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Sample records for activates cyclic-amp response

  1. Nucleoprotein structure influences the response of the mouse mammary tumor virus promoter to activation of the cyclic AMP signalling pathway.

    PubMed Central

    Pennie, W D; Hager, G L; Smith, C L

    1995-01-01

    Recent studies have provided evidence of crosstalk between steroid receptors and cyclic AMP (cAMP) signalling pathways in the regulation of gene expression. A synergism between intracellular phosphorylation inducers and either glucocorticoids or progestins has been shown to occur during activation of the mouse mammary tumor virus (MMTV) promoter. We have investigated the effect of 8-Br-cAMP and okadaic acid, modulators of cellular kinases and phosphatases, on the hormone-induced activation of the MMTV promoter in two forms: a transiently transfected template with a disorganized, accessible nucleoprotein structure and a stably replicating template with an ordered, inaccessible nucleoprotein structure. Both okadaic acid and 8-Br-cAMP synergize significantly with either glucocorticoids or progestins in activating the transiently transfected MMTV template. In contrast, 8-Br-cAMP, but not okadaic acid, is antagonistic to hormone-induced activation of the stably replicating MMTV template. Nuclear run-on experiments demonstrate that this inhibition is a transcriptional effect on both hormone-induced transcription and basal transcription. Surprisingly, 8-Br-cAMP does not inhibit glucocorticoid-induced changes in restriction enzyme access and nuclear factor 1 binding. However, association of a complex with the TATA box region is inhibited in the presence of 8-Br-cAMP. Thus, cAMP treatment interferes with the initiation process but does not inhibit interaction of the receptor with the template. Since the replicated, ordered MMTV templates and the transfected, disorganized templates show opposite responses to 8-Br-cAMP treatment, we conclude that chromatin structure can influence the response of a promoter to activation of the cAMP signalling pathway. PMID:7891707

  2. Cyclic AMP-dependent protein kinase activity in Trypanosoma cruzi.

    PubMed Central

    Ulloa, R M; Mesri, E; Esteva, M; Torres, H N; Téllez-Iñón, M T

    1988-01-01

    A cyclic AMP-dependent protein kinase activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns, giving two peaks of kinase activity, which were eluted at 0.15 M- and 0.32 M-NaCl respectively. The second activity peak was stimulated by nanomolar concentrations of cyclic AMP. In addition, a cyclic AMP-binding protein co-eluted with the second kinase activity peak. Cyclic AMP-dependent protein kinase activity was further purified by gel filtration, affinity chromatography on histone-agarose and cyclic AMP-agarose, as well as by chromatography on CM-Sephadex. The enzyme ('holoenzyme') could be partially dissociated into two different components: 'catalytic' and 'regulatory'. The 'regulatory' component had specific binding for cyclic AMP, and it inhibited phosphotransferase activity of the homologous 'catalytic component' or of the 'catalytic subunit' from bovine heart. Cyclic AMP reversed these inhibitions. A 'holoenzyme preparation' was phosphorylated in the absence of exogenous phosphate acceptor and analysed by polyacrylamide-gel electrophoresis. A 56 kDa band was phosphorylated. The same preparation was analysed by Western blotting, by using polyclonal antibodies to the regulatory subunits of protein kinases type I or II. Both antibodies reacted with the 56 kDa band. Images Fig. 7. Fig. 8. PMID:2848508

  3. Induction of cyclooxygenase-2 by ginsenoside Rd via activation of CCAAT-enhancer binding proteins and cyclic AMP response binding protein

    SciTech Connect

    Jeong, Hye Gwang; Pokharel, Yuba Raj; Han, Eun Hee; Kang, Keon Wook . E-mail: kwkang@chosun.ac.kr

    2007-07-20

    Panax ginseng is a widely used herbal medicine in East Asia and is reported to have a variety of pharmacological effects against cardiovascular diseases and cancers. Here we show a unique effect of ginsenoside Rd (Rd) on cyclooxygenase-2 (COX-2) expression in RAW264.7 macrophages. Rd (100 {mu}g/ml), but not other ginsenosides induced COX-2 and increased prostaglandin E{sub 2} production. Gel shift and Western blot analyses using nuclear fractions revealed that Rd increased both the DNA binding of and the nuclear levels of CCAAT/enhancer binding protein (C/EBP){alpha}/{beta} and cyclic AMP response element binding protein (CREB), but not of p65, in RAW264.7 cells. Moreover, Rd increased the luciferase reporter gene activity in cells transfected with a 574-bp mouse COX-2 promoter construct. Site-specific mutation analyses confirmed that Rd-mediated transcriptional activation of COX-2 gene was regulated by C/EBP and CREB. These results provide evidence that Rd activated C/EBP and CREB, and that the activation of C/EBP and CREB appears to be essential for induction of COX-2 in RAW264.7 cells.

  4. Low-Power Laser Irradiation Suppresses Inflammatory Response of Human Adipose-Derived Stem Cells by Modulating Intracellular Cyclic AMP Level and NF-κB Activity

    PubMed Central

    Wang, Chau-Zen; Ho, Mei-Ling; Yeh, Ming-Long; Wang, Yan-Hsiung

    2013-01-01

    Mesenchymal stem cell (MSC)-based tissue regeneration is a promising therapeutic strategy for treating damaged tissues. However, the inflammatory microenvironment that exists at a local injury site might restrict reconstruction. Low-power laser irradiation (LPLI) has been widely applied to retard the inflammatory reaction. The purpose of this study was to investigate the anti-inflammatory effect of LPLI on human adipose-derived stem cells (hADSCs) in an inflammatory environment. We showed that the hADSCs expressed Toll-like Receptors (TLR) 1, TLR2, TLR3, TLR4, and TLR6 and that lipopolysaccharide (LPS) significantly induced the production of pro-inflammatory cytokines (Cyclooxygenase-2 (Cox-2), Interleukin-1β (IL-1β), Interleukin-6 (IL-6), and Interleukin-8 (IL-8)). LPLI markedly inhibited LPS-induced, pro-inflammatory cytokine expression at an optimal dose of 8 J/cm2. The inhibitory effect triggered by LPLI might occur through an increase in the intracellular level of cyclic AMP (cAMP), which acts to down-regulate nuclear factor kappa B (NF-κB) transcriptional activity. These data collectively provide insight for further investigations of the potential application of anti-inflammatory treatment followed by stem cell therapy. PMID:23342077

  5. Low-power laser irradiation suppresses inflammatory response of human adipose-derived stem cells by modulating intracellular cyclic AMP level and NF-κB activity.

