi, cAMP may have a role in light signaling and the regulation of photosynthesis as well as responses to temperature and we speculate that ACs could act as light and/or temperature sensors in higher plants. Biological significance: This current study is significant since it presents the first proteomic response to cAMP, a novel and key second messenger in plants. It will be relevant to researchers in plant physiology and in particular those with an interest in second messengers and their role in biotic and abiotic stress responses. © 2013 Elsevier B.V.

  20. cAMP level modulates scleral collagen remodeling, a critical step in the development of myopia.

    Directory of Open Access Journals (Sweden)

    Yijin Tao

    Full Text Available The development of myopia is associated with decreased ocular scleral collagen synthesis in humans and animal models. Collagen synthesis is, in part, under the influence of cyclic adenosine monophosphate (cAMP. We investigated the associations between cAMP, myopia development in guinea pigs, and collagen synthesis by human scleral fibroblasts (HSFs. Form-deprived myopia (FDM was induced by unilateral masking of guinea pig eyes. Scleral cAMP levels increased selectively in the FDM eyes and returned to normal levels after unmasking and recovery. Unilateral subconjunctival treatment with the adenylyl cyclase (AC activator forskolin resulted in a myopic shift accompanied by reduced collagen mRNA levels, but it did not affect retinal electroretinograms. The AC inhibitor SQ22536 attenuated the progression of FDM. Moreover, forskolin inhibited collagen mRNA levels and collagen secretion by HSFs. The inhibition was reversed by SQ22536. These results demonstrate a critical role of cAMP in control of myopia development. Selective regulation of cAMP to control scleral collagen synthesis may be a novel therapeutic strategy for preventing and treating myopia.

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

    Science.gov (United States)

    Ashton, A R; Polya, G M

    1977-01-01

    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. PMID:196595

  2. A multi-angular mass spectrometric view at cyclic nucleotide signaling proteins : Structure/function and protein interactions of cAMP- and cGMP-dependent protein kinase

    NARCIS (Netherlands)

    Scholten, A.

    2006-01-01

    The primary focus of this thesis is the two kinases PKA and PKG, cAMP and cGMP dependent protein kinase respectively. PKA and PKG are studied both at structure/function level as well as at the level of interaction with other proteins in tissue. Our primary methods are all based on mass spectrometry.

  3. Ser/Thr Phosphorylation Regulates the Fatty Acyl-AMP Ligase Activity of FadD32, an Essential Enzyme in Mycolic Acid Biosynthesis*

    Science.gov (United States)

    Le, Nguyen-Hung; Molle, Virginie; Eynard, Nathalie; Miras, Mathieu; Stella, Alexandre; Bardou, Fabienne; Galandrin, Ségolène; Guillet, Valérie; André-Leroux, Gwenaëlle; Bellinzoni, Marco; Alzari, Pedro; Mourey, Lionel; Burlet-Schiltz, Odile; Daffé, Mamadou; Marrakchi, Hedia

    2016-01-01

    Mycolic acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is a well known source of antituberculous drug targets. Among the promising new targets in the pathway, FadD32 is an essential enzyme required for the activation of the long meromycolic chain of mycolic acids and is essential for mycobacterial growth. Following the in-depth biochemical, biophysical, and structural characterization of FadD32, we investigated its putative regulation via post-translational modifications. Comparison of the fatty acyl-AMP ligase activity between phosphorylated and dephosphorylated FadD32 isoforms showed that the native protein is phosphorylated by serine/threonine protein kinases and that this phosphorylation induced a significant loss of activity. Mass spectrometry analysis of the native protein confirmed the post-translational modifications and identified Thr-552 as the phosphosite. Phosphoablative and phosphomimetic FadD32 mutant proteins confirmed both the position and the importance of the modification and its correlation with the negative regulation of FadD32 activity. Investigation of the mycolic acid condensation reaction catalyzed by Pks13, involving FadD32 as a partner, showed that FadD32 phosphorylation also impacts the condensation activity. Altogether, our results bring to light FadD32 phosphorylation by serine/threonine protein kinases and its correlation with the enzyme-negative regulation, thus shedding a new horizon on the mycolic acid biosynthesis modulation and possible inhibition strategies for this promising drug target. PMID:27590338

  4. Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance

    Energy Technology Data Exchange (ETDEWEB)

    Arana, Maite Rocío, E-mail: arana@ifise-conicet.gov.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Tocchetti, Guillermo Nicolás, E-mail: gtocchetti@live.com.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Domizi, Pablo, E-mail: domizi@ibr-conicet.gov.ar [Instituto de Biología Molecular y Celular de Rosario (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Arias, Agostina, E-mail: agoarias@yahoo.com.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Rigalli, Juan Pablo, E-mail: jprigalli@gmail.com [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Ruiz, María Laura, E-mail: ruiz@ifise-conicet.gov.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); and others

    2015-09-01

    The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 μM) for 48 h exhibited a dose–response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics. - Highlights: • cAMP positively modulates the expression and activity of GST and MRP2 in Caco-2 cells. • Such induction resulted in increased cytoprotection against chemical injury. • PKA

  5. Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance

    International Nuclear Information System (INIS)

    Arana, Maite Rocío; Tocchetti, Guillermo Nicolás; Domizi, Pablo; Arias, Agostina; Rigalli, Juan Pablo; Ruiz, María Laura

    2015-01-01

    The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 μM) for 48 h exhibited a dose–response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics. - Highlights: • cAMP positively modulates the expression and activity of GST and MRP2 in Caco-2 cells. • Such induction resulted in increased cytoprotection against chemical injury. • PKA

  6. The antimicrobial peptide derived from insulin-like growth factor-binding protein 5, AMP-IBP5, regulates keratinocyte functions through Mas-related gene X receptors.

    Science.gov (United States)

    Chieosilapatham, Panjit; Niyonsaba, François; Kiatsurayanon, Chanisa; Okumura, Ko; Ikeda, Shigaku; Ogawa, Hideoki

    2017-10-01

    In addition to their microbicidal properties, host defense peptides (HDPs) display various immunomodulatory functions, including keratinocyte production of cytokines/chemokines, proliferation, migration and wound healing. Recently, a novel HDP named AMP-IBP5 (antimicrobial peptide derived from insulin-like growth factor-binding protein 5) was shown to exhibit antimicrobial activity against numerous pathogens, even at concentrations comparable to those of human β-defensins and LL-37. However, the immunomodulatory role of AMP-IBP5 in cutaneous tissue remains unknown. To investigate whether AMP-IBP5 triggers keratinocyte activation and to clarify its mechanism. Production of cytokines/chemokines and growth factors was determined by appropriate ELISA kits. Cell migration was assessed by in vitro wound closure assay, whereas cell proliferation was analyzed using BrdU incorporation assay complimented with XTT assay. MAPK and NF-κB activation was determined by Western blotting. Intracellular cAMP levels were assessed using cAMP enzyme immunoassay kit. Among various cytokines/chemokines and growth factors tested, AMP-IBP5 selectively increased the production of IL-8 and VEGF. Moreover, AMP-IBP5 markedly enhanced keratinocyte migration and proliferation. AMP-IBP5-induced keratinocyte activation was mediated by Mrg X1-X4 receptors with MAPK and NF-κB pathways working downstream, as evidenced by the inhibitory effects of MrgX1-X4 siRNAs and ERK-, JNK-, p38- and NF-κB-specific inhibitors. We confirmed that AMP-IBP5 indeed induced MAPK and NF-κB activation. Furthermore, AMP-IBP5-induced VEGF but not IL-8 production correlated with an increase in intracellular cAMP. Our findings suggest that in addition to its antimicrobial function, AMP-IBP5 might contribute to wound healing process through activation of keratinocytes. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

  7. Regulation of energy metabolism during social interactions in rainbow trout: a role for AMP-activated protein kinase.

    Science.gov (United States)

    Gilmour, K M; Craig, P M; Dhillon, R S; Lau, G Y; Richards, J G

    2017-11-01

    Rainbow trout ( Oncorhynchus mykiss ) confined in pairs form social hierarchies in which subordinate fish typically experience fasting and high circulating cortisol levels, resulting in low growth rates. The present study investigated the role of AMP-activated protein kinase (AMPK) in mediating metabolic adjustments associated with social status in rainbow trout. After 3 days of social interaction, liver AMPK activity was significantly higher in subordinate than dominant or sham (fish handled in the same fashion as paired fish but held individually) trout. Elevated liver AMPK activity in subordinate fish likely reflected a significantly higher ratio of phosphorylated AMPK (phospho-AMPK) to total AMPK protein, which was accompanied by significantly higher AMPKα 1 relative mRNA abundance. Liver ATP and creatine phosphate concentrations in subordinate fish also were elevated, perhaps as a result of AMPK activity. Sham fish that were fasted for 3 days exhibited effects parallel to those of subordinate fish, suggesting that low food intake was an important trigger of elevated AMPK activity in subordinate fish. Effects on white muscle appeared to be influenced by the physical activity associated with social interaction. Overall, muscle AMPK activity was significantly higher in dominant and subordinate than sham fish. The ratio of phospho-AMPK to total AMPK protein in muscle was highest in subordinate fish, while muscle AMPKα 1 relative mRNA abundance was elevated by social dominance. Muscle ATP and creatine phosphate concentrations were high in dominant and subordinate fish at 6 h of interaction and decreased significantly thereafter. Collectively, the findings of the present study support a role for AMPK in mediating liver and white muscle metabolic adjustments associated with social hierarchy formation in rainbow trout. Copyright © 2017 the American Physiological Society.

  8. Up-regulated Ectonucleotidases in Fas-Associated Death Domain Protein- and Receptor-Interacting Protein Kinase 1-Deficient Jurkat Leukemia Cells Counteract Extracellular ATP/AMP Accumulation via Pannexin-1 Channels during Chemotherapeutic Drug-Induced Apoptosis.

    Science.gov (United States)

    Boyd-Tressler, Andrea M; Lane, Graham S; Dubyak, George R

    2017-07-01

    Pannexin-1 (Panx1) channels mediate the efflux of ATP and AMP from cancer cells in response to induction of extrinsic apoptosis by death receptors or intrinsic apoptosis by chemotherapeutic agents. We previously described the accumulation of extracellular ATP /AMP during chemotherapy-induced apoptosis in Jurkat human leukemia cells. In this study, we compared how different signaling pathways determine extracellular nucleotide pools in control Jurkat cells versus Jurkat lines that lack the Fas-associated death domain (FADD) or receptor-interacting protein kinase 1 (RIP1) cell death regulatory proteins. Tumor necrosis factor- α induced extrinsic apoptosis in control Jurkat cells and necroptosis in FADD-deficient cells; treatment of both lines with chemotherapeutic drugs elicited similar intrinsic apoptosis. Robust extracellular ATP/AMP accumulation was observed in the FADD-deficient cells during necroptosis, but not during apoptotic activation of Panx1 channels. Accumulation of extracellular ATP/AMP was similarly absent in RIP1-deficient Jurkat cells during apoptotic responses to chemotherapeutic agents. Apoptotic activation triggered equivalent proteolytic gating of Panx1 channels in all three Jurkat cell lines. The differences in extracellular ATP/AMP accumulation correlated with cell-line-specific expression of ectonucleotidases that metabolized the released ATP/AMP. CD73 mRNA, and α β -methylene-ADP-inhibitable ecto-AMPase activity were elevated in the FADD-deficient cells. In contrast, the RIP1-deficient cells were defined by increased expression of tartrate-sensitive prostatic acid phosphatase as a broadly acting ectonucleotidase. Thus, extracellular nucleotide accumulation during regulated tumor cell death involves interplay between ATP/AMP efflux pathways and different cell-autonomous ectonucleotidases. Differential expression of particular ectonucleotidases in tumor cell variants will determine whether chemotherapy-induced activation of Panx1 channels

  9. Schwann Cells Metabolize Extracellular 2′,3′-cAMP to 2′-AMP

    Science.gov (United States)

    Verrier, Jonathan D.; Kochanek, Patrick M.

    2015-01-01

    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

  10. Biosynthesis of the antimicrobial cyclic lipopeptides nunamycin and nunapeptin by Pseudomonas fluorescens strain In5 is regulated by the LuxR-type transcriptional regulator NunF

    DEFF Research Database (Denmark)

    Hennessy, Rosanna Catherine; Phippen, Christopher; Nielsen, Kristian Fog

    2017-01-01

    -producing pseudomonads except for the border regions where putative LuxR-type regulators are located. This study focuses on understanding the regulatory role of the LuxR-type-encoding gene nunF in CLP production of P. fluorescens In5. Functional analysis of nunF coupled with liquid chromatography-high-resolution mass......Nunamycin and nunapeptin are two antimicrobial cyclic lipopeptides (CLPs) produced by Pseudomonas fluorescens In5 and synthesized by nonribosomal synthetases (NRPS) located on two gene clusters designated the nun-nup regulon. Organization of the regulon is similar to clusters found in other CLP...... spectrometry (LC-HRMS) showed that CLP biosynthesis is regulated by nunF. Quantitative real-time PCR analysis indicated that transcription of the NRPS genes catalyzing CLP production is strongly reduced when nunF is mutated indicating that nunF is part of the nun-nup regulon. Swarming and biofilm formation...

  11. Direct Light-up of cAMP Derivatives in Living Cells by Click Reactions

    Directory of Open Access Journals (Sweden)

    Yan Xu

    2013-10-01

    Full Text Available 8-Azidoadenosine 3′,5′-cyclic monophosphate (8-azido cAMP was directly detected in living cells, by applying Cu-free azide-alkyne cycloaddition to probe cAMP derivatives by fluorescence light-up. Fluorescence emission was generated by two non-fluorescent molecules, 8-azido cAMP as a model target and difluorinated cyclooctyne (DIFO reagent as a probe. The azide-alkyne cycloaddition reaction between 8-azido cAMP and DIFO induces fluorescence in 8-azido cAMP. The fluorescence emission serves as a way to probe 8-azido cAMP in cells.

  12. The crystal structures of apo and cAMP-bound GlxR from Corynebacterium glutamicum reveal structural and dynamic changes upon cAMP binding in CRP/FNR family transcription factors.

    Directory of Open Access Journals (Sweden)

    Philip D Townsend

    Full Text Available The cyclic AMP-dependent transcriptional regulator GlxR from Corynebacterium glutamicum is a member of the super-family of CRP/FNR (cyclic AMP receptor protein/fumarate and nitrate reduction regulator transcriptional regulators that play central roles in bacterial metabolic regulatory networks. In C. glutamicum, which is widely used for the industrial production of amino acids and serves as a non-pathogenic model organism for members of the Corynebacteriales including Mycobacterium tuberculosis, the GlxR homodimer controls the transcription of a large number of genes involved in carbon metabolism. GlxR therefore represents a key target for understanding the regulation and coordination of C. glutamicum metabolism. Here we investigate cylic AMP and DNA binding of GlxR from C. glutamicum and describe the crystal structures of apo GlxR determined at a resolution of 2.5 Å, and two crystal forms of holo GlxR at resolutions of 2.38 and 1.82 Å, respectively. The detailed structural analysis and comparison of GlxR with CRP reveals that the protein undergoes a distinctive conformational change upon cyclic AMP binding leading to a dimer structure more compatible to DNA-binding. As the two binding sites in the GlxR homodimer are structurally identical dynamic changes upon binding of the first ligand are responsible for the allosteric behavior. The results presented here show how dynamic and structural changes in GlxR lead to optimization of orientation and distance of its two DNA-binding helices for optimal DNA recognition.

  13. Two CGTCA motifs and a GHF1/Pit1 binding site mediate cAMP-dependent protein kinase A regulation of human growth hormone gene expression in rat anterior pituitary GC cells.

    Science.gov (United States)

    Shepard, A R; Zhang, W; Eberhardt, N L

    1994-01-21

    We established the cis-acting elements which mediate cAMP responsiveness of the human growth hormone (hGH) gene in transiently transfected rat anterior pituitary tumor GC cells. Analysis of the intact hGH gene or hGH 5'-flanking DNA (5'-FR) coupled to the hGh cDNA or chloramphenicol acetyltransferase or luciferase genes, indicated that cAMP primarily stimulated hGH promoter activity. Cotransfection of a protein kinase A inhibitory protein cDNA demonstrated that the cAMP response was mediated by protein kinase A. Mutational analysis of the hGH promoter identified two core cAMP response element motifs (CGTCA) located at nucleotides -187/-183 (distal cAMP response element; dCRE) and -99/-95 (proximal cAMP response element; pCRE) and a pituitary-specific transcription factor (GHF1/Pit1) binding site at nucleotides -123/-112 (dGHF1) which were required for cAMP responsiveness. GHF1 was not a limiting factor, since overexpression of GHF1 in cotransfections increased basal but not forskolin induction levels. Gel shift analyses indicated that similar, ubiquitous, thermostable protein(s) specifically bound the pCRE and dCRE motifs. The CGTCA motif-binding factors were cAMP response element binding protein (CREB)/activating transcription factor-1 (ATF-1)-related, since the DNA-protein complex was competed by unlabeled CREB consensus oligonucleotide, specifically supershifted by antisera to CREB and ATF-1 but not ATF-2, and was bound by purified CREB with the same relative binding affinity (pCRE < dCRE < CREB) and mobility as the GC nuclear extract. UV cross-linking and Southwestern blot analyses revealed multiple DNA-protein interactions of which approximately 100- and approximately 45-kDa proteins were predominant; the approximately 45-kDa protein may represent CREB. These results indicate that CREB/ATF-1-related factors act coordinately with the cell-specific factor GHF1 to mediate cAMP-dependent regulation of hGH-1 gene transcription in anterior pituitary somatotrophs.

  14. Cyclic glycine-proline regulates IGF-1 homeostasis by altering the binding of IGFBP-3 to IGF-1

    Science.gov (United States)

    Guan, Jian; Gluckman, Peter; Yang, Panzao; Krissansen, Geoff; Sun, Xueying; Zhou, Yongzhi; Wen, Jingyuan; Phillips, Gemma; Shorten, Paul R.; McMahon, Chris D.; Wake, Graeme C.; Chan, Wendy H. K.; Thomas, Mark F.; Ren, April; Moon, Steve; Liu, Dong-Xu

    2014-03-01

    The homeostasis of insulin-like growth factor-1 (IGF-1) is essential for metabolism, development and survival. Insufficient IGF-1 is associated with poor recovery from wounds whereas excessive IGF-1 contributes to growth of tumours. We have shown that cyclic glycine-proline (cGP), a metabolite of IGF-1, can normalise IGF-1 function by showing its efficacy in improving the recovery from ischemic brain injury in rats and inhibiting the growth of lymphomic tumours in mice. Further investigation in cell culture suggested that cGP promoted the activity of IGF-1 when it was insufficient, but inhibited the activity of IGF-1 when it was excessive. Mathematical modelling revealed that the efficacy of cGP was a modulated IGF-1 effect via changing the binding of IGF-1 to its binding proteins, which dynamically regulates the balance between bioavailable and non-bioavailable IGF-1. Our data reveal a novel mechanism of auto-regulation of IGF-1, which has physiological and pathophysiological consequences and potential pharmacological utility.