    PubMed

    Wu, Jyun-Yi; Chen, Chia-Hsin; Wang, Chau-Zen; Ho, Mei-Ling; Yeh, Ming-Long; Wang, Yan-Hsiung

    2013-01-01

    Mesenchymal stem cell (MSC)-based tissue regeneration is a promising therapeutic strategy for treating damaged tissues. However, the inflammatory microenvironment that exists at a local injury site might restrict reconstruction. Low-power laser irradiation (LPLI) has been widely applied to retard the inflammatory reaction. The purpose of this study was to investigate the anti-inflammatory effect of LPLI on human adipose-derived stem cells (hADSCs) in an inflammatory environment. We showed that the hADSCs expressed Toll-like Receptors (TLR) 1, TLR2, TLR3, TLR4, and TLR6 and that lipopolysaccharide (LPS) significantly induced the production of pro-inflammatory cytokines (Cyclooxygenase-2 (Cox-2), Interleukin-1β (IL-1β), Interleukin-6 (IL-6), and Interleukin-8 (IL-8)). LPLI markedly inhibited LPS-induced, pro-inflammatory cytokine expression at an optimal dose of 8 J/cm². The inhibitory effect triggered by LPLI might occur through an increase in the intracellular level of cyclic AMP (cAMP), which acts to down-regulate nuclear factor kappa B (NF-κB) transcriptional activity. These data collectively provide insight for further investigations of the potential application of anti-inflammatory treatment followed by stem cell therapy. PMID:23342077

  6. Activation of pp70/85 S6 kinases in interleukin-2-responsive lymphoid cells is mediated by phosphatidylinositol 3-kinase and inhibited by cyclic AMP.

    PubMed Central

    Monfar, M; Lemon, K P; Grammer, T C; Cheatham, L; Chung, J; Vlahos, C J; Blenis, J

    1995-01-01

    Activation of phosphatidylinositol 3-kinase (PI3K) and activation of the 70/85-kDa S6 protein kinases (alpha II and alpha I isoforms, referred to collectively as pp70S6k) have been independently linked to the regulation of cell proliferation. We demonstrate that these kinases lie on the same signalling pathway and that PI3K mediates the activation of pp70 by the cytokine interleukin-2 (IL-2). We also show that the activation of pp70S6k can be blocked at different points along the signalling pathway by using specific inhibitors of T-cell proliferation. Inhibition of PI3K activity with structurally unrelated but highly specific PI3K inhibitors (wortmannin or LY294002) results in inhibition of IL-2-dependent but not phorbol ester (conventional protein kinase C [cPKC])-dependent pp70S6k activation. The T-cell immunosuppressant rapamycin potently antagonizes IL-2-(PI3K)- and phorbol ester (cPKC)-mediated activation of pp70S6k. Thus, wortmannin and rapamycin antagonize IL-2-mediated activation of pp70S6k at distinct points along the PI3K-regulated signalling pathway, or rapamycin antagonizes another pathway required for pp70S6k activity. Agents that raise the concentration of intracellular cyclic AMP (cAMP) and activate cAMP-dependent protein kinase (PKA) also inhibit IL-2-dependent activation of pp70S6k. In this case, inhibition appears to occur at least two points in this signalling path. Like rapamycin, PKA appears to act downstream of cPKC-mediated pp70S6k activation, and like wortmannin, PKA antagonizes IL-2-dependent activation of PI3K. The results with rapamycin and wortmannin are of added interest since the yeast and mammalian rapamycin targets resemble PI3K in the catalytic domain. PMID:7528328

  7. Mitochondrial Cyclic AMP Response Element-binding Protein (CREB) Mediates Mitochondrial Gene Expression and Neuronal Survival*S

    PubMed Central

    Lee, Junghee; Kim, Chun-Hyung; Simon, David K.; Aminova, Lyaylya R.; Andreyev, Alexander Y.; Kushnareva, Yulia E.; Murphy, Anne N.; Lonze, Bonnie E.; Kim, Kwang-Soo; Ginty, David D.; Ferrante, Robert J.; Ryu, Hoon; Ratan, Rajiv R.

    2008-01-01

    Cyclic AMP response element-binding protein (CREB) is a widely expressed transcription factor whose role in neuronal protection is now well established. Here we report that CREB is present in the mitochondrial matrix of neurons and that it binds directly to cyclic AMP response elements (CREs) found within the mitochondrial genome. Disruption of CREB activity in the mitochondria decreases the expression of a subset of mitochondrial genes, including the ND5 subunit of complex I, down-regulates complex I-dependent mitochondrial respiration, and increases susceptibility to 3-nitropropionic acid, a mitochondrial toxin that induces a clinical and pathological phenotype similar to Huntington disease. These results demonstrate that regulation of mitochondrial gene expression by mitochondrial CREB, in part, underlies the protective effects of CREB and raise the possibility that decreased mitochondrial CREB activity contributes to the mitochondrial dysfunction and neuronal loss associated with neurodegenerative disorders. PMID:16207717

  8. Cyclic Amp phosphodiesterase activity in normal and inflamed human dental pulp.

    PubMed

    Spoto, G; Menna, V; Serra, E; Santoleri, F; Perfetti, G; Ciavarelli, L; Trentini, P

    2004-01-01

    Cyclic AMP phosphodiesterase (cAMP PDE) seems to be important in pulp tissues. High levels of cAMP PDE have been demonstrated to be in dental pulp cells. In the present study cAMP PDE activity was analyzed in normal healthy human dental pulps, in reversible pulpitis and in irreversible pulpitis. Enzymatic cAMP PDE control values for normal healthy pulps were 12.14 +/- 3.74 nmols/mg of proteins. In reversible pulpitis the cAMP PDE activity increased almost 2.5 times. In irreversible pulpitis specimens the values increased 4.5 times compared with normal healthy pulps activity. The differences between the groups (control vs. reversible pulpitis and vs. irreversible pulpitis) were statistically significant. These results could point to a role of cAMP PDE in the initial pulp response after injury. PMID:16857100

  9. Role of the Cyclic AMP Response Element Binding Complex and Activation of Mitogen-Activated Protein Kinases in Synergistic Activation of the Glycoprotein Hormone α Subunit Gene by Epidermal Growth Factor and Forskolin

    PubMed Central

    Roberson, Mark S.; Ban, Makiko; Zhang, Tong; Mulvaney, Jennifer M.

    2000-01-01

    The aim of these studies was to elucidate a role for epidermal growth factor (EGF) signaling in the transcriptional regulation of the glycoprotein hormone α subunit gene, a subunit of chorionic gonadotropin. Studies examined the effects of EGF and the adenylate cyclase activator forskolin on the expression of a transfected α subunit reporter gene in a human choriocarcinoma cell line (JEG3). At maximal doses, administration of EGF resulted in a 50% increase in a subunit reporter activity; forskolin administration induced a fivefold activation; the combined actions of EGF and forskolin resulted in synergistic activation (greater than eightfold) of the α subunit reporter. Mutagenesis studies revealed that the cyclic AMP response elements (CRE) were required and sufficient to mediate EGF-forskolin-induced synergistic activation. The combined actions of EGF and forskolin resulted in potentiated activation of extracellular signal-regulated kinase (ERK) enzyme activity compared with EGF alone. Specific blockade of ERK activation was sufficient to block EGF-forskolin-induced synergistic activation of the α subunit reporter. Pretreatment of JEG3 cells with a p38 mitogen-activated protein kinase inhibitor did not influence activation of the α reporter. However, overexpression of c-Jun N-terminal kinase (JNK)-interacting protein 1 as a dominant interfering molecule abolished the synergistic effects of EGF and forskolin on the α subunit reporter. CRE binding studies suggested that the CRE complex consisted of CRE binding protein and EGF-ERK-dependent recruitment of c-Jun–c-Fos (AP-1) to the CRE. A dominant negative form of c-Fos (A-Fos) that specifically disrupts c-Jun–c-Fos DNA binding inhibited synergistic activation of the α subunit. Thus, synergistic activation of the α subunit gene induced by EGF-forskolin requires the ERK and JNK cascades and the recruitment of AP-1 to the CRE binding complex. PMID:10779323

  10. Cyclic AMP-and beta-agonist-activated chloride conductance of a toad skin epithelium.

    PubMed

    Willumsen, N J; Vestergaard, L; Larsen, E H

    1992-04-01

    1. The control by intracellular cyclic AMP and beta-adrenergic stimulation of chloride conductance was studied in toad skin epithelium mounted in a chamber on the stage of an upright microscope. Impalement of identified principal cells from the serosal side with single-barrelled conventional or double-barrelled Cl(-)-sensitive microelectrodes was performed at x500 magnification. For blocking the active sodium current 50 microM-amiloride was present in the mucosal bath. 2. When clamped at transepithelial potential difference V = 0 mV, the preparations generated clamping currents of 0.9 +/- 1 microA/cm2 (mean +/- S.E.M.; number of observations n = 55). The intracellular potential of principal cells (Vb) was -96 +/- 2 mV with a fractional resistance of the basolateral membrane (fRb) of 0.016 +/- 0.003 (n = 54), and an intracellular Cl- activity of 40 +/- 2 mM (n = 24). 3. At V = 0 mV, serosal application of a cyclic AMP analogue, dibutyryl cyclic AMP (500 microM) or a beta-adrenergic agonist, isoprenaline (5 microM) resulted in a sixfold increase in transepithelial Cl- conductance identified by standard 36Cl- tracer technique. 4. The clamping current at V = 0 mV was unaffected by cyclic AMP (short-circuit current Isc = 0.1 +/- 0.3 microA/cm2, n = 16) indicating that subepidermal Cl(-)-secreting glands are not functioning in our preparations obtained by collagenase treatment. 5. Cyclic AMP- or isoprenaline-induced chloride conductance (Gcl) activation (V = 0 mV) was not reflected in membrane potential and intracellular Cl- activity in principal cells. Intracellular chloride activity was constant at approximately 40 mM at membrane potentials between -90 and -100 mV. Therefore, it can be concluded that the principal cells are not contributing to activated Cl- currents. 6. At V = -100 mV where the voltage-dependent chloride conductance of mitochondria-rich (MR) cells was already fully activated, GCl was unaffected by cyclic AMP or isoprenaline. The major effect of these

  11. Role of Tissue-Specific Transcription Factor LFB3 in a Cyclic AMP-Responsive Enhancer of the Urokinase-Type Plasminogen Activator Gene in LLC-PK1 Cells