  15. Protein kinase N2 regulates AMP kinase signaling and insulin responsiveness of glucose metabolism in skeletal muscle.

    Science.gov (United States)

    Ruby, Maxwell A; Riedl, Isabelle; Massart, Julie; Åhlin, Marcus; Zierath, Juleen R

    2017-10-01

    Insulin resistance is central to the development of type 2 diabetes and related metabolic disorders. Because skeletal muscle is responsible for the majority of whole body insulin-stimulated glucose uptake, regulation of glucose metabolism in this tissue is of particular importance. Although Rho GTPases and many of their affecters influence skeletal muscle metabolism, there is a paucity of information on the protein kinase N (PKN) family of serine/threonine protein kinases. We investigated the impact of PKN2 on insulin signaling and glucose metabolism in primary human skeletal muscle cells in vitro and mouse tibialis anterior muscle in vivo. PKN2 knockdown in vitro decreased insulin-stimulated glucose uptake, incorporation into glycogen, and oxidation. PKN2 siRNA increased 5'-adenosine monophosphate-activated protein kinase (AMPK) signaling while stimulating fatty acid oxidation and incorporation into triglycerides and decreasing protein synthesis. At the transcriptional level, PKN2 knockdown increased expression of PGC-1α and SREBP-1c and their target genes. In mature skeletal muscle, in vivo PKN2 knockdown decreased glucose uptake and increased AMPK phosphorylation. Thus, PKN2 alters key signaling pathways and transcriptional networks to regulate glucose and lipid metabolism. Identification of PKN2 as a novel regulator of insulin and AMPK signaling may provide an avenue for manipulation of skeletal muscle metabolism. Copyright © 2017 the American Physiological Society.

  16. Identification of a specific assembly of the G protein Golf as a critical and regulated module of dopamine and adenosine-activated cAMP pathways in the striatum

    Directory of Open Access Journals (Sweden)

    Denis eHervé

    2011-08-01

    Full Text Available In the principal neurons of striatum (medium spiny neurons, MSNs, cAMP pathway is primarily activated through the stimulation of dopamine D1 and adenosine A2A receptors, these receptors being mainly expressed in striatonigral and striatopallidal MSNs, respectively. Since cAMP signaling pathway could be altered in various physiological and pathological situations, including drug addiction and Parkinson’s disease, it is of crucial importance to identify the molecular components involved in the activation of this pathway. In MSNs, cAMP pathway activation is not dependent on the classical Gs GTP-binding protein but requires a specific G protein subunit heterotrimer containing Galpha-olf/beta2/gamma7 in particular association with adenylate cyclase type 5. This assembly forms an authentic functional signaling unit since loss of one of its members leads to defects of cAMP pathway activation in response to D1 or A2A receptor stimulation, inducing dramatic impairments of behavioral responses dependent on these receptors. Interestingly, D1 receptor-dependent cAMP signaling is modulated by the neuronal levels of Galpha-olf, indicating that Galpha-olf represents the rate-limiting step in this signaling cascade and could constitute a critical element for regulation of D1 receptor responses. In both Parkinsonian patients and several animal models of Parkinson’s disease, the lesion of dopamine neurons produces a prolonged elevation of Galpha-olf levels. This observation gives an explanation for the cAMP pathway hypersensitivity to D1 stimulation, occurring despite an unaltered D1 receptor density. In conclusion, alterations in the highly specialized assembly of Galpha-olf/beta2/gamma7 subunits can happen in pathological conditions, such as Parkinson’s disease, and it could have important functional consequences in relation to changes in D1 receptor signaling in the striatum.

  17. Synthesis, characterization and inhibitory activities of (4-N3[3,5-3H]Phe10)PKI(6-22)amide and its precursors: photoaffinity labeling peptides for the active site of cyclic AMP-dependent protein kinase.

    Science.gov (United States)

    Katz, B M; Lundquist, L J; Walsh, D A; Glass, D B

    1989-06-01

    PKI(6-22)amide is a 17 residue peptide corresponding to the active portion of the heat-stable inhibitor of cAMP-dependent protein kinase. The peptide is a potent (Ki = 1.6 nM), competitive inhibitor of the enzyme. The photoreactive peptide analog (4-azidophenylalanine10)PKI(6-22)amide was synthesized in both its non-radiolabeled and tritiated forms by chemical modification of precursor peptides that were prepared by stepwise solid-phase synthesis. (4-Amino[3,5-3H]phenylalanine10)PKI(6-22)amide, the precursor for the radiolabeled arylazide peptide, was obtained by catalytic reduction of the corresponding peptide containing the 3,5-diiodo-4-aminophenylalanine residue at position 10. The purified PKI peptides were analyzed by HPLC, amino acid analysis, and u.v. spectra. In the dark, (4-azidophenylalanine10)PKI(6-22)amide inhibited the catalytic subunit of cAMP-dependent protein kinase with a Ki value of 2.8 nM. The photoreactivity of the arylazide peptide was demonstrated by time-dependent u.v. spectral changes on exposure to light. Photolysis of the catalytic subunit (4-azido[3,5-3H]phenylalanine10)PKI(6-22)amide complex resulted in specific covalent labeling of the enzyme. The data indicate that this peptide is a useful photoaffinity labeling reagent for the active site of the protein kinase.

  18. Comparison of thyroid stimulating activities measured by cyclic AMP production, those by radioiodine uptake in FRTL-5 cells and TSH-binding inhibitory activities in patients with hyperthyroid and euthyroid Graves' diseases

    International Nuclear Information System (INIS)

    Kasagi, Kanji; Hatabu, Hiroto; Tokuda, Yasutaka; Arai, Keisuke; Iida, Yasuhiro; Konishi, Junji

    1988-01-01

    By using an assay measuring cAMP production in FRTL-5 thyroid cells, thyroid stimulating antibodies (TSab) were detected in all of 15 patients with euthyroid Graves' disease (EG) and of 26 patients with hyperthyroid Graves' disease (HG). There was no signicant difference between TSab activities in Eg and in HG. In an effort to elucidate why EG patients remain euthyroid in spite of having TSab, we investigated the effect of the patient's crude immunoglobulin fractions 125 I uptake in FRTL-5 thyroid cells, one of the indices of stimulation subsequent to cAMP production. The 125 Iuptake stimulation (IUS) activity was positive in 46,7% (7/15) of EG patients and 88.5% (23/26) of HG patients, being significantly lower in the former than in the latter (P 99m Tc thyroid uptake (r = 0.401, P 99m Tc thyroid uptake in comparison to 19 HG patients with a similar range of IUS activities. There was a good correlation between thyroid weight and 99m Tc thyroid uptake (r = 8.827, P 99m Tc and presumably radioiodine in vivo, might be a factor responsible for keeping EG patients euthyroid despite the presence of TSab. (author)

  19. Biosynthesis of the antimicrobial cyclic lipopeptides nunamycin and nunapeptin by Pseudomonas fluorescens strain In5 is regulated by the LuxR-type transcriptional regulator NunF.

    Science.gov (United States)

    Hennessy, Rosanna C; Phippen, Christopher B W; Nielsen, Kristian F; Olsson, Stefan; Stougaard, Peter

    2017-12-01

    Nunamycin and nunapeptin are two antimicrobial cyclic lipopeptides (CLPs) produced by Pseudomonas fluorescens In5 and synthesized by nonribosomal synthetases (NRPS) located on two gene clusters designated the nun-nup regulon. Organization of the regulon is similar to clusters found in other CLP-producing pseudomonads except for the border regions where putative LuxR-type regulators are located. This study focuses on understanding the regulatory role of the LuxR-type-encoding gene nunF in CLP production of P. fluorescens In5. Functional analysis of nunF coupled with liquid chromatography-high-resolution mass spectrometry (LC-HRMS) showed that CLP biosynthesis is regulated by nunF. Quantitative real-time PCR analysis indicated that transcription of the NRPS genes catalyzing CLP production is strongly reduced when nunF is mutated indicating that nunF is part of the nun-nup regulon. Swarming and biofilm formation was reduced in a nunF knockout mutant suggesting that these CLPs may also play a role in these phenomena as observed in other pseudomonads. Fusion of the nunF promoter region to mCherry showed that nunF is strongly upregulated in response to carbon sources indicating the presence of a fungus suggesting that environmental elicitors may also influence nunF expression which upon activation regulates nunamycin and nunapeptin production required for the growth inhibition of phytopathogens. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  20. AMP-activated protein kinase plays an important evolutionary conserved role in the regulation of glucose metabolism in fish skeletal muscle cells.

    Directory of Open Access Journals (Sweden)

    Leonardo J Magnoni

    Full Text Available AMPK, a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AMPK is activated by changes in the intracellular AMP:ATP ratio when ATP consumption is stimulated by contractile activity but also by AICAR and metformin, compounds that increase glucose transport in mammalian muscle cells. However, the possible role of AMPK in the regulation of glucose metabolism in skeletal muscle has not been investigated in other vertebrates, including fish. In this study, we investigated the effects of AMPK activators on glucose uptake, AMPK activity, cell surface levels of trout GLUT4 and expression of GLUT1 and GLUT4 as well as the expression of enzymes regulating glucose disposal and PGC1α in trout myotubes derived from a primary muscle cell culture. We show that AICAR and metformin significantly stimulated glucose uptake (1.6 and 1.3 fold, respectively and that Compound C completely abrogated the stimulatory effects of the AMPK activators on glucose uptake. The combination of insulin and AMPK activators did not result in additive nor synergistic effects on glucose uptake. Moreover, exposure of trout myotubes to AICAR and metformin resulted in an increase in AMPK activity (3.8 and 3 fold, respectively. We also provide evidence suggesting that stimulation of glucose uptake by AMPK activators in trout myotubes may take place, at least in part, by increasing the cell surface and mRNA levels of trout GLUT4. Finally, AICAR increased the mRNA levels of genes involved in glucose disposal (hexokinase, 6-phosphofructokinase, pyruvate kinase and citrate synthase and mitochondrial biogenesis (PGC-1α and did not affect glycogen content or glycogen synthase mRNA levels in trout myotubes. Therefore, we provide evidence, for the first time in non-mammalian vertebrates, suggesting a potentially important role of AMPK in stimulating glucose uptake and utilization in the skeletal muscle of fish.

  1. Regulation of P450-mediated permethrin resistance in Culex quinquefasciatus by the GPCR/Gαs/AC/cAMP/PKA signaling cascade.

    Science.gov (United States)

    Li, Ting; Liu, Nannan

    2017-12-01

    This study explores the role of G-protein-coupled receptor-intracellular signaling in the development of P450-mediated insecticide resistance in mosquitoes, Culex quinquefasciatus , focusing on the essential function of the GPCRs and their downstream effectors of Gs alpha subunit protein (Gαs) and adenylyl cyclase (ACs) in P450-mediated insecticide resistance of Culex mosquitoes. Our RNAi-mediated functional study showed that knockdown of Gαs caused the decreased expression of the downstream effectors of ACs and PKAs in the GPCR signaling pathway and resistance P450 genes, whereas knockdown of ACs decreased the expression of PKAs and resistance P450 genes. Knockdown of either Gαs or ACs resulted in an increased susceptibility of mosquitoes to permethrin. These results add significantly to our understanding of the molecular basis of resistance P450 gene regulation through GPCR/Gαs/AC/cAMP-PKA signaling pathways in the insecticide resistance of mosquitoes. The temporal and spatial dynamic analyses of GPCRs, Gαs, ACs, PKAs, and P450s in two insecticide resistant mosquito strains revealed that all the GPCR signaling pathway components tested, namely GPCRs, Gαs, ACs and PKAs, were most highly expressed in the brain for both resistant strains, suggesting the role played by these genes in signaling transduction and regulation. The resistance P450 genes were mainly expressed in the brain, midgut and malpighian tubules (MTs), suggesting their critical function in the central nervous system and importance for detoxification. The temporal dynamics analysis for the gene expression showed a diverse expression profile during mosquito development, indicating their initially functional importance in response to exposure to insecticides during their life stages.

  2. Ga and Gß Proteins Regulate the Cyclic AMP Pathway That Is Required for Development and Pathogenicity of the Phytopathogen Mycosphaerella graminicola

    NARCIS (Netherlands)

    Mehrabi, A.; M'Barek, Ben S.; Lee, van der T.A.J.; Waalwijk, C.; Wit, de P.J.G.M.; Kema, G.H.J.

    2009-01-01

    We identified and functionally characterized genes encoding three G alpha proteins and one G beta protein in the dimorphic fungal wheat pathogen Mycosphaerella graminicola, which we designated MgGpa1, MgGpa2, MgGpa3, and MgGpb1, respectively. Sequence comparisons and phylogenetic analyses showed

  3. Cyclic Nucleotide Signalling in Kidney Fibrosis

    Directory of Open Access Journals (Sweden)

    Elisabeth Schinner

    2015-01-01

    Full Text Available Kidney fibrosis is an important factor for the progression of kidney diseases, e.g., diabetes mellitus induced kidney failure, glomerulosclerosis and nephritis resulting in chronic kidney disease or end-stage renal disease. Cyclic adenosine monophosphate (cAMP and cyclic guanosine monophosphate (cGMP were implicated to suppress several of the above mentioned renal diseases. In this review article, identified effects and mechanisms of cGMP and cAMP regarding renal fibrosis are summarized. These mechanisms include several signalling pathways of nitric oxide/ANP/guanylyl cyclases/cGMP-dependent protein kinase and cAMP/Epac/adenylyl cyclases/cAMP-dependent protein kinase. Furthermore, diverse possible drugs activating these pathways are discussed. From these diverse mechanisms it is expected that new pharmacological treatments will evolve for the therapy or even prevention of kidney failure.

  4. The Bacterial Second Messenger Cyclic di-GMP Regulates Brucella Pathogenesis and Leads to Altered Host Immune Response.

    Science.gov (United States)

    Khan, Mike; Harms, Jerome S; Marim, Fernanda M; Armon, Leah; Hall, Cherisse L; Liu, Yi-Ping; Banai, Menachem; Oliveira, Sergio C; Splitter, Gary A; Smith, Judith A

    2016-12-01

    Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host-Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a ΔbpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the ΔbpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase ΔcgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, ΔbpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  5. Flavonoid Regulation of HCN2 Channels*

    Science.gov (United States)

    Carlson, Anne E.; Rosenbaum, Joel C.; Brelidze, Tinatin I.; Klevit, Rachel E.; Zagotta, William N.

    2013-01-01

    The hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are pacemaker channels whose currents contribute to rhythmic activity in the heart and brain. HCN channels open in response to hyperpolarizing voltages, and the binding of cAMP to their cyclic nucleotide-binding domain (CNBD) facilitates channel opening. Here, we report that, like cAMP, the flavonoid fisetin potentiates HCN2 channel gating. Fisetin sped HCN2 activation and shifted the conductance-voltage relationship to more depolarizing potentials with a half-maximal effective concentration (EC50) of 1.8 μm. When applied together, fisetin and cAMP regulated HCN2 gating in a nonadditive fashion. Fisetin did not potentiate HCN2 channels lacking their CNBD, and two independent fluorescence-based binding assays reported that fisetin bound to the purified CNBD. These data suggest that the CNBD mediates the fisetin potentiation of HCN2 channels. Moreover, binding assays suggest that fisetin and cAMP partially compete for binding to the CNBD. NMR experiments demonstrated that fisetin binds within the cAMP-binding pocket, interacting with some of the same residues as cAMP. Together, these data indicate that fisetin is a partial agonist for HCN2 channels. PMID:24085296

  6. MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish

    DEFF Research Database (Denmark)

    Lundegaard, Pia R.; Anastasaki, Corina; Grant, Nicola J.

    2015-01-01

    Altered phosphodiesterase (PDE)-cyclic AMP (cAMP) activity is frequently associated with anxiety disorders, but current therapies act by reducing neuronal excitability rather than targeting PDE-cAMP-mediated signaling pathways. Here, we report the novel repositioning of anti-cancer MEK inhibitors...... as anxiolytics in a zebrafish model of anxiety-like behaviors. PDE inhibitors or activators of adenylate cyclase cause behaviors consistent with anxiety in larvae and adult zebrafish. Small-molecule screening identifies MEK inhibitors as potent suppressors of cAMP anxiety behaviors in both larvae and adult...... zebrafish, while causing no anxiolytic behavioral effects on their own. The mechanism underlying cAMP-induced anxiety is via crosstalk to activation of the RAS-MAPK signaling pathway. We propose that targeting crosstalk signaling pathways can be an effective strategy for mental health disorders, and advance...