    PubMed Central

    Soubt, Mazin Khalil; Marksitzer, René; Menoud, Pierre-Alain; Nagamine, Yoshikuni

    1998-01-01

    A cyclic AMP (cAMP)-inducible enhancer in the pig urokinase-type plasminogen activator gene located 3.4 kb upstream of the transcription initiation site is composed of three protein-binding domains, A, B, and C. Domains A and B each contain a CRE (cAMP response element)-like sequence but require the adjoining C domain for full cAMP responsiveness. A tissue-specific transcription factor, LFB3/HNF1β/vHNF1, binds to the C domain. Mutation analyses suggest that the imperfect CRE and LFB3-binding sequences are required for tight coupling of hormonal and tissue-specific regulation. CREB and ATF1 bind to domains A and B, and this binding is enhanced upon phosphorylation by cAMP-dependent protein kinase (protein kinase A [PKA]). Analysis in a mammalian two-hybrid system revealed that CREB/ATF1 and LFB3 interact and that transactivation potential is enhanced by PKA activation. Interestingly, however, phosphorylation of CREB at Ser-133 does not contribute to its interaction with LFB3. The region of LFB3 involved in its interaction with CREB/ATF1 lies, at least partly, between amino acids 400 and 450. Deletion of this region removed the ability of LFB3 to mediate cAMP induction of the ABC enhancer but did not impair its basal transactivation activity on the albumin promoter. Thus, the two activities are distinct functions of LFB3. PMID:9671480

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

    PubMed

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

    2015-09-01

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

  13. Spatial Memory in the Morris Water Maze and Activation of Cyclic AMP Response Element-Binding (CREB) Protein within the Mouse Hippocampus

    ERIC Educational Resources Information Center

    Porte, Yves; Buhot, Marie Christine; Mons, Nicole E.

    2008-01-01

    We investigated the spatio-temporal dynamics of learning-induced cAMP response element-binding protein activation/phosphorylation (pCREB) in mice trained in a spatial reference memory task in the water maze. Using immunohistochemistry, we examined pCREB immunoreactivity (pCREB-ir) in hippocampal CA1 and CA3 and related brain structures. During the…

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

    PubMed

    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. PMID:22538662

  15. Role of cyclic AMP in promoting the thromboresistance of human endothelial cells by enhancing thrombomodulin and decreasing tissue factor activities.

    PubMed Central

    Archipoff, G.; Beretz, A.; Bartha, K.; Brisson, C.; de la Salle, C.; Froget-Léon, C.; Klein-Soyer, C.; Cazenave, J. P.

    1993-01-01

    1. The effects of forskolin, prostaglandin E1 (PGE1), dibutyryl cyclic AMP (db cyclic AMP), dibutyryl cyclic GMP (db cyclic GMP) and 3-isobutyl-l-methyl-xanthine (IBMX) were investigated on the expression of tissue factor and thrombomodulin activities on the surface of human saphenous vein endothelial cells (HSVEC) in culture. 2. Forskolin (10(-6) to 10(-4) M), PGE1 (10(-7) to 10(-5) M) and db cyclic AMP (10(-4) to 10(-3) M) caused a concentration-dependent decrease of cytokine-induced tissue factor activity. 3. Similar concentrations of forskolin, PGE1 and db cyclic AMP enhanced significantly constitutive thrombomodulin activity and reversed the decrease of this activity caused by interleukin-1 (IL-1). 4. IBMX (10(-4) M) decreased tissue factor activity and enhanced the effect of forskolin on tissue factor and thrombomodulin activities. 5. Forskolin (10(-4) M) decreased the IL-1-induced tissue factor mRNA and increased the thrombomodulin mRNA level. IL-1 did not change the thrombomodulin mRNA level after 2 h of incubation with HSVEC in culture. 6. Dibutyryl cyclic GMP (10(-4) M to 10(-3) M) did not influence tissue factor or thrombomodulin activity. 7. Our data suggest that elevation of intracellular cyclic AMP levels may participate in the regulation of tissue factor and thrombomodulin expression, thus contributing to promote or restore antithrombotic properties of the endothelium. Images Figure 5 Figure 6 PMID:7684300

  16. Evolution of motif variants and positional bias of the cyclic-AMP response element

    PubMed Central

    Smith, Brandon; Fang, Hung; Pan, Youlian; Walker, P Roy; Famili, A Fazel; Sikorska, Marianna

    2007-01-01

    Background Transcription factors regulate gene expression by interacting with their specific DNA binding sites. Some transcription factors, particularly those involved in transcription initiation, always bind close to transcription start sites (TSS). Others have no such preference and are functional on sites even tens of thousands of base pairs (bp) away from the TSS. The Cyclic-AMP response element (CRE) binding protein (CREB) binds preferentially to a palindromic sequence (TGACGTCA), known as the canonical CRE, and also to other CRE variants. CREB can activate transcription at CREs thousands of bp away from the TSS, but in mammals CREs are found far more frequently within 1 to 150 bp upstream of the TSS than in any other region. This property is termed positional bias. The strength of CREB binding to DNA is dependent on the sequence of the CRE motif. The central CpG dinucleotide in the canonical CRE (TGACGTCA) is critical for strong binding of CREB dimers. Methylation of the cytosine in the CpG can inhibit binding of CREB. Deamination of the methylated cytosines causes a C to T transition, resulting in a functional, but lower affinity CRE variant, TGATGTCA. Results We performed genome-wide surveys of CREs in a number of species (from worm to human) and showed that only vertebrates exhibited a CRE positional bias. We performed pair-wise comparisons of human CREs with orthologous sequences in mouse, rat and dog genomes and found that canonical and TGATGTCA variant CREs are highly conserved in mammals. However, when orthologous sequences differ, canonical CREs in human are most frequently TGATGTCA in the other species and vice-versa. We have identified 207 human CREs showing such differences. Conclusion Our data suggest that the positional bias of CREs likely evolved after the separation of urochordata and vertebrata. Although many canonical CREs are conserved among mammals, there are a number of orthologous genes that have canonical CREs in one species but the