  7. Piperidine alkaloids from Piperretrofractum Vahl. protect against high-fat diet-induced obesity by regulating lipid metabolism and activating AMP-activated protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kyung Jin [Department of Biomaterials Science and Engineering, Yonsei University, Seoul 120-749 (Korea, Republic of); Lee, Myoung-Su; Jo, Keunae [Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Hwang, Jae-Kwan, E-mail: jkhwang@yonsei.ac.kr [Department of Biomaterials Science and Engineering, Yonsei University, Seoul 120-749 (Korea, Republic of); Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Translational Research Center for Protein Functional Control, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2011-07-22

    Highlights: {yields} Piperidine alkaloids from Piperretrofractum Vahl. (PRPAs), including piperine, pipernonaline, and dehydropipernonaline, are isolated as the anti-obesity constituents. {yields} PRPA administration significantly reduces body weight gain without altering food intake and fat pad mass. {yields} PRPA reduces high-fat diet-induced triglyceride accumulation in liver. {yields} PRPAs attenuate HFD-induced obesity by activating AMPK and PPAR{delta}, and regulate lipid metabolism, suggesting their potential anti-obesity effects. -- Abstract: The fruits of Piperretrofractum Vahl. have been used for their anti-flatulent, expectorant, antitussive, antifungal, and appetizing properties in traditional medicine, and they are reported to possess gastroprotective and cholesterol-lowering properties. However, their anti-obesity activity remains unexplored. The present study was conducted to isolate the anti-obesity constituents from P. retrofractum Vahl. and evaluate their effects in high-fat diet (HFD)-induced obese mice. Piperidine alkaloids from P. retrofractum Vahl. (PRPAs), including piperine, pipernonaline, and dehydropipernonaline, were isolated as the anti-obesity constituents through a peroxisome proliferator-activated receptor {delta} (PPAR{delta}) transactivation assay. The molecular mechanism was investigated in 3T3-L1 adipocytes and L6 myocytes. PRPA treatment activated AMP-activated protein kinase (AMPK) signaling and PPAR{delta} protein and also regulated the expression of lipid metabolism-related proteins. In the animal model, oral PRPA administration (50, 100, or 300 mg/kg/day for 8 weeks) significantly reduced HFD-induced body weight gain without altering the amount of food intake. Fat pad mass was reduced in the PRPA treatment groups, as evidenced by reduced adipocyte size. In addition, elevated serum levels of total cholesterol, low-density lipoprotein cholesterol, total lipid, leptin, and lipase were suppressed by PRPA treatment. PRPA also

  8. Piperidine alkaloids from Piperretrofractum Vahl. protect against high-fat diet-induced obesity by regulating lipid metabolism and activating AMP-activated protein kinase

    International Nuclear Information System (INIS)

    Kim, Kyung Jin; Lee, Myoung-Su; Jo, Keunae; Hwang, Jae-Kwan

    2011-01-01

    Highlights: → Piperidine alkaloids from Piperretrofractum Vahl. (PRPAs), including piperine, pipernonaline, and dehydropipernonaline, are isolated as the anti-obesity constituents. → PRPA administration significantly reduces body weight gain without altering food intake and fat pad mass. → PRPA reduces high-fat diet-induced triglyceride accumulation in liver. → PRPAs attenuate HFD-induced obesity by activating AMPK and PPARδ, and regulate lipid metabolism, suggesting their potential anti-obesity effects. -- Abstract: The fruits of Piperretrofractum Vahl. have been used for their anti-flatulent, expectorant, antitussive, antifungal, and appetizing properties in traditional medicine, and they are reported to possess gastroprotective and cholesterol-lowering properties. However, their anti-obesity activity remains unexplored. The present study was conducted to isolate the anti-obesity constituents from P. retrofractum Vahl. and evaluate their effects in high-fat diet (HFD)-induced obese mice. Piperidine alkaloids from P. retrofractum Vahl. (PRPAs), including piperine, pipernonaline, and dehydropipernonaline, were isolated as the anti-obesity constituents through a peroxisome proliferator-activated receptor δ (PPARδ) transactivation assay. The molecular mechanism was investigated in 3T3-L1 adipocytes and L6 myocytes. PRPA treatment activated AMP-activated protein kinase (AMPK) signaling and PPARδ protein and also regulated the expression of lipid metabolism-related proteins. In the animal model, oral PRPA administration (50, 100, or 300 mg/kg/day for 8 weeks) significantly reduced HFD-induced body weight gain without altering the amount of food intake. Fat pad mass was reduced in the PRPA treatment groups, as evidenced by reduced adipocyte size. In addition, elevated serum levels of total cholesterol, low-density lipoprotein cholesterol, total lipid, leptin, and lipase were suppressed by PRPA treatment. PRPA also protected against the development of

  9. Aspirin-induced AMP-activated protein kinase activation regulates the proliferation of vascular smooth muscle cells from spontaneously hypertensive rats

    International Nuclear Information System (INIS)

    Sung, Jin Young; Choi, Hyoung Chul

    2011-01-01

    Highlights: → Aspirin-induced AMPK phosphorylation was greater in VSMC from SHR than WKY. → Aspirin-induced AMPK phosphorylation inhibited proliferation of VSMC from SHR. → Low basal AMPK phosphorylation in SHR elicits increased VSMC proliferation. → Inhibition of AMPK restored decreased VSMC proliferation by aspirin in SHR. → Aspirin exerts anti-proliferative effect through AMPK activation in VSMC from SHR. -- Abstract: Acetylsalicylic acid (aspirin), used to reduce risk of cardiovascular disease, plays an important role in the regulation of cellular proliferation. However, mechanisms responsible for aspirin-induced growth inhibition are not fully understood. Here, we investigated whether aspirin may exert therapeutic effects via AMP-activated protein kinase (AMPK) activation in vascular smooth muscle cells (VSMC) from wistar kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Aspirin increased AMPK and acetyl-CoA carboxylase phosphorylation in a time- and dose-dependent manner in VSMCs from WKY and SHR, but with greater efficacy in SHR. In SHR, a low basal phosphorylation status of AMPK resulted in increased VSMC proliferation and aspirin-induced AMPK phosphorylation inhibited proliferation of VSMCs. Compound C, an AMPK inhibitor, and AMPK siRNA reduced the aspirin-mediated inhibition of VSMC proliferation, this effect was more pronounced in SHR than in WKY. In VSMCs from SHR, aspirin increased p53 and p21 expression and inhibited the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. These results indicate that in SHR VSMCs aspirin exerts anti-proliferative effects through the induction of AMPK phosphorylation.

  10. Aspirin-induced AMP-activated protein kinase activation regulates the proliferation of vascular smooth muscle cells from spontaneously hypertensive rats

    Energy Technology Data Exchange (ETDEWEB)

    Sung, Jin Young [Department of Pharmacology, College of Medicine, Yeungnam University, Daegu 705-717 (Korea, Republic of); Choi, Hyoung Chul, E-mail: hcchoi@med.yu.ac.kr [Department of Pharmacology, College of Medicine, Yeungnam University, Daegu 705-717 (Korea, Republic of)

    2011-05-06

    Highlights: {yields} Aspirin-induced AMPK phosphorylation was greater in VSMC from SHR than WKY. {yields} Aspirin-induced AMPK phosphorylation inhibited proliferation of VSMC from SHR. {yields} Low basal AMPK phosphorylation in SHR elicits increased VSMC proliferation. {yields} Inhibition of AMPK restored decreased VSMC proliferation by aspirin in SHR. {yields} Aspirin exerts anti-proliferative effect through AMPK activation in VSMC from SHR. -- Abstract: Acetylsalicylic acid (aspirin), used to reduce risk of cardiovascular disease, plays an important role in the regulation of cellular proliferation. However, mechanisms responsible for aspirin-induced growth inhibition are not fully understood. Here, we investigated whether aspirin may exert therapeutic effects via AMP-activated protein kinase (AMPK) activation in vascular smooth muscle cells (VSMC) from wistar kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Aspirin increased AMPK and acetyl-CoA carboxylase phosphorylation in a time- and dose-dependent manner in VSMCs from WKY and SHR, but with greater efficacy in SHR. In SHR, a low basal phosphorylation status of AMPK resulted in increased VSMC proliferation and aspirin-induced AMPK phosphorylation inhibited proliferation of VSMCs. Compound C, an AMPK inhibitor, and AMPK siRNA reduced the aspirin-mediated inhibition of VSMC proliferation, this effect was more pronounced in SHR than in WKY. In VSMCs from SHR, aspirin increased p53 and p21 expression and inhibited the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. These results indicate that in SHR VSMCs aspirin exerts anti-proliferative effects through the induction of AMPK phosphorylation.

  11. The importance of regulation of blood glucose levels through activation of peripheral 5'-AMP-activated protein kinase on ischemic neuronal damage.

    Science.gov (United States)

    Harada, Shinichi; Fujita-Hamabe, Wakako; Tokuyama, Shogo

    2010-09-10

    5'-AMP-activated protein kinase (AMPK) is a serine/threonine kinase that plays a key role in energy homeostasis. Recently, it was reported that centrally activated AMPK is involved in the development of ischemic neuronal damage, while the effect of peripherally activated AMPK on ischemic neuronal damage is not known. In addition, we have previously reported that the development of post-ischemic glucose intolerance could be one of the triggers for the aggravation of neuronal damage. In this study, we focused on effect of activation of peripheral or central AMPK on the development of ischemic neuronal damage. Male ddY mice were subjected to 2 h of middle cerebral artery occlusion (MCAO). Neuronal damage was estimated by histological and behavioral analysis after MCAO. In the liver and skeletal muscle, AMPK activity was not affected by MCAO. But, application of intraperitoneal metformin (250 mg/kg), an AMPK activator, significantly suppressed the development of post-ischemic glucose intolerance and ischemic neuronal damage without alteration of central AMPK activity. On the other hand, application of intracerebroventricular metformin (25, 100 microg/mouse) significantly exacerbated the development of neuronal damage observed on day 1 after MCAO, in a dose-dependent manner. These effects were significantly blocked by compound C, a specific AMPK inhibitor. These results suggest that central AMPK was activated by ischemic stress per se, however, peripheral AMPK was not altered. Furthermore, the regulation of post-ischemic glucose intolerance by activation of peripheral AMPK is of assistance for the suppression of cerebral ischemic neuronal damage. 2010 Elsevier B.V. All rights reserved.

  12. The Popeye Domain Containing Genes and cAMP Signaling

    Directory of Open Access Journals (Sweden)

    Thomas Brand

    2014-05-01

    Full Text Available 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.

  13. Improvements in the automated radioimmunoassay for cAMP or cGMP

    International Nuclear Information System (INIS)

    Brooker, G.

    1988-01-01

    The work others in developing antibodies and the original radioimmunoassay for cyclic nucleotides provides the basis for these sensitive assays. The acetylation radioimmunoassay for cyclic nucleotides has enabled the measurement of cyclic AMP and cyclic GMP in very small biological samples. This is because accurate determinations can be made in samples containing less than 1 fmol of cyclic AMP or cyclic GMP. The Gamma-Flo automated radioimmunoassay system has been adapted to these assays such that cyclic nucleotides can be automatically measured at a rate of about 60 samples/hr. The Gamma-Flo instrument provides high-precision assays and eliminates human intervention in all steps of the radioimmunoassay. The automated assay has been in continuous operation in our laboratory over the last 10 years and this chapter summarizes the methodology and delineates improvements which have occurred over that time frame. Details for the preparation of the radioligands apply also to the manual acetylated radioimmunoassay for cyclic nucleotides

  14. Novel Radioligands for Cyclic Nucleotide Phosphodiesterase Imaging with Positron Emission Tomography: An Update on Developments Since 2012

    Directory of Open Access Journals (Sweden)

    Susann Schröder

    2016-05-01

    Full Text Available Cyclic nucleotide phosphodiesterases (PDEs are a class of intracellular enzymes that inactivate the secondary messenger molecules, cyclic adenosine monophosphate (cAMP and cyclic guanosine monophosphate (cGMP. Thus, PDEs regulate the signaling cascades mediated by these cyclic nucleotides and affect fundamental intracellular processes. Pharmacological inhibition of PDE activity is a promising strategy for treatment of several diseases. However, the role of the different PDEs in related pathologies is not completely clarified yet. PDE-specific radioligands enable non-invasive visualization and quantification of these enzymes by positron emission tomography (PET in vivo and provide an important translational tool for elucidation of the relationship between altered expression of PDEs and pathophysiological effects as well as (pre-clinical evaluation of novel PDE inhibitors developed as therapeutics. Herein we present an overview of novel PDE radioligands for PET published since 2012.

  15. DAF-16/FoxO directly regulates an atypical AMP-activated protein kinase gamma isoform to mediate the effects of insulin/IGF-1 signaling on aging in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Jennifer M A Tullet

    2014-02-01

    Full Text Available The DAF-16/FoxO transcription factor controls growth, metabolism and aging in Caenorhabditis elegans. The large number of genes that it regulates has been an obstacle to understanding its function. However, recent analysis of transcript and chromatin profiling implies that DAF-16 regulates relatively few genes directly, and that many of these encode other regulatory proteins. We have investigated the regulation by DAF-16 of genes encoding the AMP-activated protein kinase (AMPK, which has α, β and γ subunits. C. elegans has 5 genes encoding putative AMP-binding regulatory γ subunits, aakg-1-5. aakg-4 and aakg-5 are closely related, atypical isoforms, with orthologs throughout the Chromadorea class of nematodes. We report that ∼75% of total γ subunit mRNA encodes these 2 divergent isoforms, which lack consensus AMP-binding residues, suggesting AMP-independent kinase activity. DAF-16 directly activates expression of aakg-4, reduction of which suppresses longevity in daf-2 insulin/IGF-1 receptor mutants. This implies that an increase in the activity of AMPK containing the AAKG-4 γ subunit caused by direct activation by DAF-16 slows aging in daf-2 mutants. Knock down of aakg-4 expression caused a transient decrease in activation of expression in multiple DAF-16 target genes. This, taken together with previous evidence that AMPK promotes DAF-16 activity, implies the action of these two metabolic regulators in a positive feedback loop that accelerates the induction of DAF-16 target gene expression. The AMPK β subunit, aakb-1, also proved to be up-regulated by DAF-16, but had no effect on lifespan. These findings reveal key features of the architecture of the gene-regulatory network centered on DAF-16, and raise the possibility that activation of AMP-independent AMPK in nutritionally replete daf-2 mutant adults slows aging in C. elegans. Evidence of activation of AMPK subunits in mammals suggests that such FoxO-AMPK interactions may be

  16. DAF-16/FoxO directly regulates an atypical AMP-activated protein kinase gamma isoform to mediate the effects of insulin/IGF-1 signaling on aging in Caenorhabditis elegans.

    Science.gov (United States)

    Tullet, Jennifer M A; Araiz, Caroline; Sanders, Matthew J; Au, Catherine; Benedetto, Alexandre; Papatheodorou, Irene; Clark, Emily; Schmeisser, Kathrin; Jones, Daniel; Schuster, Eugene F; Thornton, Janet M; Gems, David

    2014-02-01

    The DAF-16/FoxO transcription factor controls growth, metabolism and aging in Caenorhabditis elegans. The large number of genes that it regulates has been an obstacle to understanding its function. However, recent analysis of transcript and chromatin profiling implies that DAF-16 regulates relatively few genes directly, and that many of these encode other regulatory proteins. We have investigated the regulation by DAF-16 of genes encoding the AMP-activated protein kinase (AMPK), which has α, β and γ subunits. C. elegans has 5 genes encoding putative AMP-binding regulatory γ subunits, aakg-1-5. aakg-4 and aakg-5 are closely related, atypical isoforms, with orthologs throughout the Chromadorea class of nematodes. We report that ∼75% of total γ subunit mRNA encodes these 2 divergent isoforms, which lack consensus AMP-binding residues, suggesting AMP-independent kinase activity. DAF-16 directly activates expression of aakg-4, reduction of which suppresses longevity in daf-2 insulin/IGF-1 receptor mutants. This implies that an increase in the activity of AMPK containing the AAKG-4 γ subunit caused by direct activation by DAF-16 slows aging in daf-2 mutants. Knock down of aakg-4 expression caused a transient decrease in activation of expression in multiple DAF-16 target genes. This, taken together with previous evidence that AMPK promotes DAF-16 activity, implies the action of these two metabolic regulators in a positive feedback loop that accelerates the induction of DAF-16 target gene expression. The AMPK β subunit, aakb-1, also proved to be up-regulated by DAF-16, but had no effect on lifespan. These findings reveal key features of the architecture of the gene-regulatory network centered on DAF-16, and raise the possibility that activation of AMP-independent AMPK in nutritionally replete daf-2 mutant adults slows aging in C. elegans. Evidence of activation of AMPK subunits in mammals suggests that such FoxO-AMPK interactions may be evolutionarily conserved.

  17. Study on the change of cyclic nucleotide in mice with yang vacuity disease

    International Nuclear Information System (INIS)

    Zhu Xinhua; Shen Ling; Wang Shuguang

    2002-01-01

    To study the relation between Yang Vacuity disease happening, development and cyclic nucleotide response, and prove curative effects of some assisting Yang drug, the plasma cAMP, cGMP and cAMP/cGMP levels were detected by radioimmunoassay in the Yang Vacuity group and curing group. Results: showed: (1) Yang Vacuity group: the symptoms were clear, death rate was high, the plasma cAMP and cAMP/cGMP increased obviously, it suggests that cyclic nucleotide was imbalance. (2) Curing group: the symptoms of Yang Vacuity disease were improved obviously, death rate dropped, cAMP declined, cGMP increased, while cAMP/cGMP reached the normal level, it showed that cyclic nucleotide of the body had altered greatly. (3) It is a reference target for Yang Vacuity. (4) Assisting yang drug (Sini Decoction) had a close relation with correcting imbalance of cyclic nucleotide

  18. Regulation of the MAP kinase cascade in PC12 cells: B-Raf activates MEK-1 (MAP kinase or ERK kinase) and is inhibited by cAMP

    DEFF Research Database (Denmark)

    Peraldi, P; Frödin, M; Barnier, J V

    1995-01-01

    AMP inhibits B-Raf autokinase activity as well as its ability to phosphorylate and activate MEK-1. This inhibition is likely to be due to a direct effect since we found that PKA phosphorylates B-Raf in vitro. Further, we show that B-Raf binds to p21ras, but more important, this binding to p21ras is virtually...... abolished with B-Raf from PC12 cells treated with CPT-cAMP. Hence, these data indicate that the PKA-mediated phosphorylation of B-Raf hampers its interaction with p21ras, which is responsible for the PKA-mediated decrease in B-Raf activity. Finally, our work suggests that in PC12 cells, cAMP stimulates MAP...

  19. Modulation of phenotype and function of human CD4+CD25+ T regulatory lymphocytes mediated by cAMP elevating agents

    Directory of Open Access Journals (Sweden)

    Antonella Riccomi

    2016-09-01

    Full Text Available We have shown that Cholera Toxin (CT and other cyclic AMP (cAMP elevating agents induce up-regulation of the inhibitory molecule CTLA-4 in human resting CD4+ T lymphocytes, which following the treatment acquired suppressive functions. In this study, we evaluated the effect of cAMP elevating agents on human CD4+CD25+ T cells, which include the T regulatory (Treg cells that play a pivotal role in the maintenance of immunological tolerance. We found that cAMP elevating agents induce up-regulation of CTLA-4 in CD4+CD25- and further enhance its expression in CD4+CD25+ T cells. We observed an increase of two isoforms of mRNA coding for the membrane and the soluble CTLA-4 molecules, suggesting that the regulation of CTLA-4 expression by cAMP is at the transcriptional level. In addition, we found that the increase of cAMP in CD4+CD25+ T cells converts the CD4+CD25+Foxp3- T cells in CD4+CD25+Foxp3+ T cells, whereas the increase of cAMP in CD4+CD25- T cells did not up-regulate Foxp3 in the absence of activation stimuli. To investigate the function of these cells, we performed an in vitro suppression assay by culturing CD4+CD25+ T cells untreated or pre-treated with CT with anti-CD3 mAbs-stimulated autologous PBMC. We found that CT enhances the inhibitory function of CD4+CD25+ T cells, CD4+ and CD8+ T cell proliferation and IFNγ production are strongly inhibited by CD4+CD25+ T cells pre-treated with cAMP elevating agents. Furthermore, we found that CD4+CD25+ T lymphocytes pre-treated with cAMP elevating agents induce the up-regulation of CD80 and CD86 co-stimulatory molecules on immature dendritic cells (DCs in the absence of antigenic stimulation, however without leading to full DC maturation. These data show that the increase of intracellular cAMP modulates the phenotype and function of human CD4+CD25+ T cells.

  20. Cyclic adenosine 3:5-monophosphate binding proteins in Hartmannella culbertsoni

    International Nuclear Information System (INIS)

    Verma, A.K.; Krishna Murti, C.R.

    1976-01-01

    When 100, 000 g supernatant fractions of homogenates of Hartmannella culbertsoni were incubated with ('- 3 H)-cyclic adenosine 3 : 5 monophosphate and passed through a sephadex G-100 column, radioactivity appeared with protein fractions eluted after the void colume. About 75% radioactivity bound to these fractions was recovered as cyclic adenosine 3 : 5 monophosphate. Unlabelled cAMP diluted the amount of radioactivity bound. Adenosine, deoxyadenosine, 5-AMP, 3-AMP, ADP and ATP did not inhibit binding. (author)

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

    Science.gov (United States)

    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...

  2. Peroxisome proliferator-activated receptor delta (PPARdelta )-mediated regulation of preadipocyte proliferation and gene expression is dependent on cAMP signaling

    DEFF Research Database (Denmark)

    Hansen, Jacob B.; Zhang, H; Rasmussen, T H

    2001-01-01

    of a PPARdelta ligand and methylisobutylxanthine (MIX) or other cAMP elevating agents. We further show that ligands and MIX synergistically stimulated PPARdelta-mediated transactivation. In 3T3-L1 preadipocytes, simultaneous administration of a PPARdelta-selective ligand and MIX significantly enhanced the early...

  3. AMP activated protein kinase α2 controls substrate metabolism during post-exercise recovery via regulation of pyruvate dehydrogenase kinase 4

    DEFF Research Database (Denmark)

    Fritzen, Andreas Mæchel; Lundsgaard, Annemarie; Jeppesen, Jacob

    2015-01-01

    after prolonged exercise and during the following six hours post exercise in 5´AMP activated protein kinase (AMPK)α2 and α1 knock-out (KO) and wild type (WT) mice with free access to food. Substrate oxidation was similar during exercise at the same relative intensity between genotypes. During post...

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

    Science.gov (United States)

    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…

  5. The cAMP Signaling and MAP Kinase Pathways in Plant Pathogenic Fungi

    NARCIS (Netherlands)

    Mehrabi, R.; Zhao, X.; Kim, Y.; Xu, J.R.

    2009-01-01

    The key components of the well conserved cyclic AMP signaling and MAP kinase pathways have been functionally characterized in the corn smut Ustilago maydis, rice blast fungus Magnaporthe grisea, and a few other fungal pathogens. In general, the cAMP signaling and the MAP kinase cascade homologous to

  6. Linderane Suppresses Hepatic Gluconeogenesis by Inhibiting the cAMP/PKA/CREB Pathway Through Indirect Activation of PDE 3 via ERK/STAT3

    Directory of Open Access Journals (Sweden)

    Wei Xie

    2018-05-01

    Full Text Available The role of phosphodiesterase 3 (PDE3, a cyclic AMP (cAMP-degrading enzyme, in modulating gluconeogenesis remains unknown. Here, linderane, a natural compound, was found to inhibit gluconeogenesis by activating hepatic PDE3 in rat primary hepatocytes. The underlying molecular mechanism and its effects on whole-body glucose and lipid metabolism were investigated. The effect of linderane on gluconeogenesis, cAMP content, phosphorylation of cAMP-response element-binding protein (CREB and PDE activity were examined in cultured primary hepatocytes and C57BL/6J mice. The precise mechanism by which linderane activates PDE3 and inhibits the cAMP pathway was explored using pharmacological inhibitors. The amelioration of metabolic disorders was observed in ob/ob mice. Linderane inhibited gluconeogenesis, reduced phosphoenolpyruvate carboxykinase (Pck1 and glucose-6-phosphatase (G6pc gene expression, and decreased intracellular cAMP concentration and CREB phosphorylation in rat primary hepatocytes under both basal and forskolin-stimulated conditions. In rat primary hepatocytes, it also increased total PDE and PDE3 activity but not PDE4 activity. The suppressive effect of linderane on the cAMP pathway and gluconeogenesis was abolished by the non-specific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX and the specific PDE3 inhibitor cilostazol. Linderane indirectly activated PDE3 through extracellular regulated protein kinase 1/2 (ERK1/2 and signal transducer and activator of transcription 3 (STAT3 activation. Linderane improved glucose and lipid metabolism after chronic oral administration in ob/ob mice. Our findings revealed linderane as an indirect PDE3 activator that suppresses gluconeogenesis through cAMP pathway inhibition and has beneficial effects on metabolic syndromes in ob/ob mice. This investigation highlighted the potential for PDE3 activation in the treatment of type 2 diabetes.

  7. Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs) Effects on AMP-Activated Protein Kinase (AMPK) Regulation of Chicken Sperm Functions.

    Science.gov (United States)

    Nguyen, Thi Mong Diep; Combarnous, Yves; Praud, Christophe; Duittoz, Anne; Blesbois, Elisabeth

    2016-01-01

    Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca(2+), or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca(2+) but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca(2+) than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca(2+). Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca(2+) entry in sperm through the Ca(2+)/CaM/CaMKKs/CaMKI pathway. The Ca(2+)/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca(2+) entry

  8. Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs Effects on AMP-Activated Protein Kinase (AMPK Regulation of Chicken Sperm Functions.

    Directory of Open Access Journals (Sweden)

    Thi Mong Diep Nguyen

    Full Text Available Sperm require high levels of energy to ensure motility and acrosome reaction (AR accomplishment. The AMP-activated protein kinase (AMPK has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca(2+/calmodulin-dependent protein kinase kinases (CaMKKs mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca(2+, or of CaMKKs inhibitor (STO-609. Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β, CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca(2+ but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca(2+ than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca(2+. Our results show for the first time the presence of CaMKKs (α and β and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca(2+ entry in sperm through the Ca(2+/CaM/CaMKKs/CaMKI pathway. The Ca(2+/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca(2

  9. CRIS-a novel cAMP-binding protein controlling spermiogenesis and the development of flagellar bending.

    Directory of Open Access Journals (Sweden)

    Anke Miriam Krähling

    Full Text Available The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD. Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca(2+ regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca(2+-regulated proteins that--in mature sperm--are involved in flagellar bending.

  10. Assisted Medical Procedures (AMP)

    Data.gov (United States)

    National Aeronautics and Space Administration — DOCUMENTATION, DEVELOPMENT, AND PROGRESS The AMP was initially being developed as part the Advanced Integrated Clinical System (AICS)-Guided Medical Procedure System...

  11. Antimicrobial Peptides (AMPs

    Directory of Open Access Journals (Sweden)

    Mehrzad Sadredinamin

    2016-04-01

    Full Text Available Antimicrobial peptides (AMPs are extensive group of molecules that produced by variety tissues of invertebrate, plants, and animal species which play an important role in their immunity response. AMPs have different classifications such as; biosynthetic machines, biological sources, biological functions, molecular properties, covalent bonding patterns, three dimensional structures, and molecular targets.These molecules have multidimensional properties including antimicrobial activity, antiviral activity, antifungal activity, anti-parasite activity, biofilm control, antitumor activity, mitogens activity and linking innate to adaptive immunity that making them promising agents for therapeutic drugs. In spite of this advantage of AMPs, their clinical developments have some limitation for commercial development. But some of AMPs are under clinical trials for the therapeutic purpose such as diabetic foot ulcers, different bacterial infections and tissue damage. In this review, we emphasized on the source, structure, multidimensional properties, limitation and therapeutic applications of various antimicrobial peptides.

  12. An Improved Targeted cAMP Sensor to Study the Regulation of Adenylyl Cyclase 8 by Ca2+ Entry through Voltage-Gated Channels

    Science.gov (United States)

    Everett, Katy L.; Cooper, Dermot M. F.

    2013-01-01

    Here we describe an improved sensor with reduced pH sensitivity tethered to adenylyl cyclase (AC) 8. The sensor was used to study cAMP dynamics in the AC8 microdomain of MIN6 cells, a pancreatic β-cell line. In these cells, AC8 was activated by Ca2+ entry through L-type voltage-gated channels following depolarisation. This activation could be reconstituted in HEK293 cells co-expressing AC8 and either the α1C or α1D subunit of L-type voltage-gated Ca2+ channels. The development of this improved sensor opens the door to the study of cAMP microdomains in excitable cells that have previously been challenging due to the sensitivity of fluorescent proteins to pH changes. PMID:24086669

  13. An improved targeted cAMP sensor to study the regulation of adenylyl cyclase 8 by Ca2+ entry through voltage-gated channels.

    Directory of Open Access Journals (Sweden)

    Katy L Everett

    Full Text Available Here we describe an improved sensor with reduced pH sensitivity tethered to adenylyl cyclase (AC 8. The sensor was used to study cAMP dynamics in the AC8 microdomain of MIN6 cells, a pancreatic β-cell line. In these cells, AC8 was activated by Ca(2+ entry through L-type voltage-gated channels following depolarisation. This activation could be reconstituted in HEK293 cells co-expressing AC8 and either the α1C or α1D subunit of L-type voltage-gated Ca(2+ channels. The development of this improved sensor opens the door to the study of cAMP microdomains in excitable cells that have previously been challenging due to the sensitivity of fluorescent proteins to pH changes.

  14. The solvent effect on the enthalpy of hydrolysis of cyclic adenosine 3',5'-monophosphate : a quantum chemical study

    NARCIS (Netherlands)

    Scheffers - Sap, Miek; Buck, H.M.

    1978-01-01

    The solvent effect on the enthalpy of hydrolysis has been studied by the Extended-Hückel method for the hydrolysis reactions of cyclic adenosine 3',5'-monophosphate (cyclic 3',5'-AMP) and related cyclic phosphate diesters. The results show that the difference in enthalpy of hydrolysis between cyclic

  15. Axon Regeneration Is Regulated by Ets-C/EBP Transcription Complexes Generated by Activation of the cAMP/Ca2+ Signaling Pathways.

    Directory of Open Access Journals (Sweden)

    Chun Li

    2015-10-01

    Full Text Available The ability of specific neurons to regenerate their axons after injury is governed by cell-intrinsic regeneration pathways. In Caenorhabditis elegans, the JNK and p38 MAPK pathways are important for axon regeneration. Axonal injury induces expression of the svh-2 gene encoding a receptor tyrosine kinase, stimulation of which by the SVH-1 growth factor leads to activation of the JNK pathway. Here, we identify ETS-4 and CEBP-1, related to mammalian Ets and C/EBP, respectively, as transcriptional activators of svh-2 expression following axon injury. ETS-4 and CEBP-1 function downstream of the cAMP and Ca2+-p38 MAPK pathways, respectively. We show that PKA-dependent phosphorylation of ETS-4 promotes its complex formation with CEBP-1. Furthermore, activation of both cAMP and Ca2+ signaling is required for activation of svh-2 expression. Thus, the cAMP/Ca2+ signaling pathways cooperatively activate the JNK pathway, which then promotes axon regeneration.

  16. Cyclic glycine-proline regulates IGF-1 homeostasis by altering the binding of IGFBP-3 to IGF-1

    OpenAIRE

    Guan, Jian; Gluckman, Peter; Yang, Panzao; Krissansen, Geoff; Sun, Xueying; Zhou, Yongzhi; Wen, Jingyuan; Phillips, Gemma; Shorten, Paul R.; McMahon, Chris D.; Wake, Graeme C.; Chan, Wendy H. K.; Thomas, Mark F.; Ren, April; Moon, Steve

    2014-01-01

    The homeostasis of insulin-like growth factor-1 (IGF-1) is essential for metabolism, development and survival. Insufficient IGF-1 is associated with poor recovery from wounds whereas excessive IGF-1 contributes to growth of tumours. We have shown that cyclic glycine-proline (cGP), a metabolite of IGF-1, can normalise IGF-1 function by showing its efficacy in improving the recovery from ischemic brain injury in rats and inhibiting the growth of lymphomic tumours in mice. Further investigation ...

  17. The reciprocal interaction of sympathetic nervous system and cAMP-PKA-NF-kB pathway in immune suppression after experimental stroke.

    Science.gov (United States)

    Zuo, Lei; Shi, Luhang; Yan, Fuling

    2016-08-03

    Sympathetic nervous system(SNS) is involved in the mechanism of immune suppression after stroke. Furthermore, as the pro-inflammatory effect of nuclear factor kappa B(NF-kB) is inhibited after stroke, which is regulated by cyclic adenosine monophosphate(cAMP) and proteinkinase A(PKA). The cAMP-PKA-NF-kB pathway might play an important role in noradrenergic-mediated immune dysfunction. The purpose of our research is to analyze how SNS interfere with the immune system after acute stroke and the underlying mechanism of cAMP-PKA-NF-kB pathway in regulating the inflammation. 32 healthy male Sprague-Dawley rats were divided into 4 groups equally and randomly (1) Sham operation group; (2) middle cerebral artery occlusion; (MCAO) control group; (3) propranolol MCAO group; (4) isopropylarterenol sham group. 72h later after MCAO or sham operation, tumor necrosis factor-α(TNF-α)and interleukine-10(IL-10) in serum as well as cAMP, PKA and NF-kB in spleen cells were tested. TNF-α decreased while IL-10 increased in serum after acute ischemia stroke (pkB was inhibited (pkB is down-regulated. Since the pro-inflammatory effect of NF-kB slacked, the immune system may be inhibited after stroke. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. Critical Role of Nitric Oxide-cGMP Cascade in the Formation of cAMP-Dependent Long-Term Memory

    Science.gov (United States)

    Aonuma, Hitoshi; Mizunami, Makoto; Matsumoto, Yukihisa; Unoki, Sae

    2006-01-01

    Cyclic AMP pathway plays an essential role in formation of long-term memory (LTM). In some species, the nitric oxide (NO)-cyclic GMP pathway has been found to act in parallel and complementary to the cAMP pathway for LTM formation. Here we describe a new role of the NO-cGMP pathway, namely, stimulation of the cAMP pathway to induce LTM. We have…

  19. Octopamine regulates antennal sensory neurons via daytime-dependent changes in cAMP and IP3 levels in the hawkmoth Manduca sexta.

    Directory of Open Access Journals (Sweden)

    Thomas Schendzielorz

    Full Text Available The biogenic amine octopamine (OA mediates reward signals in olfactory learning and memory as well as circadian rhythms of sleep and activity. In the crepuscular hawkmoth Manduca sexta, OA changed pheromone detection thresholds daytime-dependently, suggesting that OA confers circadian control of olfactory transduction. Thus, with enzyme-linked immunosorbent assays we searched hawkmoth antennae for daytime-dependent changes in the concentration of OA and its respective second messengers. Antennal stimulation with OA raised cAMP- and IP3 levels. Furthermore, antennae expressed daytime-dependent changes in the concentration of OA, with maxima at Zeitgebertime (ZT 20 when moths were active and also maximal concentrations of cAMP occurred. Maximal IP3 levels at ZT 18 and 23 correlated with maximal flight activity of male moths, while minimal IP3 levels at dusk correlated with peaks of feeding activity. Half maximal effective concentration (EC50 for activation of the OA-receptor decreased during the moth's activity phase suggesting daytime-dependent changes in OA receptor sensitivity. With an antiserum against tyramine, the precursor of OA, two centrifugal neurons were detected projecting out into the sensory cell layer of the antenna, possibly mediating more rapid stimulus-dependent OA actions. Indeed, in fast kinetic assays OA receptor stimulation increased cAMP concentrations within 50 msec. Thus, we hypothesize that fast, stimulus-dependent centrifugal control of OA-release in the antenna occurs. Additional slow systemic OA actions might be based upon circadian release of OA into the hemolymph mediating circadian rhythms of antennal second messenger levels. The resulting rhythms of odor sensitivity are suggested to underlie circadian rhythms in odor-mediated behavior.

  20. Duodenal activation of cAMP-dependent protein kinase induces vagal afferent firing and lowers glucose production in rats.

    Science.gov (United States)

    Rasmussen, Brittany A; Breen, Danna M; Luo, Ping; Cheung, Grace W C; Yang, Clair S; Sun, Biying; Kokorovic, Andrea; Rong, Weifang; Lam, Tony K T

    2012-04-01

    The duodenum senses nutrients to maintain energy and glucose homeostasis, but little is known about the signaling and neuronal mechanisms involved. We tested whether duodenal activation of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) is sufficient and necessary for cholecystokinin (CCK) signaling to trigger vagal afferent firing and regulate glucose production. In rats, we selectively activated duodenal PKA and evaluated changes in glucose kinetics during the pancreatic (basal insulin) pancreatic clamps and vagal afferent firing. The requirement of duodenal PKA signaling in glucose regulation was evaluated by inhibiting duodenal activation of PKA in the presence of infusion of the intraduodenal PKA agonist (Sp-cAMPS) or CCK1 receptor agonist (CCK-8). We also assessed the involvement of a neuronal network and the metabolic impact of duodenal PKA activation in rats placed on high-fat diets. Intraduodenal infusion of Sp-cAMPS activated duodenal PKA and lowered glucose production, in association with increased vagal afferent firing in control rats. The metabolic and neuronal effects of duodenal Sp-cAMPS were negated by coinfusion with either the PKA inhibitor H89 or Rp-CAMPS. The metabolic effect was also negated by coinfusion with tetracaine, molecular and pharmacologic inhibition of NR1-containing N-methyl-d-aspartate (NMDA) receptors within the dorsal vagal complex, or hepatic vagotomy in rats. Inhibition of duodenal PKA blocked the ability of duodenal CCK-8 to reduce glucose production in control rats, whereas duodenal Sp-cAMPS bypassed duodenal CCK resistance and activated duodenal PKA and lowered glucose production in rats on high-fat diets. We identified a neural glucoregulatory function of duodenal PKA signaling. Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

  1. The Autonomic Nervous System Regulates the Heart Rate through cAMP-PKA Dependent and Independent Coupled-Clock Pacemaker Cell Mechanisms.

    Science.gov (United States)

    Behar, Joachim; Ganesan, Ambhighainath; Zhang, Jin; Yaniv, Yael

    2016-01-01

    Sinoatrial nodal cells (SANCs) generate spontaneous action potentials (APs) that control the cardiac rate. The brain modulates SANC automaticity, via the autonomic nervous system, by stimulating membrane receptors that activate (adrenergic) or inactivate (cholinergic) adenylyl cyclase (AC). However, these opposing afferents are not simply additive. We showed that activation of adrenergic signaling increases AC-cAMP/PKA signaling, which mediates the increase in the SANC AP firing rate (i.e., positive chronotropic modulation). However, there is a limited understanding of the underlying internal pacemaker mechanisms involved in the crosstalk between cholinergic receptors and the decrease in the SANC AP firing rate (i.e., negative chronotropic modulation). We hypothesize that changes in AC-cAMP/PKA activity are crucial for mediating either decrease or increase in the AP firing rate and that the change in rate is due to both internal and membrane mechanisms. In cultured adult rabbit pacemaker cells infected with an adenovirus expressing the FRET sensor AKAR3, PKA activity and AP firing rate were tightly linked in response to either adrenergic receptor stimulation (by isoproterenol, ISO) or cholinergic stimulation (by carbachol, CCh). To identify the main molecular targets that mediate between PKA signaling and pacemaker function, we developed a mechanistic computational model. The model includes a description of autonomic-nervous receptors, post- translation signaling cascades, membrane molecules, and internal pacemaker mechanisms. Yielding results similar to those of the experiments, the model simulations faithfully reproduce the changes in AP firing rate in response to CCh or ISO or a combination of both (i.e., accentuated antagonism). Eliminating AC-cAMP-PKA signaling abolished the core effect of autonomic receptor stimulation on the AP firing rate. Specifically, disabling the phospholamban modulation of the SERCA activity resulted in a significantly reduced effect

  2. The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Eun-Ah [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From

  3. AMP language reference manual

    International Nuclear Information System (INIS)

    Drouffe, J.M.

    1982-06-01

    The use of a program for symbolic calculations named AMP (Algebraic Manipulation Program) is described. Its main features are: high speed heart for usual algebraic calculations; conversational capability; derivation, substitutions, matricial calculus, expansions, non commutative algebrae...; possibility to define new symbols and associated rules; possibility to create and use external libraries; written for IBM/370 like computers

  4. Hypoxia induces cancer-associated cAMP/PKA signalling through HIF-mediated transcriptional control of adenylyl cyclases VI and VII.