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

    PubMed Central

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

    1995-01-01

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

  18. Cyclic AMP-Responsive Element Modulator α Polymorphisms Are Potential Genetic Risks for Systemic Lupus Erythematosus

    PubMed Central

    Guo, Qian; Chen, Xuyong; Du, Yan; Guo, Jianping; Su, Yin

    2015-01-01

    To investigate whether the cyclic AMP-responsive element modulator α (CREMα) polymorphisms are novel susceptibility factors for systemic lupus erythematosus (SLE), four tag SNPs, rs1057108, rs2295415, rs11592925, and rs1148247, were genotyped in 889 SLE cases and 825 healthy controls. Association analyses were performed on whole dataset or clinical/serologic subsets. Association statistics were calculated by age and sex adjusted logistic regression. The G allele frequencies of rs2295415 and rs1057108 were increased in SLE patients, compared with healthy controls (rs2295415: 21.2% versus 17.8%, OR 1.244, P = 0.019; rs1057108: 30.8% versus 27.7%, OR 1.165, P = 0.049). The haplotype constituted by the two risk alleles “G-G” from rs1057108 and rs2295415 displayed strong association with SLE susceptibility (OR 1.454, P = 0.00056). Following stratification by clinical/serologic features, a suggestive association was observed between rs2295415 and anti-Sm antibodies-positive SLE (OR 1.382, P = 0.044). Interestingly, a potential protective effect of rs2295415 was observed for SLE patients with renal disorder (OR 0.745, P = 0.032). Our data provide first evidence that CREMα SNPs rs2295415 and rs1057108 maybe novel genetic susceptibility factors for SLE. SNP rs2295415 appears to confer higher risk to develop anti-Sm antibodies-positive SLE and may play a protective role against lupus nephritis. PMID:26601115

  19. Phorbol esters modulate cyclic AMP accumulation in porcine thyroid cells

    SciTech Connect

    Emoto, T.; Kasai, K.; Hiraiwa, M.; Shimoda, S.

    1988-01-01

    In cultured porcine thyroid cells, during 60 min incubation phorbol 12-myristate 13-acetate (PMA) had no effect on basal cyclic AMP accumulation and slightly stimulated cyclic AMP accumulation evoked by thyroid stimulating hormone (TSH) or forskolin. Cholera toxin-induced cyclic AMP accumulation was significantly stimulated by PMA. On the other hand, cyclic AMP accumulation evoked by prostaglandin E/sub 1/ or E/sub 2/ (PGE/sub 1/ and PGE/sub 2/) was markedly depressed by simultaneous addition of PMA. These opposing effects of PMA on cyclic AMP accumulation evoked by PGE and cholera toxin were observed in a dose-related fashion, with half-maximal effect of around 10/sup -9/ M in either case. The almost same effects of PMA on cyclic AMP accumulation in basal and stimulated conditions were also observed in freshly prepared thyroid cells. The present study was performed in the presence of phosphodiesterase inhibitor, 3-iso-butyl-1-methylxanthine (IBMX), indicating that PMA affected adenylate cyclase activity. Therefore, it is suggested that PMA may modulate the production of cyclic AMP in response to different stimuli, possibly by affecting several sites in the adenylate cyclase complex in thyroid cells.

  20. Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription.

    PubMed

    Bell, A; Gaston, K; Williams, R; Chapman, K; Kolb, A; Buc, H; Minchin, S; Williams, J; Busby, S

    1990-12-25

    The effects of a number of mutations in the E. coli cyclic AMP receptor protein (CRP) have been determined by monitoring the in vivo expression and in vitro open complex formation at two semi-synthetic promoters that are totally CRP-dependent. At one promoter the CRP-binding site is centered around 41.5 base pairs upstream from the transcription start whilst at the other promoter it is 61.5 base pairs upstream. The CRP mutation E171K reduces expression from both promoters whilst H159L renders CRP totally inactive: neither mutation stops CRP binding at either promoter. The mutations K52N and K52Q reverse the effect of H159L and 'reeducate' CRP to activate transcription. CRP carrying both H159L and K52N activates transcription from the promoter with the CRP site at -41.5 better than wild type CRP. In sharp contrast, this doubly changed CRP is totally inactive with respect to the activation of transcription from the promoter carrying the CRP site at -61.5. Our results suggest that CRP can use different contacts and/or conformations during transcription activation at promoters with different architectures. PMID:2259621

  1. Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription.

    PubMed Central

    Bell, A; Gaston, K; Williams, R; Chapman, K; Kolb, A; Buc, H; Minchin, S; Williams, J; Busby, S

    1990-01-01

    The effects of a number of mutations in the E. coli cyclic AMP receptor protein (CRP) have been determined by monitoring the in vivo expression and in vitro open complex formation at two semi-synthetic promoters that are totally CRP-dependent. At one promoter the CRP-binding site is centered around 41.5 base pairs upstream from the transcription start whilst at the other promoter it is 61.5 base pairs upstream. The CRP mutation E171K reduces expression from both promoters whilst H159L renders CRP totally inactive: neither mutation stops CRP binding at either promoter. The mutations K52N and K52Q reverse the effect of H159L and 'reeducate' CRP to activate transcription. CRP carrying both H159L and K52N activates transcription from the promoter with the CRP site at -41.5 better than wild type CRP. In sharp contrast, this doubly changed CRP is totally inactive with respect to the activation of transcription from the promoter carrying the CRP site at -61.5. Our results suggest that CRP can use different contacts and/or conformations during transcription activation at promoters with different architectures. Images PMID:2259621

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

    SciTech Connect

    Mukai, Atsushi; Hashimoto, Naohiro

    2008-01-15

    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.

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

    PubMed Central

    Wang, Jiali; Liu, Chengzhi; Lu, Huizhi; Liu, Mengjia; Zhao, Ye; Tian, Bing; Wang, Liangyan; Hua, Yuejin

    2016-01-01

    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. PMID:27182600

  4. Cholesterol ester hydrolase in pig liver is activated by cyclic AMP-dependent protein kinase

    SciTech Connect

    Chen, J.J.S.; Dubin, E.; Margolis, S.