    Science.gov (United States)

    Simko, Veronika; Iuliano, Filippo; Sevcikova, Andrea; Labudova, Martina; Barathova, Monika; Radvak, Peter; Pastorekova, Silvia; Pastorek, Jaromir; Csaderova, Lucia

    2017-08-31

    Hypoxia is a phenomenon often arising in solid tumours, linked to aggressive malignancy, bad prognosis and resistance to therapy. Hypoxia-inducible factor-1 has been identified as a key mediator of cell and tissue adaptation to hypoxic conditions through transcriptional activation of many genes involved in glucose metabolism and other cancer-related processes, such as angiogenesis, cell survival and cell invasion. Cyclic adenosine 3'5'-monophosphate is one of the most ancient and evolutionarily conserved signalling molecules and the cAMP/PKA signalling pathway plays an important role in cellular adaptation to hypoxia. We have investigated possible new mechanisms behind hypoxic activation of the cAMP/PKA pathway. For the first time, we have shown that hypoxia induces transcriptional up-regulation of the system of adenylyl cyclases, enzymes responsible for cAMP production, in a panel of carcinoma cell lines of various origin. Our data prove functional relevance of the hypoxic increase of adenylyl cyclases VI and VII at least partially mediated by HIF-1 transcription factor. We have identified adenylyl cyclase VI and VII isoforms as mediators of cellular response to hypoxia, which led to the elevation of cAMP levels and enhanced PKA activity, with an impact on cell migration and pH regulation.

  5. GSKIP- and GSK3-mediated anchoring strengthens cAMP/PKA/Drp1 axis signaling in the regulation of mitochondrial elongation.

    Science.gov (United States)

    Loh, Joon-Khim; Lin, Ching-Chih; Yang, Ming-Chang; Chou, Chia-Hua; Chen, Wan-Shia; Hong, Ming-Chang; Cho, Chung-Lung; Hsu, Ching-Mei; Cheng, Jiin-Tsuey; Chou, An-Kuo; Chang, Chung-Hsing; Tseng, Chao-Neng; Wang, Chi-Huei; Lieu, Ann-Shung; Howng, Shen-Long; Hong, Yi-Ren

    2015-08-01

    GSK3β binding of GSKIP affects neurite outgrowth, but the physiological significance of PKA binding to GSKIP remains to be determined. We hypothesized that GSKIP and GSK3β mediate cAMP/PKA/Drp1 axis signaling and modulate mitochondrial morphology by forming a working complex comprising PKA/GSKIP/GSK3β/Drp1. We demonstrated that GSKIP wild-type overexpression increased phosphorylation of Drp1 S637 by 7-8-fold compared to PKA kinase-inactive mutants (V41/L45) and a GSK3β binding-defective mutant (L130) under H2O2 and forskolin challenge in HEK293 cells, indicating that not only V41/L45, but also L130 may be involved in Drp1-associated protection of GSKIP. Interestingly, silencing either GSKIP or GSK3β but not GSK3α resulted in a dramatic decrease in Drp1 S637 phosphorylation, revealing that both GSKIP and GSK3β are required in this novel PKA/GSKIP/GSK3β/Drp1 complex. Moreover, overexpressed kinase-dead GSK3β-K85R, which retains the capacity to bind GSKIP, but not K85M which shows total loss of GSKIP-binding, has a higher Drp1 S637 phosphorylation similar to the GSKIP wt overexpression group, indicating that GSK3β recruits Drp1 by anchoring rather than in a kinase role. With further overexpression of either V41/L45P or the L130P GSKIP mutant, the elongated mitochondrial phenotype was lost; however, ectopically expressed Drp1 S637D, a phosphomimetic mutant, but not S637A, a non-phosphorylated mutant, restored the elongated mitochondrial morphology, indicating that Drp1 is a downstream effector of direct PKA signaling and possibly has an indirect GSKIP function involved in the cAMP/PKA/Drp1 signaling axis. Collectively, our data revealed that both GSKIP and GSK3β function as anchoring proteins in the cAMP/PKA/Drp1 signaling axis modulating Drp1 phosphorylation. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. AMP Deaminase 3 Deficiency Enhanced 5′-AMP Induction of Hypometabolism

    Science.gov (United States)

    Daniels, Isadora Susan; O′Brien, William G.; Nath, Vinay; Zhao, Zhaoyang; Lee, Cheng Chi

    2013-01-01

    A hypometabolic state can be induced in mice by 5′-AMP administration. Previously we proposed that an underlying mechanism for this hypometabolism is linked to reduced erythrocyte oxygen transport function due to 5′-AMP uptake altering the cellular adenylate equilibrium. To test this hypothesis, we generated mice deficient in adenosine monophosphate deaminase 3 (AMPD3), the key catabolic enzyme for 5′-AMP in erythrocytes. Mice deficient in AMPD3 maintained AMPD activities in all tissues except erythrocytes. Developmentally and morphologically, the Ampd3−/− mice were indistinguishable from their wild type siblings. The levels of ATP, ADP but not 5′-AMP in erythrocytes of Ampd3−/− mice were significantly elevated. Fasting blood glucose levels of the Ampd3−/− mice were comparable to wild type siblings. In comparison to wild type mice, the Ampd3−/− mice displayed a deeper hypometabolism with a significantly delayed average arousal time in response to 5′-AMP administration. Together, these findings demonstrate a central role of AMPD3 in the regulation of 5′-AMP mediated hypometabolism and further implicate erythrocytes in this behavioral response. PMID:24066180

  7. Endogenous Parathyroid Hormone Promotes Fracture Healing by Increasing Expression of BMPR2 through cAMP/PKA/CREB Pathway in Mice

    Directory of Open Access Journals (Sweden)

    Wei Zhou

    2017-06-01

    Full Text Available Background/Aims: Endogenous parathyroid hormone (PTH plays an important role in fracture healing. This study investigated whether endogenous PTH regulates fracture healing by bone morphogenetic protein (BMP and/or the transforming growth factor-β (TGF-β signaling pathway. Methods: Eight-week-old wild-type (WT and PTH-knockout (PTH KO male mice were selected, and models of open right-femoral fracture were constructed. Fracture healing and callus characteristics of mice in the two groups were compared by X-ray, micro-computed tomography, histological, and immunohistochemical examinations. Bone marrow mesenchymal stem cells (BMMSCs of 8-week-old WT and PTHKO male mice were obtained and induced into osteoblasts and chondrocytes. Results: We found that expression levels of Runt-related transcription factor (RUNX2, bone morphogenetic protein-receptor-type Ⅱ (BMPR2, phosphorylated Smad 1/5/8, and phosphorylated cyclic adenosine monophosphate-responsive element binding protein (CREB in the callus of PTHKO mice were significantly decreased, whereas no significant difference in expression of SOX9, TGF-βR2,or pSMAD2/3 was observed between PTHKO and WT mice. Additionally, the activity of osteoblast alkaline phosphatase was low at 7 days post-induction, and was upregulated by addition of PTH or dibutyryl cyclic adenosine monophosphate (dbcAMP to the cell culture. Furthermore, H89 (protein kinase A inhibitoreliminated the simulating effects of PTH and dbcAMP, and a low concentration of cyclic adenosine monophosphate (cAMP was observed in PTHKO mouse BMMSCs. Conclusion: These results suggested that endogenous PTH enhanced BMPR2 expression by a cAMP/PKA/CREB pathway in osteoblasts, and increased RUNX2 expression through transduction of the BMP/pSMAD1/5/8 signaling pathway.

  8. Linoleic acid enhance the production of moncolin K and red pigments in Monascus ruber by activating mokH and mokA, and by accelerating cAMP-PkA pathway.

    Science.gov (United States)

    Huang, Jing; Liao, NanQing; Li, HaoMing

    2018-04-01

    Monacolin K, an inhibitor of HMG-CoA reductase, is a secondary metabolite synthesized by polyketide synthases (PKS) from Monascus ruber. The mokH gene encoding Zn(II)2Cys6 binding protein and mokA gene encoding polyketide synthase are presumed to activate monacolin K production. In this study, linoleic acid could be a quorum sensing signaling molecule to increase monacolin K production in the cyclic AMP(cAMP)-protein kinase A(PKA) signaling pathway. Analysis of the PKA activity and the cAMP concentration shows that linoleic acid could increase cAMP concentration and activate PKA. Analysis of the RT-qPCR products demonstrates that 256μM and 512μM linoleic acid can up-regulate mokH and mokA gene transcript levels. Especially with 512μM linoleic acid addition, linoleic acid increase 1.35 folds of monacolin K production, but 64μM linoleic acid increase 1.94 folds of red pigment production in Monascus ruber. These results show the cAMP-PkA pathway activity can up-regulate mokA and mokH gene, which enhance the yield of Monacolin K. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Flitney, F W; Singh, J

    1980-07-01

    complex, but the accompanying change in isometric twitch tension is paralleled closely by corresponding changes in the ratio 3',5'cyclic AMP:3',5'-cyclic GMP. 5. It is concluded that ATP exerts a dual effect on the ventricle and that the contractile response is regulated by changes in the metabolism of 3',5'-cyclic nucleotides. The effects of indomethacin indicate a possible involvement of prostaglandins in mediating the ATP response. It is suggested that the initial effect of ATP on the ventricle is to increase the permeability of the fibres to Ca2+. 6. The relationship between 3',5' cyclic nucleotide levels and ventricular contractility is discussed. It is postulated that the antagonistic effects of 3',5'-cyclic AMP and 3',5'-cyclic GMP are expressed at the level of certain phosphoproteins which regulate both the availability of Ca2+ and the sensitivity of the contractile proteins to Ca2+.

  10. Regulation of Hxt3 and Hxt7 turnover converges on the Vid30 complex and requires inactivation of the Ras/cAMP/PKA pathway in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Chris Snowdon

    Full Text Available Eukaryotic cells adjust their intracellular protein complement as a mechanism to adapt to changing environmental signals. In Saccharomyces cerevisiae the hexose transporters Hxt3 and Hxt7 are expressed and function on the plasma membrane in high and low glucose abundance, respectively. By contrast, Hxt3 is endocytosed and degraded in the vacuole when cells are starved of glucose and Hxt7 in response to rapamycin treatment or when nitrogen is limiting. Yeast uses several signaling pathways, including the TORC1 and Ras/cAMP/Protein Kinase A (PKA pathways, to adapt to nutrient changes in the environment. The multi-protein Vid30 complex (Vid30c, an E3 ubiquitin ligase required for the degradation of FBPase, assists in this adaptation process in a mechanism that is poorly understood. Here we show the endocytosis and the subsequent degradation of both Hxt3 and Hxt7, in response to different nutrient signals, is dependent on components of the Vid30c. Additionally, we define the signaling events required for the turnover of Hxt3 and Hxt7 by showing that Hxt3 turnover requires Ras2 and PKA inactivation, whereas Hxt7 turnover requires TORC1 and Ras2 inactivation. Further investigation led us to identify Rim15, a kinase that is inhibited by both the TORC1 and Ras/cAMP/PKA pathways, as a key downstream effector in signaling both turnover events. Finally, we show that the turnover of both Hxt3 and Hxt7 is dependent on the essential E3 ubiquitin ligase, Rsp5, indicating that the role of the Vid30c might be indirect of Hxt ubiquitylation.

  11. Regulation of Pancreatic β Cell Mass by Cross-Interaction between CCAAT Enhancer Binding Protein β Induced by Endoplasmic Reticulum Stress and AMP-Activated Protein Kinase Activity.

    Directory of Open Access Journals (Sweden)

    Tomokazu Matsuda

    Full Text Available During the development of type 2 diabetes, endoplasmic reticulum (ER stress leads to not only insulin resistance but also to pancreatic beta cell failure. Conversely, cell function under various stressed conditions can be restored by reducing ER stress by activating AMP-activated protein kinase (AMPK. However, the details of this mechanism are still obscure. Therefore, the current study aims to elucidate the role of AMPK activity during ER stress-associated pancreatic beta cell failure. MIN6 cells were loaded with 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR and metformin to assess the relationship between AMPK activity and CCAAT enhancer binding protein β (C/EBPβ expression levels. The effect of C/EBPβ phosphorylation on expression levels was also investigated. Vildagliptin and metformin were administered to pancreatic beta cell-specific C/EBPβ transgenic mice to investigate the relationship between C/EBPβ expression levels and AMPK activity in the pancreatic islets. When pancreatic beta cells are exposed to ER stress, the accumulation of the transcription factor C/EBPβ lowers the AMP/ATP ratio, thereby decreasing AMPK activity. In an opposite manner, incubation of MIN6 cells with AICAR or metformin activated AMPK, which suppressed C/EBPβ expression. In addition, administration of the dipeptidyl peptidase-4 inhibitor vildagliptin and metformin to pancreatic beta cell-specific C/EBPβ transgenic mice decreased C/EBPβ expression levels and enhanced pancreatic beta cell mass in proportion to the recovery of AMPK activity. Enhanced C/EBPβ expression and decreased AMPK activity act synergistically to induce ER stress-associated pancreatic beta cell failure.

  12. Further studies on the effect of adenosine cyclic monophosphate derivatives on cell proliferation in the jejunal crypts of rat.

    Science.gov (United States)

    Tutton, P J; Barkla, D H

    1982-01-01

    1. Cell proliferation in the jejunal crypt epithelium of rat was measured using a stathmokinetic technique. 2. Sodium butyrate was found to promote jejunal crypt cell proliferation. 3. N6, O2'-Dibutyryl cyclic adenosine monophosphate (cAMP), N6-monobutyryl-cAMP and N6-monobutyryl-8-bromo-cAMP were found to inhibit cell proliferation when compared to sodium butyrate treated tissues. 4. 8-Chlorophenylthio-cAMP was found to inhibit cell division when compared to untreated animals. 5. O2'-Monobutyryl cAMP and 8-bromo-cAMP were not found to inhibit cell proliferation.

  13. Dysfunctional Hyperpolarization-Activated Cyclic Nucleotide-gated Ion Channels in Cardiac Diseases

    Directory of Open Access Journals (Sweden)

    Xiaoqi Zhao

    Full Text Available Abstract Hyperpolarization-activated cyclic nucleotide-gated (HCN channels are reverse voltage-dependent, and their activation depends on the hyperpolarization of the membrane and may be directly or indirectly regulated by the cyclic adenosine monophosphate (cAMP or other signal-transduction cascades. The distribution, quantity and activation states of HCN channels differ in tissues throughout the body. Evidence exhibits that HCN channels play critical roles in the generation and conduction of the electrical impulse and the physiopathological process of some cardiac diseases. They may constitute promising drug targets in the treatment of these cardiac diseases. Pharmacological treatment targeting HCN channels is of benefit to these cardiac conditions.

  14. Cyclic AMP response element binding protein and brain-derived ...

    Indian Academy of Sciences (India)

    Madhu

    learning tasks and memory (Bramham and Messaoudi 2005). Thus, BDNF is involved in structural remodeling, neuronal .... in hippocampal-dependent learning and memory (Tyler et al 2002; Mizuno and Giese 2005) may play a ..... neurons in the olfactory bulb; J. Neurosci. 25 10105–10118. Gould E and Tanapat P 1999 ...

  15. Non-Enzymatic Oligomerization of 3 ', 5 ' Cyclic AMP

    Czech Academy of Sciences Publication Activity Database

    Costanzo, G.; Pino, S.; Timperio, A.M.; Šponer, Judit E.; Sponer, Jiri; Nováková, Olga; Šedo, O.; Zdráhal, Z.; Di Mauro, E.

    2016-01-01

    Roč. 11, č. 11 (2016), č. článku e0165723. E-ISSN 1932-6203 R&D Projects: GA ČR(CZ) GA14-12010S; GA ČR GBP206/12/G151 Institutional support: RVO:68081707 Keywords : template-directed synthesis * nucleoside phosphorylation * montmorillonite catalysis * molecular-structure Subject RIV: BO - Biophysics Impact factor: 2.806, year: 2016

  16. Review Cyclic AMP response element binding protein and brain ...

    Indian Academy of Sciences (India)

    Madhu

    Several types of adult-onset stressors, including physical and psychosocial stressors ... stress-induced atrophy with antidepressant therapy in animal models (Watanabe et ..... (Strauss et al 2005) and geriatric depression (Hwang et al. 2005).

  17. A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium.

    Science.gov (United States)

    Lee, S; Parent, C A; Insall, R; Firtel, R A

    1999-09-01

    We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced approximately 60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated

  18. AMP-activated protein kinase regulates lymphocyte responses to metabolic stress but is largely dispensable for immune cell development and function.

    Science.gov (United States)

    Mayer, Alice; Denanglaire, Sébastien; Viollet, Benoit; Leo, Oberdan; Andris, Fabienne

    2008-04-01

    AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, represents an energy sensor able to adapt cellular metabolism in response to nutritional environmental variations. TCR stimulation activates AMPK, a regulatory event that is known to stimulate ATP-producing processes, possibly in anticipation of the increased energetic needs associated with cell division and expression of effector function. Taking advantage of the selective expression of the AMPKalpha1 catalytic subunit in lymphoid cells, we have analyzed the in vitro and in vivo capacity of lymphocytes lacking AMPK activity (AMPKalpha1-KO cells) to respond to metabolic stress and to initiate and sustain an immune response. AMPKalpha1-KO cells displayed increasing sensitivity to energetic stress in vitro, and were found unable to maintain adequate ATP levels in response to ATP synthase inhibition. These cells were, however, able to respond to antigen stimulation in vitro, as shown by optimal proliferation and cytokine production. Similarly, AMPKalpha1-KO mice were fully immunocompetent in vivo and displayed normal cell proliferation, humoral, cytotoxic and delayed-type hypersensitivity (DTH) responses following antigen injection. In conclusion, AMPK represents an important enzyme allowing lymphocytes to resist a mild energy crisis in vitro, but is largely dispensable for activation and expression of effector function in response to antigen stimulation.

  19. AMP-activated protein kinase and adenosine are both metabolic modulators that regulate chloride secretion in the shark rectal gland ( Squalus acanthias).

    Science.gov (United States)

    Neuman, Rugina I; van Kalmthout, Juliette A M; Pfau, Daniel J; Menendez, Dhariyat M; Young, Lawrence H; Forrest, John N

    2018-04-01

    The production of endogenous adenosine during secretagogue stimulation of CFTR leads to feedback inhibition limiting further chloride secretion in the rectal gland of the dogfish shark (Squalus acanthias). In the present study, we examined the role of AMP-kinase (AMPK) as an energy sensor also modulating chloride secretion through CFTR. We found that glands perfused with forskolin and isobutylmethylxanthine (F + I), potent stimulators of chloride secretion in this ancient model, caused significant phosphorylation of the catalytic subunit Thr 172 of AMPK. These findings indicate that AMPK is activated during energy-requiring stimulated chloride secretion. In molecular studies, we confirmed that the activating Thr 172 site is indeed present in the α-catalytic subunit of AMPK in this ancient gland, which reveals striking homology to AMPKα subunits sequenced in other vertebrates. When perfused rectal glands stimulated with F + I were subjected to severe hypoxic stress or perfused with pharmacologic inhibitors of metabolism (FCCP or oligomycin), phosphorylation of AMPK Thr 172 was further increased and chloride secretion was dramatically diminished. The pharmacologic activation of AMPK with AICAR-inhibited chloride secretion, as measured by short-circuit current, when applied to the apical side of shark rectal gland monolayers in primary culture. These results indicate that that activated AMPK, similar to adenosine, transmits an inhibitory signal from metabolism, that limits chloride secretion in the shark rectal gland.