    1986-05-01

    To examine whether hepatic neutral cholesterol ester hydrolase (CEH) is regulated by phosphorylation, the authors have assayed CEH activity from pig liver cytosol by measuring /sup 14/C-oleate release from labeled cholesteryl oleate at pH 7.4. When pig liver cytosol was incubated with 2 mM Mg and 0.5 mM ATP, CEH activity was increased (141 +/- 8% of control, mean +/- SEM). Addition of 25..mu..M cyclic AMP (cAMP) further activated CEH activity (164 +/- 4% of control) as compared to incubation with Mg and ATP (p < 0.02). In the presence of 5 mM EDTA or in the absence of either Mg or ATP, no activation of CEH was observed. The activation was completely abolished by further incubation of activated cytosol with E. coli alkaline phosphatase. Activation of CEH activity was partially prevented by the addition of protein kinase inhibitor (p < 0.02) and this effect was completely reversed in the presence of exogenous cAMP-dependent protein kinase (p < 0.05). To examine further the role of the cAMP-dependent protein kinase, CEH activity was purified 240-fold by 35% (NH/sub 4/)/sub 2/SO/sub 4/ precipitation and Sepharose 4B chromatography. Incubation of partially purified CEH fractions with Mg, ATP and cAMP did not increase CEH activity. Addition of exogenous cAMP-dependent protein kinase activated CEH activity of partially purified fractions. The authors observations indicate that pig liver CEH is activated by phosphorylation mediated by cAMP-dependent protein kinase.

  5. Hypoxia induces phosphorylation of the cyclic AMP response element-binding protein by a novel signaling mechanism.

    PubMed

    Beitner-Johnson, D; Millhorn, D E

    1998-07-31

    To investigate signaling mechanisms by which hypoxia regulates gene expression, we examined the effect of hypoxia on the cyclic AMP response element-binding protein (CREB) in PC12 cells. Exposure to physiological levels of hypoxia (5% O2, approximately 50 mm Hg) rapidly induced a persistent phosphorylation of CREB on Ser133, an event that is required for CREB-mediated transcriptional activation. Hypoxia-induced phosphorylation of CREB was more robust than that induced by any other stimulus tested, including forskolin, depolarization, and osmotic stress. Furthermore, this effect was not mediated by any of the previously known signaling pathways that lead to phosphorylation of CREB, including protein kinase A, calcium/calmodulin-dependent protein kinase, protein kinase C, ribosomal S6 kinase-2, and mitogen-activated protein kinase-activated protein kinase-2. Hypoxic activation of a CRE-containing reporter (derived from the 5'-flanking region of the tyrosine hydroxylase gene) was attenuated markedly by mutation of the CRE. Thus, a physiological reduction in O2 levels induces a functional phosphorylation of CREB at Ser133 via a novel signaling pathway. PMID:9677418

  6. Stimulus-specific deactivation of chemotactic factor-induced cyclic AMP response and superoxide generation by human neutrophils.

    PubMed Central

    Simchowitz, L; Atkinson, J P; Spilberg, I

    1980-01-01

    The responses of isolated human peripheral neutrophils to either simultaneous or sequential additions of two chemotactic factors were studied. Simultaneous additions of formyl-methionyl-leucyl-phenylalanine (10-100 nM) and the fifth component of complement, C5a (1-10 microliters/ml), evoked partially additive responses of membrane depolarization as measured by the fluorescent dye 3,3'-dipropyl-thiocarbocyanine, a transient elevation of intracellular cyclic AMP (cAMP), and superoxide (O2-) generation as assessed by ferricytochrome c reduction. Preincubation of the cells with either formyl-methionyl-leucyl-phenylalanine or C5a alone caused dose-dependent inhibition of the depolarization, the cAMP increase, and O2- release induced by a subsequent exposure to an optimal dose of the same stimulus, i.e., deactivation occurred. In contrast, when cells were treated with one chemotactic factor and then exposed to the other stimulus, the cells exhibited a normal response of peak depolarization, the rise in cAMP, and O2-0 production i.e., cross-deactivation failed to occur. The results imply that deactivation of these phenomena is stimulus specific. Further, these observations are consistent with the hypothesis that cross-deactivation of chemotaxis is mediated by one or more processes that are irrelevant to O2- generation, and that occur distal to the depolarization and cAMP steps in the sequence of neutrophil activation: possibly microtubule polymerization and orientation. PMID:6252250

  7. Long-range signaling in growing neurons after local elevation of cyclic AMP-dependent activity

    PubMed Central

    1994-01-01

    Cyclic AMP-dependent activity at the growth cone or the soma of cultured Xenopus spinal neurons was elevated by local extracellular perfusion of the neuron with culture medium containing 8-bromoadenosine 3',5'-cyclic monophosphate (8-br-cAMP) or forskolin. During local perfusion of one of the growth cones of multipolar neurons with these drugs, the perfused growth cone showed further extension, while the distant, unperfused growth cones were inhibited in their growth. Local perfusion of the growth cone with culture medium or local perfusion with 8-br-cAMP at a cell-free region 100 microns away from the growth cone did not produce any effect on the extension of the growth cone. Reduced extension of all growth cones was observed when the perfusion with 8-br-cAMP was restricted to the soma. The distant inhibitory effect does not depend on the growth of the perfused growth cone since local coperfusion of the growth cone with 8-br-cAMP and colchicine inhibited growth on both perfused and unperfused growth cones, while local perfusion with colchicine alone inhibited only the perfused growth cone. The distant inhibitory effect was abolished when the perfusion of 8-br-cAMP was carried out together with kinase inhibitor H- 8, suggesting the involvement of cAMP-dependent protein kinase and/or its downstream factors in the long-range inhibitory signaling. Uniform exposure of the entire neuron to bath-applied 8-br-cAMP, however, led to enhanced growth activity at all growth cones. Thus, local elevation of cAMP-dependent activity produces long-range and opposite effects on distant parts of the neuron, and a cytosolic gradient of second messengers may produce effects distinctly different from those following uniform global elevation of the messenger, leading to differential growth regulation at different regions of the same neuron. PMID:7798321

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

    SciTech Connect

    Gao Yanan; Gao Ge; Long Caixia; Han Song; Zu Pengyu; Fang Li . E-mail: lfang@utmb.edu; Li Junfa . E-mail: junfali@cpums.edu.cn

    2006-02-10

    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.

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

    NASA Technical Reports Server (NTRS)

    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.

  10. Glial potassium channels activated by neuronal firing or intracellular cyclic AMP in Helix.

    PubMed Central

    Gommerat, I; Gola, M

    1996-01-01

    1. Cell-attached and whole cell patch clamp experiments were performed on satellite glial cells adhering to the cell body of neurones in situ within the nervous system of the snail Helix pomatia. The underlying neurone was under current or voltage-clamp control. 2. Neuronal firing induced a delayed (20-30 s) persistent (3-4 min) increase in the opening probability of glial K+ channels. The channels were also activated by perfusing the ganglion with a depolarizing high-K+ saline, except when the underlying neurone was prevented from depolarizing under voltage-clamp conditions. 3. Two K(+)-selective channels were detected in the glial membrane. The channel responding to neuronal firing was present in 95% of the patches (n = 393). It had a unitary conductance of 56 pS, a Na+ :K+ permeability ratio < 0.02 and displayed slight inward rectification in symmetrical [K+] conditions. It was sensitive to TEA, Ba2+ and Cs+. The following results refer to this channel as studied in the cell-attached configuration. 4. The glial K+ channel was activated by bath application of the membrane-permeant cyclic AMP derivatives 8-bromo-cAMP and dibutyryl-cAMP, the adenylyl cyclase activator forskolin and the diesterase inhibitors IBMX, theophylline and caffeine. It was insensitive to cyclic GMP activators and to conditions that might alter the intracellular [Ca2+] (ionomycin, low-Ca2+ saline and Ca2+ channel blockers). 5. The forskolin-induced changes in channel behaviour (open and closed time distributions, burst duration, short and long gaps within bursts) could be accounted for by a four-state model (3 closed states, 1 open state) by simply changing one of the six rate parameters. 6. The present results suggest that the signal sent by an active neurone to satellite glial cells is confined to the glial cells round that neurone. The effect of this signal on the class of glial K+ channels studied can be mimicked by an increase in glial cAMP concentration. The subsequent delayed opening

  11. Cyclic AMP enhances agonist-induced Ca2+ entry into endothelial cells by activation of potassium channels and membrane hyperpolarization.

    PubMed Central

    Graier, W F; Kukovetz, W R; Groschner, K

    1993-01-01

    The mechanism underlying cyclic AMP (cAMP)-mediated amplification of agonist-induced Ca2+ responses in endothelial cells was investigated in pig endothelial cells. Forskolin, adenosine and isoprenaline, as well as the membrane-permeant cAMP analogue dibutyryl cAMP, enhanced bradykinin-induced rises in intracellular free Ca2+ as well as bradykinin-induced Mn2+ entry. These agents were also found to hyperpolarize endothelial cells without increasing intracellular Ca2+ by itself, i.e. in the absence of bradykinin. Both amplification of bradykinin effects and the hyperpolarizing action was blocked by the protein kinase inhibitor H-8. The involvement of K+ channels in the hyperpolarizing effects of forskolin was consequently studied in perforated outside-out vesicles. Two different types of K+ channels were recorded, one of which had a large conductance (170 pS) and was activated by forskolin. We suggest that stimulation of endothelial adenylate cyclase results in activation of large-conductance K+ channels and consequently in membrane hyperpolarization, which in turn enhances bradykinin-induced entry of Ca2+ by increasing its electrochemical gradient. PMID:8385935

  12. Pituitary adenylate cyclase-activating peptide induces long-lasting neuroprotection through the induction of activity-dependent signaling via the cyclic AMP response element-binding protein-regulated transcription co-activator 1

    PubMed Central

    Baxter, Paul S; Martel, Marc-Andre; McMahon, Aoife; Kind, Peter C; Hardingham, Giles E

    2011-01-01

    Pituitary adenylate cyclase-activating peptide (PACAP) is a neuroprotective peptide which exerts its effects mainly through the cAMP-protein kinase A (PKA) pathway. Here, we show that in cortical neurons, PACAP-induced PKA signaling exerts a major part of its neuroprotective effects indirectly, by triggering action potential (AP) firing. Treatment of cortical neurons with PACAP induces a rapid and sustained PKA-dependent increase in AP firing and associated intracellular Ca2+ transients, which are essential for the anti-apoptotic actions of PACAP. Transient exposure to PACAP induces long-lasting neuroprotection in the face of apoptotic insults which is reliant on AP firing and the activation of cAMP response element (CRE) binding protein (CREB)-mediated gene expression. Although direct, activity-independent PKA signaling is sufficient to trigger phosphorylation on CREB’s activating serine-133 site, this is insufficient for activation of CREB-mediated gene expression. Full activation is dependent on CREB-regulated transcription co-activator 1 (CRTC1), whose PACAP-induced nuclear import is dependent on firing activity-dependent calcineurin signaling. Over-expression of CRTC1 is sufficient to rescue PACAP-induced CRE-mediated gene expression in the face of activity-blockade, while dominant negative CRTC1 interferes with PACAP-induced, CREB-mediated neuroprotection. Thus, the enhancement of AP firing may play a significant role in the neuroprotective actions of PACAP and other adenylate cyclase-coupled ligands. PMID:21623792

  13. Repression of protein kinase C and stimulation of cyclic AMP response elements by fumonisin, a fungal encoded toxin which is a carcinogen.