  20. Negative regulation of AMP-activated protein kinase (AMPK) activity by macrophage migration inhibitory factor (MIF) family members in non-small cell lung carcinomas.

    Science.gov (United States)

    Brock, Stephanie E; Rendon, Beatriz E; Yaddanapudi, Kavitha; Mitchell, Robert A

    2012-11-02

    AMP-activated protein kinase (AMPK) is a nutrient- and metabolic stress-sensing enzyme activated by the tumor suppressor kinase, LKB1. Because macrophage migration inhibitory factor (MIF) and its functional homolog, d-dopachrome tautomerase (d-DT), have protumorigenic functions in non-small cell lung carcinomas (NSCLCs) but have AMPK-activating properties in nonmalignant cell types, we set out to investigate this apparent paradox. Our data now suggest that, in contrast to MIF and d-DTs AMPK-activating properties in nontransformed cells, MIF and d-DT act cooperatively to inhibit steady-state phosphorylation and activation of AMPK in LKB1 wild type and LKB1 mutant human NSCLC cell lines. Our data further indicate that MIF and d-DT, acting through their shared cell surface receptor, CD74, antagonize NSCLC AMPK activation by maintaining glucose uptake, ATP production, and redox balance, resulting in reduced Ca(2+)/calmodulin-dependent kinase kinase β-dependent AMPK activation. Combined, these studies indicate that MIF and d-DT cooperate to inhibit AMPK activation in an LKB1-independent manner.

  1. A cAMP/PKA/Kinesin-1 Axis Promotes the Axonal Transport of Mitochondria in Aging Drosophila Neurons.

    Science.gov (United States)

    Vagnoni, Alessio; Bullock, Simon L

    2018-04-23

    Mitochondria play fundamental roles within cells, including energy provision, calcium homeostasis, and the regulation of apoptosis. The transport of mitochondria by microtubule-based motors is critical for neuronal structure and function. This process allows local requirements for mitochondrial functions to be met and also facilitates recycling of these organelles [1, 2]. An age-related reduction in mitochondrial transport has been observed in neurons of mammalian and non-mammalian organisms [3-6], and has been proposed to contribute to the broader decline in neuronal function that occurs during aging [3, 5-7]. However, the factors that influence mitochondrial transport in aging neurons are poorly understood. Here we provide evidence using the tractable Drosophila wing nerve system that the cyclic AMP/protein kinase A (cAMP/PKA) pathway promotes the axonal transport of mitochondria in adult neurons. The level of the catalytic subunit of PKA decreases during aging, and acute activation of the cAMP/PKA pathway in aged flies strongly stimulates mitochondrial motility. Thus, the age-related impairment of transport is reversible. The expression of many genes is increased by PKA activation in aged flies. However, our results indicate that elevated mitochondrial transport is due in part to upregulation of the heavy chain of the kinesin-1 motor, the level of which declines during aging. Our study identifies evolutionarily conserved factors that can strongly influence mitochondrial motility in aging neurons. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  2. Cyclic multiverses

    Science.gov (United States)

    Marosek, Konrad; Dąbrowski, Mariusz P.; Balcerzak, Adam

    2016-09-01

    Using the idea of regularization of singularities due to the variability of the fundamental constants in cosmology we study the cyclic universe models. We find two models of oscillating and non-singular mass density and pressure (`non-singular' bounce) regularized by varying gravitational constant G despite the scale factor evolution is oscillating and having sharp turning points (`singular' bounce). Both violating (big-bang) and non-violating (phantom) null energy condition models appear. Then, we extend this idea on to the multiverse containing cyclic individual universes with either growing or decreasing entropy though leaving the net entropy constant. In order to get an insight into the key idea, we consider the doubleverse with the same geometrical evolution of the two `parallel' universes with their physical evolution [physical coupling constants c(t) and G(t)] being different. An interesting point is that there is a possibility to exchange the universes at the point of maximum expansion - the fact which was already noticed in quantum cosmology. Similar scenario is also possible within the framework of Brans-Dicke theory where varying G(t) is replaced by the dynamical Brans-Dicke field φ(t) though these theories are slightly different.

  3. Oxyresveratrol ameliorates nonalcoholic fatty liver disease by regulating hepatic lipogenesis and fatty acid oxidation through liver kinase B1 and AMP-activated protein kinase.

    Science.gov (United States)

    Lee, Ju-Hee; Baek, Su Youn; Jang, Eun Jeong; Ku, Sae Kwang; Kim, Kyu Min; Ki, Sung Hwan; Kim, Chang-Eop; Park, Kwang Il; Kim, Sang Chan; Kim, Young Woo

    2018-06-01

    Oxyresveratrol (OXY) is a naturally occurring polyhydroxylated stilbene that is abundant in mulberry wood (Morus alba L.), which has frequently been supplied as a herbal medicine. It has been shown that OXY has regulatory effects on inflammation and oxidative stress, and may have potential in preventing or curing nonalcoholic fatty liver disease (NAFLD). This study examined the effects of OXY on in vitro model of NAFLD in hepatocyte by the liver X receptor α (LXRα)-mediated induction of lipogenic genes and in vivo model in mice along with its molecular mechanism. OXY inhibited the LXRα agonists-mediated sterol regulatory element binding protein-1c (SREBP-1c) induction and expression of the lipogenic genes and upregulated the mRNA of fatty acid β-oxidation-related genes in hepatocytes, which is more potent than genistein and daidzein. OXY also induced AMP-activated protein kinase (AMPK) activation in a time-dependent manner. Moreover, AMPK activation by the OXY treatment helped inhibit SREBP-1c using compound C as an AMPK antagonist. Oral administration of OXY decreased the Oil Red O stained-positive areas significantly, indicating lipid droplets and hepatic steatosis regions, as well as the serum parameters, such as fasting glucose, total cholesterol, and low density lipoprotein-cholesterol in high fat diet fed-mice, as similar with orally treatment of atorvastatin. Overall, this result suggests that OXY has the potency to inhibit hepatic lipogenesis through the AMPK/SREBP-1c pathway and can be used in the development of pharmaceuticals to prevent a fatty liver. Copyright © 2018. Published by Elsevier B.V.

  4. Assessing Self-Regulation as a Cyclical, Context-Specific Phenomenon: Overview and Analysis of SRL Microanalytic Protocols

    Directory of Open Access Journals (Sweden)

    Timothy J. Cleary

    2012-01-01

    Full Text Available The primary purpose of this paper is to review relevant research related to the use of an assessment technique, called Self-Regulated Learning (SRL Microanalysis. This structured interview is grounded in social-cognitive theory and research and thus seeks to evaluate students' regulatory processes as they engage in well-defined academic or nonacademic tasks and activities. We illustrate the essential features of this contextualized assessment approach and detail a simple five-step process that researchers can use to apply this approach to their work. Example questions and administration procedures for five key self-regulation subprocesses (i.e., including goal-setting, strategic planning, monitoring, self-evaluation, and attributions are highlighted, with particular emphasis placed on causal attributions. The psychometric properties of SRL microanalytic assessment protocols and potential areas of future research are presented.

  5. Role of AC-cAMP-PKA Cascade in Antidepressant Action of Electroacupuncture Treatment in Rats

    Directory of Open Access Journals (Sweden)

    Jian-hua Liu

    2012-01-01

    Full Text Available Adenylyl cyclase (AC-cyclic adenosine monophosphate (cAMP-cAMP-dependent protein kinase A (PKA cascade is considered to be associated with the pathogenesis and treatment of depression. The present study was conducted to explore the role of the cAMP cascade in antidepressant action of electroacupuncture (EA treatment for chronic mild stress (CMS-induced depression model rats. The results showed that EA improved significantly behavior symptoms in depression and dysfunction of AC-cAMP-PKA signal transduction pathway induced by CMS, which was as effective as fluoxetine. Moreover, the antidepressant effects of EA rather than Fluoxetine were completely abolished by H89, a specific PKA inhibitor. Consequently, EA has a significant antidepressant treatment in CMS-induced depression model rats, and AC-cAMP-PKA signal transduction pathway is crucial for it.

  6. Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

    Science.gov (United States)

    Chiavegatti, T; Costa, V L; Araújo, M S; Godinho, R O

    2007-01-01

    Background and purpose: cAMP is a key intracellular signalling molecule that regulates multiple processes of the vertebrate skeletal muscle. We have shown that cAMP can be actively pumped out from the skeletal muscle cell. Since in other tissues, cAMP efflux had been associated with extracellular generation of adenosine, in the present study we have assessed the fate of interstitial cAMP and the existence of an extracellular cAMP-adenosine signalling pathway in skeletal muscle. Experimental approach: cAMP efflux and/or its extracellular degradation were analysed by incubating rat cultured skeletal muscle with exogenous cAMP, forskolin or isoprenaline. cAMP and its metabolites were quantified by radioassay or HPLC, respectively. Key results: Incubation of cells with exogenous cAMP was followed by interstitial accumulation of 5′-AMP and adenosine, a phenomenon inhibited by selective inhibitors of ecto-phosphodiesterase (DPSPX) and ecto-nucleotidase (AMPCP). Activation of adenylyl cyclase (AC) in cultured cells with forskolin or isoprenaline increased cAMP efflux and extracellular generation of 5′-AMP and adenosine. Extracellular cAMP-adenosine pathway was also observed after direct and receptor-dependent stimulation of AC in rat extensor muscle ex vivo. These events were attenuated by probenecid, an inhibitor of ATP binding cassette family transporters. Conclusions and implications: Our results show the existence of an extracellular biochemical cascade that converts cAMP into adenosine. The functional relevance of this extracellular signalling system may involve a feedback modulation of cellular response initiated by several G protein-coupled receptor ligands, amplifying cAMP influence to a paracrine mode, through its metabolite, adenosine. PMID:18157164

  7. Hormone regulation system and cyclic nucleotids in the Chernobyl accident liquidators with doses absorbed less then 1 Gy

    International Nuclear Information System (INIS)

    Kovalenko, A.N.

    1997-01-01

    During 6 years after the accident (1987-1992) a functional state of endocrine system that regulate the adaptation, reproduction, metabolism, vessels tonicity and water-electrolyte balance were investigated in 249 liquidators with doses absorbed less then 1 Gy. The changes of these systems activity in state of basal secretion and peculiarities of their reactions under influence of perturbation (adrenaline, insulin) were revealed. Post-irradiation endocrinopathy was characterized and its role in decrease of the organism's adaptation and in mechanism of sanogenesis and pathogenesis was found. (author)

  8. The participation of elevated levels of cyclic GMP in the recovery from radiation-induced mitotic delay

    International Nuclear Information System (INIS)

    Daniel, J.W.; Oleinick, N.L.

    1984-01-01

    The levels of cyclic AMP and cyclic GMP have been measured in Physarum plasmodia before and after treatment with gamma-radiation, 2 mM caffeine, or combinations of the two agents compared to the length of the radiation-induced mitotic delay. Caffeine alone produces a rapid transient elevation of cyclic AMP and a slower delayed elevation of cyclic GMP. Irradiation elicits an immediate transient increase in cyclic AMP and a later cyclic GMP increase which accompanies or precedes the delayed mitosis. A composite pattern is produced by combinations of radiation and caffeine, a distinctive feature of which is an elevated level of cyclic GMP near the time of the radiation-delayed and caffeine-promoted mitosis. With pretreatment by caffeine, the least radiation-induced mitotic delay occurs when plasmodia are irradiated during the caffeine-elicited increase in cyclic GMP. The plasmodium becomes refractory to the reduction of mitotic delay by caffeine at approximately the time it becomes refractory to the further elevation of cyclic GMP by caffeine. The data support a role for cyclic AMP in the onset of and for cyclic GMP in the recovery from mitotic delay induced by ionizing radiation. (author)

  9. The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

    International Nuclear Information System (INIS)

    Zuloaga, R.; Fuentes, E.N.; Molina, A.; Valdés, J.A.

    2013-01-01

    Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP 3 /calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation

  10. The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

    Energy Technology Data Exchange (ETDEWEB)

    Zuloaga, R. [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Fuentes, E.N.; Molina, A. [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción (Chile); Valdés, J.A., E-mail: jvaldes@unab.cl [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción (Chile)

    2013-10-18

    Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP{sub 3}/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.

  11. AMP (Activity Manipulation Program)

    International Nuclear Information System (INIS)

    Engle, W.W. Jr.

    1976-03-01

    AMP is a FORTRAN IV program written to handle energy-group structured activity factors such as sources, conversion factors, and response functions, as used by ANISN, DOT III, and other nuclear reactor and shielding codes. Activities may be retrieved from ANISN-type cross-section and activity sets found on cards and tapes, and from tabular-type sets on cards. They may be altered by change of group structure, multiplication by a constant, or multiplication by delta E (the group-energy interval), and then output to ANISN-type cards or tape and tabular-type cards. A full edit of input and output activities is always printed by group and activity number

  12. Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle

    DEFF Research Database (Denmark)

    Treebak, Jonas Thue; Taylor, Eric B.; Witczak, Carol A.

    2010-01-01

    TBC1D4 (also known as AS160) regulates GLUT4 translocation and glucose uptake in adipocytes and skeletal muscle. Its mode of action involves phosphorylation of Serine (S)/Threonine (T) residues by upstream kinases resulting in inactivation of Rab-GAP activity leading to GLUT4 mobilization...

  13. Biosynthesis of the antimicrobial cyclic lipopeptides nunamycin and nunapeptin by Pseudomonas fluorescens strain In5 is regulated by the LuxR-type transcriptional regulator NunF

    DEFF Research Database (Denmark)

    Hennessy, Rosanna Catherine; Phippen, Christopher; Nielsen, Kristian Fog

    2017-01-01

    -producing pseudomonads except for the border regions where putative LuxR-type regulators are located. This study focuses on understanding the regulatory role of the LuxR-type-encoding gene nunF in CLP production of P. fluorescens In5. Functional analysis of nunF coupled with liquid chromatography-high-resolution mass...... spectrometry (LC-HRMS) showed that CLP biosynthesis is regulated by nunF. Quantitative real-time PCR analysis indicated that transcription of the NRPS genes catalyzing CLP production is strongly reduced when nunF is mutated indicating that nunF is part of the nun-nup regulon. Swarming and biofilm formation...... that environmental elicitors may also influence nunF expression which upon activation regulates nunamycin and nunapeptin production required for the growth inhibition of phytopathogens....

  14. Involvement of cyclic nucleotides in locust flight muscle metabolism

    NARCIS (Netherlands)

    Worm, R.A.A.

    1980-01-01

    1. Flight had no significant effect on the levels of c-AMP of c-GMP in the flight muscles of Locusta migratoria. 2. Injections of 0.01 or 0.1 corpus cardiacum equivalents into the abdominal cavity did not elicit any effect on cyclic nucleotide levels either. 3. Injection of A23187 resulted in

  15. Cyclic di-GMP regulation of the bvg-repressed genes and the orphan response regulator RisA in Bordetella pertussis

    Science.gov (United States)

    Expression of Bordetella pertussis virulence factors is activated by the BvgAS two-component system. Under modulating growth conditions BvgAS indirectly represses another set of genes through the action of BvgR, a bvg-activated protein. BvgR blocks activation of the response regulator RisA which is ...

  16. The role of an ancestral hyperpolarization-activated cyclic nucleotide-gated K+ channel in branchial acid-base regulation in the green crab, Carcinus maenas.

    Science.gov (United States)

    Fehsenfeld, Sandra; Weihrauch, Dirk

    2016-03-01

    Numerous electrophysiological studies on branchial K(+) transport in brachyuran crabs have established an important role for potassium channels in osmoregulatory ion uptake and ammonia excretion in the gill epithelium of decapod crustaceans. However, hardly anything is known of the actual nature of these channels in crustaceans. In the present study, the identification of a hyperpolarization-activated cyclic nucleotide-gated potassium channel (HCN) in the transcriptome of the green crab Carcinus maenas and subsequent performance of quantitative real-time PCR revealed the ubiquitous expression of this channel in this species. Even though mRNA expression levels in the cerebral ganglion were found to be approximately 10 times higher compared with all other tissues, posterior gills still expressed significant levels of HCN, indicating an important role for this transporter in branchial ion regulation. The relatively unspecific K(+)-channel inhibitor Ba(2+), as well as the HCN-specific blocker ZD7288, as applied in gill perfusion experiments and electrophysiological studies employing the split gill lamellae revealed the presence of at least two different K(+)/NH4(+)-transporting structures in the branchial epithelium of C. maenas. Furthermore, HCN mRNA levels in posterior gill 7 decreased significantly in response to the respiratory or metabolic acidosis that was induced by acclimation of green crabs to high environmental PCO2 and ammonia, respectively. Consequently, the present study provides first evidence that HCN-promoted NH4(+) epithelial transport is involved in both branchial acid-base and ammonia regulation in an invertebrate. © 2016. Published by The Company of Biologists Ltd.

  17. Prediction and characterisation of a highly conserved, remote and cAMP responsive enhancer that regulates Msx1 gene expression in cardiac neural crest and outflow tract.

    Science.gov (United States)

    Miller, Kerry Ann; Davidson, Scott; Liaros, Angela; Barrow, John; Lear, Marissa; Heine, Danielle; Hoppler, Stefan; MacKenzie, Alasdair

    2008-05-15

    Double knockouts of the Msx1 and Msx2 genes in the mouse result in severe cardiac outflow tract malformations similar to those frequently found in newborn infants. Despite the known role of the Msx genes in cardiac formation little is known of the regulatory systems (ligand receptor, signal transduction and protein-DNA interactions) that regulate the tissue-specific expression of the Msx genes in mammals during the formation of the outflow tract. In the present study we have used a combination of multi-species comparative genomics, mouse transgenic analysis and in-situ hybridisation to predict and validate the existence of a remote ultra-conserved enhancer that supports the expression of the Msx1 gene in migrating mouse cardiac neural crest and the outflow tract primordia. Furthermore, culturing of embryonic explants derived from transgenic lines with agonists of the PKC and PKA signal transduction systems demonstrates that this remote enhancer is influenced by PKA but not PKC dependent gene regulatory systems. These studies demonstrate the efficacy of combining comparative genomics and transgenic analyses and provide a platform for the study of the possible roles of Msx gene mis-regulation in the aetiology of congenital heart malformation.

  18. Down-regulation of outer membrane proteins by noncoding RNAs: unraveling the cAMP-CRP- and sigmaE-dependent CyaR-ompX regulatory case

    DEFF Research Database (Denmark)

    Johansen, Jesper; Eriksen, Maiken; Kallipolitis, Birgitte

    2008-01-01

    is sufficient to trigger the envelope stress response. Recent work indicates that small Hfq-binding RNAs play a major role in maintaining envelope homeostasis and, so far, two sigma(E)-dependent small noncoding RNAs (sRNAs), MicA and RybB, have been shown to facilitate rapid removal of multiple omp transcripts......The sigma(E) (extracytoplasmic stress response sigma factor in Escherichia coli) signaling system of Gram-negative bacteria plays an essential role in the maintenance of the extracytoplasmic compartment. Upon induction of this system, approximately 100 genes are up-regulated. The majority...... is also up-regulated, directly or indirectly, by sigma(E). In addition, this work identified MicA as a factor that cooperates in the negative control of ompX expression. The conservation of CyaR, MicA, RybB, and their targets suggests that the omp mRNA-sRNA regulatory network is an integral part...

  19. Cyclic adenosine monophosphate-dependent phosphorylation of mammalian mitochondrial proteins: enzyme and substrate characterization and functional role

    Czech Academy of Sciences Publication Activity Database

    Dobrová, Zuzana; Sardanelli, A. M.; Speranza, F.; Scacco, S.; Signorile, A.; Lorusso, V.; Papa, S.

    2001-01-01

    Roč. 40, - (2001), s. 13941-13947 ISSN 0006-2960 Institutional research plan: CEZ:AV0Z5020903 Keywords : cAMP * cyclic adenosine monophosphate Subject RIV: CE - Biochemistry Impact factor: 4.114, year: 2001

  20. Exposure to a specific time-varying electromagnetic field inhibits cell proliferation via cAMP and ERK signaling in cancer cells.

    Science.gov (United States)

    Buckner, Carly A; Buckner, Alison L; Koren, Stan A; Persinger, Michael A; Lafrenie, Robert M

    2018-04-01

    Exposure to specific electromagnetic field (EMF) patterns can affect a variety of biological systems. We have shown that exposure to Thomas-EMF, a low-intensity, frequency-modulated (25-6 Hz) EMF pattern, inhibited growth and altered cell signaling in malignant cells. Exposure to Thomas-EMF for 1 h/day inhibited the growth of malignant cells including B16-BL6 mouse melanoma cells, MDA-MB-231, MDA-MB-468, BT-20, and MCF-7 human breast cancer and HeLa cervical cancer cells but did not affect non-malignant cells. The Thomas-EMF-dependent changes in cell proliferation were mediated by adenosine 3',5'-cyclic monophosphate (cAMP) and extracellular-signal-regulated kinase (ERK) signaling pathways. Exposure of malignant cells to Thomas-EMF transiently changed the level of cellular cAMP and promoted ERK phosphorylation. Pharmacologic inhibitors (SQ22536) and activators (forskolin) of cAMP production both blocked the ability of Thomas-EMF to inhibit cell proliferation, and an inhibitor of the MAP kinase pathway (PD98059) was able to partially block Thomas-EMF-dependent inhibition of cell proliferation. Genetic modulation of protein kinase A (PKA) in B16-BL6 cells also altered the effect of Thomas-EMF on cell proliferation. Cells transfected with the constitutively active form of PKA (PKA-CA), which interfered with ERK phosphorylation, also interfered with the Thomas-EMF effect on cell proliferation. The non-malignant cells did not show any EMF-dependent changes in cAMP levels, ERK phosphorylation, or cell growth. These data indicate that exposure to the specific Thomas-EMF pattern can inhibit the growth of malignant cells in a manner dependent on contributions from the cAMP and MAP kinase pathways. Bioelectromagnetics. 39;217-230, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  1. Recent Advances in the Development of Antimicrobial Peptides (AMPs): Attempts for Sustainable Medicine?

    Science.gov (United States)

    Kokel, Anne; Torok, Marianna

    2018-01-17

    Since the first isolation of antimicrobial peptides (AMPs) they have attracted extensive interest in medicinal chemistry. However, only a few AMP-based drugs are currently available on the market. Despite their effectiveness, biodegradability, and versatile mode of action that is less likely to induce resistance compared to conventional antibiotics, AMPs suffer from major issues that need to be addressed to broaden their use. Notably, AMPs can lack selectivity leading to side effects and cytotoxicity, and also exhibit in vivo instability. Several strategies are being actively considered to overcome the limitations that restrain the success of AMPs. In the current work, recent strategies reported for improving AMPs in the context of drug design and delivery were surveyed, and also their possible impact on patients and the environment was assessed. As a major advantage AMPs possess an easily tunable skeleton offering opportunities to improve their properties. Strategic structural modifications and the beneficial properties of cyclic or branched AMPs in term of stability have been reported. The conjugation of AMPs with nanoparticles has also been explored to increase their in vivo stability. Other techniques such as the coupling of AMPs with specific antibodies aim to increase the selectivity of the potential drug towards the target. These strategies were evaluated for their effect on the environment highlighting green technologies. Although further research is needed taking into account both environmental and human health consequences of novel AMPs several of these compounds are promising drug candidates for use in sustainable medicine. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

    Directory of Open Access Journals (Sweden)

    Valeriy M Paramonov

    2015-09-01

    Full Text Available 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.

  3. Sleutelmag en amp

    Directory of Open Access Journals (Sweden)

    P. J. Rossouw

    1985-06-01

    Full Text Available As die Heidelbergse Kategismus (Sondag 31 handel oor die sleutels van die Koninkryk, dan word die betrokkenheid van die ampte slegs per implikasie veronderstel. Meer eksplisiet word die amp en sleutelmag in die Nederlandse Geloofsbelydenis gekoppel; “ons glo dat hierdie ware kerk ooreenkomstig die geestelike bestuurswyse wat ons Here ons in sy Woord geleer het, geregeer moet word... hulle (moet sorg dra dat die ware godsdiens onderhou (word, die ware leer orals versprei, die oortreders op geestelike wyse vermaan en in toom gehou (word... (N.G.B. art. 30 en: “Ons glo verder dat die regeerders van die kerk, al is dit nuttig en goed om onder mekaar ’n bepaalde orde tot instandhouding van die liggaam van die kerk in te stel en te handhaaf, tog noukeurig moet oppas om nie af te wyk van wat Christus, ons enigste Meester, vir ons ingestel het nie... Ons aanvaar derhalwe slegs wat kan dien om eendrag en eenheid te bewaar en te bevorder, en om alles in gehoorsaamheid van God te onderhou. Daarvoor is nodig die ban en alles wat daarmee saamhang, toegepas ooreenkomstig die Woord van God” (N.G.B. art. 32.

  4. Optogenetic Manipulation of Cyclic Di-GMP (c-di-GMP) Levels Reveals the Role of c-di-GMP in Regulating Aerotaxis Receptor Activity in Azospirillum brasilense.

    Science.gov (United States)

    O'Neal, Lindsey; Ryu, Min-Hyung; Gomelsky, Mark; Alexandre, Gladys

    2017-09-15

    Bacterial chemotaxis receptors provide the sensory inputs that inform the direction of navigation in changing environments. Recently, we described the bacterial second messenger cyclic di-GMP (c-di-GMP) as a novel regulator of a subclass of chemotaxis receptors. In Azospirillum brasilense , c-di-GMP binds to a chemotaxis receptor, Tlp1, and modulates its signaling function during aerotaxis. Here, we further characterize the role of c-di-GMP in aerotaxis using a novel dichromatic optogenetic system engineered for manipulating intracellular c-di-GMP levels in real time. This system comprises a red/near-infrared-light-regulated diguanylate cyclase and a blue-light-regulated c-di-GMP phosphodiesterase. It allows the generation of transient changes in intracellular c-di-GMP concentrations within seconds of irradiation with appropriate light, which is compatible with the time scale of chemotaxis signaling. We provide experimental evidence that binding of c-di-GMP to the Tlp1 receptor activates its signaling function during aerotaxis, which supports the role of transient changes in c-di-GMP levels as a means of adjusting the response of A. brasilense to oxygen gradients. We also show that intracellular c-di-GMP levels in A. brasilense change with carbon metabolism. Our data support a model whereby c-di-GMP functions to imprint chemotaxis receptors with a record of recent metabolic experience, to adjust their contribution to the signaling output, thus allowing the cells to continually fine-tune chemotaxis sensory perception to their metabolic state. IMPORTANCE Motile bacteria use chemotaxis to change swimming direction in response to changes in environmental conditions. Chemotaxis receptors sense environmental signals and relay sensory information to the chemotaxis machinery, which ultimately controls the swimming pattern of cells. In bacteria studied to date, differential methylation has been known as a mechanism to control the activity of chemotaxis receptors and

  5. Bacterial subversion of cAMP signalling inhibits cathelicidin expression, which is required for innate resistance to Mycobacterium tuberculosis

    Science.gov (United States)

    Gupta, Shashank; Winglee, Kathryn; Gallo, Richard; Bishai, William R

    2017-01-01

    Antimicrobial peptides such as cathelicidins are an important component of innate immune defence against inhaled microorganisms and have demonstrated antimicrobial activity against Mycobacterium tuberculosis with in vitro models. Despite this, little is known about the regulation and expression of cathelicidin during tuberculosis in vivo. We sought to determine whether the cathelicidin-related antimicrobial peptide (Cramp) gene, the murine functional homologue of the human cathelicidin gene (CAMP or LL-37), is required for regulating protective immunity during M. tuberculosis infection in vivo. We used Cramp−/− mice in a validated model of pulmonary tuberculosis and conducted cell-based assays with macrophages from these mice. We evaluated the in vivo susceptibility of Cramp−/− mice to infection and further dissected various pro-inflammatory immune responses against M. tuberculosis. We observed increased susceptibility of Cramp−/− mice to M. tuberculosis compared to wild type mice. Macrophages from Cramp−/− mice were unable to control M. tuberculosis growth in an in vitro infection model, were deficient in intracellular calcium influx and were defective in stimulating T-cells. Additionally, CD4 and CD8 T-cells from Cramp−/− mice produced less IFNβ upon stimulation. Furthermore, bacterial-derived cyclic-AMP modulated cathelicidin expression in macrophages. Our results demonstrate that cathelicidin is required for innate resistance to M. tuberculosis in a relevant animal model and is a key mediator in regulating the levels of pro-inflammatory cytokines by calcium and cyclic nucleotides. PMID:28097645

  6. Malonyl-CoA decarboxylase (MCD) is differentially regulated in subcellular compartments by 5'AMP-activated protein kinase (AMPK). Studies using H9c2 cells overexpressing MCD and AMPK by adenoviral gene transfer technique.

    Science.gov (United States)

    Sambandam, Nandakumar; Steinmetz, Michael; Chu, Angel; Altarejos, Judith Y; Dyck, Jason R B; Lopaschuk, Gary D

    2004-07-01

    Malonyl-CoA, a potent inhibitor of carnitine pamitoyl transferase-I (CPT-I), plays a pivotal role in fuel selection in cardiac muscle. Malonyl-CoA decarboxylase (MCD) catalyzes the degradation of malonyl-CoA, removes a potent allosteric inhibition on CPT-I and thereby increases fatty acid oxidation in the heart. Although MCD has several Ser/Thr phosphorylation sites, whether it is regulated by AMP-activated protein kinase (AMPK) has been controversial. We therefore overexpressed MCD (Ad.MCD) and constitutively active AMPK (Ad.CA-AMPK) in H9c2 cells, using an adenoviral gene delivery approach in order to examine if MCD is regulated by AMPK. Cells infected with Ad.CA-AMPK demonstrated a fourfold increase in AMPK activity as compared with control cells expressing green fluorescent protein (Ad.GFP). MCD activity increased 40- to 50-fold in Ad.MCD + Ad.GFP cells when compared with Ad.GFP control. Co-expressing AMPK with MCD further augmented MCD expression and activity in Ad.MCD + Ad.CA-AMPK cells compared with the Ad.MCD + Ad.GFP control. Subcellular fractionation further revealed that 54.7 kDa isoform of MCD expression was significantly higher in cytosolic fractions of Ad.MCD + Ad.CA-AMPK cells than of the Ad.MCD +Ad.GFP control. However, the MCD activities in cytosolic fractions were not different between the two groups. Interestingly, in the mitochondrial fractions, MCD activity significantly increased in Ad.MCD + Ad.CA-AMPK cells when compared with Ad.MCD + Ad.GFP cells. Using phosphoserine and phosphothreonine antibodies, no phosphorylation of MCD by AMPK was observed. The increase in MCD activity in mitochondria-rich fractions of Ad.MCD + Ad.CA-AMPK cells was accompanied by an increase in the level of the 50.7 kDa isoform of MCD protein in the mitochondria. This differential regulation of MCD expression and activity in the mitochondria by AMPK may potentially regulate malonyl-CoA levels at sites nearby CPT-I on the mitochondria.

  7. Cyclic adenosine monophosphate levels and the function of skin microvascular endothelial cells.

    Science.gov (United States)

    Tuder, R M; Karasek, M A; Bensch, K G

    1990-02-01

    The maintenance of the normal epithelioid morphology of human dermal microvascular endothelial cells (MEC) grown in vitro depends strongly on the presence of factors that increase intracellular levels of cyclic AMP. Complete removal of dibutyryl cAMP and isobutylmethylxanthine (IMX) from the growth medium results in a progressive transition from an epithelioid to a spindle-shaped cell line. This transition cannot be reversed by the readdition of dibutyryl cAMP and IMX to the growth medium or by addition of agonists that increase cAMP levels. Spindle-shaped MEC lose the ability to express Factor VIII rAG and DR antigens and to bind peripheral blood mononuclear leukocyte (PBML). Ultrastructural analyses of transitional cells and spindle-shaped cells show decreased numbers of Weibel-Palade bodies in transitional cells and their complete absence in spindle-shaped cells. Interferon-gamma alters several functional properties of both epithelioid and spindle-shaped cells. In the absence of dibutyryl cAMP it accelerates the transition from epithelial to spindle-shaped cells, whereas in the presence of cyclic AMP interferon-gamma increases the binding of PBMLs to both epithelioid and spindle-shaped MEC and the endocytic activity of the endothelial cells. These results suggest that cyclic AMP is an important second messenger in the maintenance of several key functions of microvascular endothelial cells. Factors that influence the levels of this messenger in vivo can be expected to influence the angiogenic and immunologic functions of the microvasculature.

  8. Glycogen Synthase Kinase 3α Is the Main Isoform That Regulates the Transcription Factors Nuclear Factor-Kappa B and cAMP Response Element Binding in Bovine Endothelial Cells Infected with Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Octavio Silva-García

    2018-01-01

    Full Text Available Glycogen synthase kinase 3 (GSK3 is a constitutive enzyme implicated in the regulation of cytokine expression and the inflammatory response during bacterial infections. Mammals have two GSK3 isoforms named GSK3α and GSK3β that plays different but often overlapping functions. Although the role of GSK3β in cytokine regulation during the inflammatory response caused by bacteria is well described, GSK3α has not been found to participate in this process. Therefore, we tested if GSK3α may act as a regulatory isoform in the cytokine expression by bovine endothelial cells infected with Staphylococcus aureus because this bacterium is one of the major pathogens that cause tissue damage associated with inflammatory dysfunction. Interestingly, although both isoforms were phosphorylated–inactivated, we consistently observed a higher phosphorylation of GSK3α at Ser21 than that of GSK3β at Ser9 after bacterial challenge. During a temporal course of infection, we characterized a molecular switch from pro-inflammatory cytokine expression (IL-8, promoted by nuclear factor-kappa B (NF-κB, at an early stage (2 h to an anti-inflammatory cytokine expression (IL-10, promoted by cAMP response element binding (CREB, at a later stage (6 h. We observed an indirect effect of GSK3α activity on NF-κB activation that resulted in a low phosphorylation of CREB at Ser133, a decreased interaction between CREB and the co-activator CREB-binding protein (CBP, and a lower expression level of IL-10. Gene silencing of GSK3α and GSK3β with siRNA indicated that GSK3α knockout promoted the interaction between CREB and CBP that, in turn, increased the expression of IL-10, reduced the interaction of NF-κB with CBP, and reduced the expression of IL-8. These results indicate that GSK3α functions as the primary isoform that regulates the expression of IL-10 in endothelial cells infected with S. aureus.

  9. cAMP/PKA signalling reinforces the LATS–YAP pathway to fully suppress YAP in response to actin cytoskeletal changes

    Science.gov (United States)

    Kim, Minchul; Kim, Miju; Lee, Seunghee; Kuninaka, Shinji; Saya, Hideyuki; Lee, Ho; Lee, Sookyung; Lim, Dae-Sik

    2013-01-01

    Actin cytoskeletal damage induces inactivation of the oncoprotein YAP (Yes-associated protein). It is known that the serine/threonine kinase LATS (large tumour suppressor) inactivates YAP by phosphorylating its Ser127 and Ser381 residues. However, the events downstream of actin cytoskeletal changes that are involved in the regulation of the LATS–YAP pathway and the mechanism by which LATS differentially phosphorylates YAP on Ser127 and Ser381 in vivo have remained elusive. Here, we show that cyclic AMP (cAMP)-dependent protein kinase (PKA) phosphorylates LATS and thereby enhances its activity sufficiently to phosphorylate YAP on Ser381. We also found that PKA activity is involved in all contexts previously reported to trigger the LATS–YAP pathway, including actin cytoskeletal damage, G-protein-coupled receptor activation, and engagement of the Hippo pathway. Inhibition of PKA and overexpression of YAP cooperate to transform normal cells and amplify neural progenitor pools in developing chick embryos. We also implicate neurofibromin 2 as an AKAP (A-kinase-anchoring protein) scaffold protein that facilitates the function of the cAMP/PKA–LATS–YAP pathway. Our study thus incorporates PKA as novel component of the Hippo pathway. PMID:23644383

  10. Effects of prostaglandin E2 and cAMP elevating drugs on GM-CSF release by cultured human airway smooth muscle cells. Relevance to asthma therapy.

    Science.gov (United States)

    Lazzeri, N; Belvisi, M G; Patel, H J; Yacoub, M H; Chung, K F; Mitchell, J A

    2001-01-01

    Human airway smooth muscle (HASM) cells release granulocyte macrophage-colony stimulating factor (GM-CSF) and express cyclooxygenase (COX)-2 (resulting in the release of prostaglandin [PG] E2) after stimulation with cytokines. Because COX-2 activity can regulate a number of inflammatory processes, we have assessed its effects, as well as those of agents that modulate cyclic adenosine monophosphate (cAMP), on GM-CSF release by HASM cells. Cells stimulated with a combination of proinflammatory cytokines (interleukin-1beta and tumor necrosis factor-alpha each at 10 ng/ml) for 24 h released significant amounts of PGE2 (measured by radioimmunoassay) and GM-CSF (measured by enzyme-linked immunosorbent assay). Indomethacin and other COX-1/COX-2 inhibitors caused concentration-dependent inhibitions of PGE2 concomitantly with increases in GM-CSF formation. Addition of exogenous PGE2 or the beta2-agonist fenoterol, which increase cAMP, to cytokine-treated HASM cells had no effect on GM-CSF release unless COX activity was first blocked with indomethacin. The type 4 phosphodiesterase inhibitors rolipram and SB 207499 both caused concentration-dependent reductions in GM-CSF production. Thus, when HASM cells are activated with cytokines they release PGE2, which acts as a "braking mechanism" to limit the coproduction of GM-CSF. Moreover, agents that elevate cAMP also reduce GM-CSF formation by these cells.

  11. Differential regulation by agonist and phorbol ester of cloned m1 and m2 muscarinic acetylcholine receptors in mouse Y1 adrenal cells and in Y1 cells deficient in cAMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Scherer, N.M.; Nathanson, N.M.

    1990-01-01

    Cloned muscarinic acetylcholine m1 and m2 receptors were expressed in stably transfected mouse Y1 adrenal cells and in a variant Y1 line, Kin-8, which is deficient in cAMP-dependent protein kinase activity (PKA - ). m1 and m2 receptors were rapidly internalized following exposure of transfected PKA + or PKA - cells to the muscarinic agonist carbachol. Thus, agonist-dependent internalization of m1 and m2 did not require PKA activity. A differential effect of PKA on regulation by agonist of the m2 receptor, but not the m1 receptor, was unmasked in PKA - cells. These data indicate that the basal activity of PKA may modulate the agonist-dependent internalization of the m2 receptor, but not the m1 receptor. The internalization of the m1 and m2 receptors in both PKA + and PKA - cells was accompanied by desensitization of functional responses. Exposure of PKA + cells to 10 -7 M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, resulted in a 30 ± 9% decrease in the number of m1 receptors on the cell surface. The m2 receptor was not internalized following treatment of either PKA + or PKA - cells with PMA. Thus, the m1 and m2 receptors show differential sensitivity to internalization by PMA. Agonist-dependent internalization of the m1 receptor appeared to be independent of activation of PKC because (1) agonist-dependent internalization of m1 was not attenuated in PKA - cells, (2) the rate and extent of internalization of m1 in cells exposed to PMA were less than those in cells exposed to agonist, and (3) treatment of cells with concanavalin A selectivity blocked internalization of m1 in cells exposed to PMA, but not to agonist. The effects of agonist and PMA on receptor internalization were not additive. Exposure of PKA + or PKA - cells to PMA reduced the magnitude of pilocarpine-stimulated PI hydrolysis by about 25%

  12. AMP-guided tumour-specific nanoparticle delivery via adenosine A1 receptor.

    Science.gov (United States)

    Dai, Tongcheng; Li, Na; Han, Fajun; Zhang, Hua; Zhang, Yuanxing; Liu, Qin

    2016-03-01

    Active targeting-ligands have been increasingly used to functionalize nanoparticles for tumour-specific clinical applications. Here we utilize nucleotide adenosine 5'-monophosphate (AMP) as a novel ligand to functionalize polymer-based fluorescent nanoparticles (NPs) for tumour-targeted imaging. We demonstrate that AMP-conjugated NPs (NPs-AMP) efficiently bind to and are following internalized into colon cancer cell CW-2 and breast cancer cell MDA-MB-468 in vitro. RNA interference and inhibitor assays reveal that the targeting effects mainly rely on the specific binding of AMP to adenosine A1 receptor (A1R), which is greatly up-regulated in cancer cells than in matched normal cells. More importantly, NPs-AMP specifically accumulate in the tumour site of colon and breast tumour xenografts and are further internalized into the tumour cells in vivo via tail vein injection, confirming that the high in vitro specificity of AMP can be successfully translated into the in vivo efficacy. Furthermore, NPs-AMP exhibit an active tumour-targeting behaviour in various colon and breast cancer cells, which is positively related to the up-regulation level of A1R in cancer cells, suggesting that AMP potentially suits for more extensive A1R-overexpressing cancer models. This work establishes AMP to be a novel tumour-targeting ligand and provides a promising strategy for future diagnostic or therapeutic applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    1982-01-01

    Freshly isolated chicken intestinal cells contain approximately 20 pmol adenosine 3',5'-cyclic monophosphate (cAMP)/mg cellular protein. Incubation with 3 μg/ml cholera toxin (CT) at 37 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 + 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 + entry. The data provide evidence for a cAMP-mediated control of intestinal cell Na + uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na + during induction of intestinal secretory activity. Studies on the time-dependent effects of chlorpromazine on both intracellular cAMP concentration and Na + influx suggest that the reactivation of the Na + transport system after cAMP-induced inhibition is slow relative to the disappearance of cAMP

  14. Cyclic ADP-ribose and heat regulate oxytocin release via CD38 and TRPM2 in the hypothalamus during social or psychological stress in mice

    Directory of Open Access Journals (Sweden)

    Jing Zhong

    2016-07-01

    Full Text Available Hypothalamic oxytocin (OT is released into the brain by cyclic ADP-ribose (cADPR with or without depolarizing stimulation. Previously, we showed that the intracellular free calcium concentration ([Ca2+]i that seems to trigger OT release can be elevated by -NAD+, cADPR, and ADP in mouse oxytocinergic neurons. As these -NAD+ metabolites activate warm-sensitive TRPM2 cation channels, when the incubation temperature is increased, the [Ca2+]i in hypothalamic neurons is elevated. However, it has not been determined whether OT release is facilitated by heat in vitro or hyperthermia in vivo in combination with cADPR. Furthermore, it has not been examined whether CD38 and TRPM2 exert their functions on OT release during stress or stress-induced hyperthermia in relation to the anxiolytic roles and social behaviors of OT under stress conditions. Here, we report that OT release from the isolated hypothalami of male mice in culture was enhanced by extracellular application of cADPR or increasing the incubation temperature from 35°C to 38.5°C, and simultaneous stimulation showed a greater effect. This release was inhibited by a cADPR-dependent ryanodine receptor inhibitor and a nonspecific TRPM2 inhibitor. The facilitated release by heat and cADPR was suppressed in the hypothalamus isolated from CD38 knockout mice and CD38- or TRPM2-knockdown mice. In the course of these experiments, we noted that OT release differed markedly between individual mice under stress with group housing. That is, when male mice received cage-switch stress and eliminated due to their social subclass, significantly higher levels of OT release were found in subordinates compared with ordinates. In mice exposed to anxiety stress in an open field, the cerebrospinal fluid (CSF OT level increased transiently at 5 minutes after exposure, and the rectal temperature also increased from 36.6°C to 37.8°C. OT levels in the CSF of mice with lipopolysaccharide-induced fever (+0.8

  15. Studies on c-AMP contents in sea urchin eggs fertilized with normal and x-irradiated sperm

    International Nuclear Information System (INIS)

    Kimura, Hiroshi

    1975-01-01

    Intracellular levels of cyclic 3', 5'-adenosine monophosphate (c-AMP) seemed to remain constant through the first cleavage cycle of sea urchin eggs. X-irradiation to the sperm, which induced the first cleavage delay, did not change this level. Although it was shown in the previous paper that X-ray-induced cleavage delay was reduced by caffeine but not by aminophyline, both caffeine and aminophyline caused an increase in c-AMP levels. These results indicated the possibility that c-AMP does not mediate this caffeine effect on cleavage delay. (auth.)

  16. Inhibition of cAMP-Activated Intestinal Chloride Secretion by Diclofenac: Cellular Mechanism and Potential Application in Cholera

    OpenAIRE

    Pongkorpsakol, Pawin; Pathomthongtaweechai, Nutthapoom; Srimanote, Potjanee; Soodvilai, Sunhapas; Chatsudthipong, Varanuj; Muanprasat, Chatchai

    2014-01-01

    Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84) cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell...

  17. A conjugate of decyltriphenylphosphonium with plastoquinone can carry cyclic adenosine monophosphate, but not cyclic guanosine monophosphate, across artificial and natural membranes.

    Science.gov (United States)

    Firsov, Alexander M; Rybalkina, Irina G; Kotova, Elena A; Rokitskaya, Tatyana I; Tashlitsky, Vadim N; Korshunova, Galina A; Rybalkin, Sergei D; Antonenko, Yuri N

    2018-02-01

    The present study demonstrated for the first time the interaction between adenosine 3',5'-cyclic monophosphate (cAMP), one of the most important signaling compounds in living organisms, and the mitochondria-targeted antioxidant plastoquinonyl-decyltriphenylphosphonium (SkQ1). The data obtained on model liquid membranes and human platelets revealed the ability of SkQ1 to selectively transport cAMP, but not guanosine 3',5'-cyclic monophosphate (cGMP), across both artificial and natural membranes. In particular, SkQ1 elicited translocation of cAMP from the source to the receiving phase of a Pressman-type cell, while showing low activity with cGMP. Importantly, only conjugate with plastoquinone, but not dodecyl-triphenylphosphonium, was effective in carrying cAMP. In human platelets, SkQ1 also appeared to serve as a carrier of cAMP, but not cGMP, from outside to inside the cell, as measured by phosphorylation of the vasodilator stimulated phosphoprotein. The SkQ1-induced transfer of cAMP across the plasma membrane found here can be tentatively suggested to interfere with cAMP signaling pathways in living cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Control of renin secretion from rat juxtaglomerular cells by cAMP-specific phosphodiesterases

    DEFF Research Database (Denmark)

    Friis, Ulla G; Jensen, Boye L; Sethi, Shala

    2002-01-01

    , and the PDE4 inhibitor rolipram enhanced cellular cAMP content. Dialysis of single JG cells with cAMP in whole-cell patch-clamp experiments led to concentration-dependent, biphasic changes in cell membrane capacitance (C(m)) with a marked increase in C(m) at 1 micromol/L, no net change at 10 micromol....../L, and a decrease at 100 micromol/L cAMP. cGMP also had a dual effect on C(m) at 10-fold higher concentration compared with cAMP. Trequinsin, milrinone, and rolipram mimicked the effect of cAMP on C(m). Trequinsin, cAMP, and cGMP enhanced outward current 2- to 3-fold at positive membrane potentials. The effects...... of cAMP, cGMP, and trequinsin on C(m) and cell currents were abolished by inhibition of protein kinase A with Rp-cAMPs. We conclude that degradation of cAMP by PDE3 and PDE4 contributes to regulation of renin release from JG cells. Our data provide evidence at the cellular level that stimulation...

  19. Ru (amp)(bipy)Cl

    Indian Academy of Sciences (India)

    Administrator

    [RuV(amp)(bipy)O]+ intermediate complex which leads to the high affinity for hydrogen atom/hydride abstraction. Acknowledgement. We gratefully acknowledge the financial support from the Department of Science &. Technology, Government of India. We are thankful to Shri Hardyal Singh for his encouragement. Reference.

  20. Atrazine acts as an endocrine disrupter by inhibiting cAMP-specific phosphodiesterase-4

    International Nuclear Information System (INIS)

    Kucka, Marek; Pogrmic-Majkic, Kristina; Fa, Svetlana; Stojilkovic, Stanko S.; Kovacevic, Radmila

    2012-01-01

    Atrazine, one of the most commonly used herbicides worldwide, acts as an endocrine disruptor, but the mechanism of its action has not been characterized. In this study, we show that atrazine rapidly increases cAMP levels in cultured rat pituitary and testicular Leydig cells in a concentration-dependent manner, but less effectively than 3-isobutyl-1-methylxanthine, a competitive non-specific inhibitor of phosphodiesterases (PDEs). In forskolin (an activator of adenylyl cyclase)- and probenecid (an inhibitor of cyclic nucleotide transporters)-treated cells, but not in 3-isobutyl-1-methylxanthine-treated cells, atrazine further increased cAMP levels, indicating that inhibition of PDEs accounts for accumulation of cAMP. In contrast to cAMP, atrazine did not alter cGMP levels, further indicating that it inhibits cAMP-specific PDEs. Atrazine-induced changes in cAMP levels were sufficient to stimulate prolactin release in pituitary cells and androgen production in Leydig cells, indicating that it acts as an endocrine disrupter both in cells that secrete by exocytosis of prestored hormones and in cells that secrete by de novo hormone synthesis. Rolipram abolished the stimulatory effect of atrazine on cAMP release in both cell types, suggesting that it acts as an inhibitor of PDE4s, isoforms whose mRNA transcripts dominate in pituitary and Leydig cells together with mRNA for PDE8A. In contrast, immortalized lacto-somatotrophs showed low expression of these mRNA transcripts and several fold higher cAMP levels compared to normal pituitary cells, and atrazine was unable to further increase cAMP levels. These results indicate that atrazine acts as a general endocrine disrupter by inhibiting cAMP-specific PDE4s. -- Highlights: ► Atrazine stimulates cAMP accumulation in pituitary and Leydig cells. ► Atrazine also stimulates PRL and androgens secretion. ► Stimulatory effects of atrazine were abolished in cells with IBMX-inhibited PDEs. ► Atrazine specificity toward cAMP

  1. AMP affects intracellular Ca2+ signaling, migration, cytokine secretion and T cell priming capacity of dendritic cells.

    Directory of Open Access Journals (Sweden)

    Elisabeth Panther

    Full Text Available The nucleotide adenosine-5'-monophosphate (AMP can be released by various cell types and has been shown to elicit different cellular responses. In the extracellular space AMP is dephosphorylated to the nucleoside adenosine which can then bind to adenosine receptors. However, it has been shown that AMP can also activate A(1 and A(2a receptors directly. Here we show that AMP is a potent modulator of mouse and human dendritic cell (DC function. AMP increased intracellular Ca(2+ concentration in a time and dose dependent manner. Furthermore, AMP stimulated actin-polymerization in human DCs and induced migration of immature human and bone marrow derived mouse DCs, both via direct activation of A(1 receptors. AMP strongly inhibited secretion of TNF-α and IL-12p70, while it enhanced production of IL-10 both via activation of A(2a receptors. Consequently, DCs matured in the presence of AMP and co-cultivated with naive CD4(+CD45RA(+ T cells inhibited IFN-γ production whereas secretion of IL-5 and IL-13 was up-regulated. An enhancement of Th2-driven immune response could also be observed when OVA-pulsed murine DCs were pretreated with AMP prior to co-culture with OVA-transgenic naïve OTII T cells. An effect due to the enzymatic degradation of AMP to adenosine could be ruled out, as AMP still elicited migration and changes in cytokine secretion in bone-marrow derived DCs generated from CD73-deficient animals and in human DCs pretreated with the ecto-nucleotidase inhibitor 5'-(alpha,beta-methylene diphosphate (APCP. Finally, the influence of contaminating adenosine could be excluded, as AMP admixed with adenosine desaminase (ADA was still able to influence DC function. In summary our data show that AMP when present during maturation is a potent regulator of dendritic cell function and point out the role for AMP in the pathogenesis of inflammatory disorders.

  2. Effects of oxytocin and methacholine on cyclic nucleotide levels of rabbit myometrium.

    Science.gov (United States)

    Schlageter, N; Janis, R A; Gualtieri, R T; Hechter, O

    1980-03-01

    The effects of oxytocin and methacholine on cyclic nucleotide levels in estrogen-primed rabbit myometrium were studied in the presence and absence of 1-methyl-3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor. In the absence of MIX, methacholine increased guanosine 3',5'-cyclic monophosphate (cGMP) levels at a time when contraction was decreasing, but had no influence on adenosine 3',5'-cyclic monophosphate (cAMP) levels. In contrast, oxytocin did not elevate cGMP, but rapidly decreased cAMP levels. MIX (1 mM) increased both cAMP and cGMP levels. Oxytocin or methacholine further increased cGMP, indicating activation of guanylate cyclase. Oxytocin- but not methacholine-induced stimulation of guanylate cyclase was abolished in Ca2+-free solution. Oxytocin increased cAMP over the levels produced by MIX alone, whereas methacholine decreased cAMP below the MIX control values; these effects were insensitive to indomethacin. Tissue levels of cGMP and cAMP did not directly correlate with isometric tension. The results also indicate that both oxytocin and methacholine stimulate guanylate cyclase but have opposing effects on adenylate cyclase of rabbit myometrium.

  3. Role of calmodulin and calcineurin in regulating flagellar motility and wave polarity in Leishmania.

    Science.gov (United States)

    Mukhopadhyay, Aakash Gautam; Dey, Chinmoy Sankar

    2017-11-01

    We have previously reported the involvement of cyclic AMP in regulating flagellar waveforms in Leishmania. Here, we investigated the roles of calcium, calmodulin, and calcineurin in flagellar motility regulation in L. donovani. Using high-speed videomicroscopy, we show that calcium-independent calmodulin and calcineurin activity is necessary for motility in Leishmania. Inhibition of calmodulin and calcineurin induced ciliary beats interrupting flagellar beating in both live (in vivo) and ATP-reactivated (in vitro) parasites. Our results indicate that signaling mediated by calmodulin and calcineurin operates antagonistically to cAMP signaling in regulating the waveforms of Leishmania flagellum. These two pathways are possibly involved in maintaining the balance between the two waveforms, essential for responding to environmental cues, survival, and infectivity.

  4. Characterization of a crp* mutant of the E. coli cAMP receptor protein

    International Nuclear Information System (INIS)

    Ren, Y.L.; Garges, S.; Adhya, S.; Krakow, J.S.

    1987-01-01

    One of the crp* mutants previously isolated to activate lac promoter in vivo has been characterized with regard to its biochemical properties. CRP*592 shows a more open conformation than CRP as indicated by its sensitivity to proteolytic attack. Dithionitrobenzoic acid mediated intersubunit crosslinking of CRP requires cAMP; this reaction occurs with unliganded CRP*592. Binding of CRP to its site on the lac promoter and activation of abortive initiation is effected by cAMP but not by cGMP. CRP*592 can activate abortive initiation in the presence of cAMP or cGMP and also at a high CRP*592 concentration in the absence of cyclic nucleotide. DNase I footprinting shows that cAMP-CRP* binds to its site on lac P + while unliganded CRP* and cGMP-CRP* form a stable complex with the [ 32 P]lac P + only in the presence of RNA polymerase. While cGMP binds to CRP it cannot replace cAMP in effecting the conformation necessary for site specific promoter binding; the weakly active unliganded CRP*592 can be shifted to a functional conformation by cAMP, cGMP and RNA polymerase

  5. Understanding cAMP-dependent allostery by NMR spectroscopy: comparative analysis of the EPAC1 cAMP-binding domain in its apo and cAMP-bound states.

    Science.gov (United States)

    Mazhab-Jafari, Mohammad T; Das, Rahul; Fotheringham, Steven A; SilDas, Soumita; Chowdhury, Somenath; Melacini, Giuseppe

    2007-11-21

    cAMP (adenosine 3',5'-cyclic monophosphate) is a ubiquitous second messenger that activates a multitude of essential cellular responses. Two key receptors for cAMP in eukaryotes are protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC), which is a recently discovered guanine nucleotide exchange factor (GEF) for the small GTPases Rap1 and Rap2. Previous attempts to investigate the mechanism of allosteric activation of eukaryotic cAMP-binding domains (CBDs) at atomic or residue resolution have been hampered by the instability of the apo form, which requires the use of mixed apo/holo systems, that have provided only a partial picture of the CBD apo state and of the allosteric networks controlled by cAMP. Here, we show that, unlike other eukaryotic CBDs, both apo and cAMP-bound states of the EPAC1 CBD are stable under our experimental conditions, providing a unique opportunity to define at an unprecedented level of detail the allosteric interactions linking two critical functional sites of this CBD. These are th