    PubMed

    Huang, C; Dickman, M; Henderson, G; Jones, C

    1995-04-15

    Fusarium moniliforme (FM) is a major fungal pathogen of corn and is involved with stalk rot disease. FM is widely spread throughout the world, including the United States. Most strains of FM produce several mycotoxins, the most prominent of which is called fumonisin. Recent epidemiological studies indicated that ingestion of fumonisin correlates with a higher incidence of esophageal cancer in Southern and Northern Africa and China. Furthermore, fumonisin causes a neurodegenerative disease in horses, induces hepatic cancer in rats, and induces pulmonary edema in swine. Considering that high levels of fumonisin have been detected in healthy and diseased corn grown in the United States, fumonisin may pose a health threat to humans and livestock animals. Structurally, fumonisin resembles sphingolipids which are present in the membranes of animal and plant cells. At the present time, very little is known concerning the mechanism by which fumonisin elicits its carcinogenic effect. Our studies indicate that fumonisin represses expression of protein kinase C and AP-1-dependent transcription. In contrast, fumonisin stimulated a simple promoter containing a single cyclic AMP response element. Since fumonisin did not alter protein kinase A activity, it appears that cyclic AMP response element activation was independent of protein kinase A. It is hypothesized that the ability of fumonisin to alter signal transduction pathways plays a role in carcinogenesis. PMID:7712470

  14. Phospholipase C in Dictyostelium discoideum. Cyclic AMP surface receptor and G-protein-regulated activity in vitro.

    PubMed

    Bominaar, A A; Kesbeke, F; Van Haastert, P J

    1994-01-01

    The cellular slime mould Dictyostelium discoideum shows several responses after stimulation with the chemoattractant cAMP, including a transient rise in cyclic AMP (cAMP), cGMP and Ins(1,4,5)P3. In this paper the regulation of phospholipase C in vitro is described. Under our experimental conditions commercial PtdIns(4,5)P2 cannot be used to analyse phospholipase C activity in Dictyostelium lysates, because it is hydrolysed mainly to glycerophosphoinositol instead of Ins(1,4,5)P3. Enzyme activity was determined with endogenous unlabelled PtdInsP2 as a substrate. The product was measured by isotope-dilution assay and identified as authentic Ins(1,4,5)P3. Since phospholipase C is strictly Ca(2+)-dependent, with an optimal concentration range of 1-100 microM, cell lysates were prepared in EGTA and the enzyme reaction was started by adding 10 microM free Ca2+. Phospholipase C activity increased 2-fold during Dictyostelium development up to 8 h of starvation, after which the activity declined to less than 10% of the vegetative level. Enzyme activity in vitro increased up to 2-fold after stimulation of cells with the agonist cAMP in vivo. Addition of 10 microM guanosine 5'-[gamma-thio]triphosphate during lysis activated the enzyme to the same extent, and this effect was antagonized by guanosine 5'-[beta-thio]diphosphate. These results strongly suggest that surface cAMP receptors and G-proteins regulate phospholipase C during Dictyostelium development. PMID:8280097

  15. Modulators of cyclic AMP systems.

    PubMed

    Hess, S M; Chasin, M; Free, C A; Harris, D N

    1975-01-01

    On the basis of the data reported here, one may conclude that although many agents that act in the central nervous system are modulators of the action of cyclic AMP, it is difficult to establish a direct connection between the pharmacologic activity and the levels of cyclic AMP in the brain. This lack of interrelation applies to the benzodiazepines as well as to the pyrazolopyridines. The data for members of the latter group are somewhat frustrating in this regard, since an excellent correlation has been shown to exist between the potency of inhibition of PDE and activity in the antianxiety test. In measurements of steroidogenesis in the isolated adrenal cell, the correlation between activity in vito and the conflict assay is even better. The data presented here and reported elsewhere (Shimizu et al., 1974; Kelly et al., 1974; Mayer and King, 1974; King and Mayer, 1974) provide evidence that agents that act as inhibitors of PDE in cell-free systems exert their influence on cyclic AMP in tissue slices of the brain of guinea pigs by mechanisms that seem not to be related to an effect on PDE. Papaverine, and possibly chlordiazepoxide, may act by releasing agonists that, in turn, stimulate the accumulation of cyclic AMP. This activity is blocked bo other inhibitors of PDE, such as theophyline. Results obtained by the use of platelets are refreshingly clear. Inhibition of aggregation has been shown to occur when the level of cyclic AMP is raised, and a suggestive exists that the most potent inhibitors of platelet PDE are the best potentiators of the action of PGE1 in blocking aggregation. The study utilizing drugs collected from a large number of therapeutic classes makes clear that it is difficult to attribute the mechanism of action for any of the classes studied to modulation of cyclic AMP. An unexpected finding of this study, however, was the fact that pharmacologic agents include an unusually large number of inhibitors of PDE as compared with agents chosen at

  16. Cyclic AMP in prokaryotes.

    PubMed Central

    Botsford, J L; Harman, J G

    1992-01-01

    Cyclic AMP (cAMP) is found in a variety of prokaryotes including both eubacteria and archaebacteria. cAMP plays a role in regulating gene expression, not only for the classic inducible catabolic operons, but also for other categories. In the enteric coliforms, the effects of cAMP on gene expression are mediated through its interaction with and allosteric modification of a cAMP-binding protein (CRP). The CRP-cAMP complex subsequently binds specific DNA sequences and either activates or inhibits transcription depending upon the positioning of the complex relative to the promoter. Enteric coliforms have provided a model to explore the mechanisms involved in controlling adenylate cyclase activity, in regulating adenylate cyclase synthesis, and in performing detailed examinations of CRP-cAMP complex-regulated gene expression. This review summarizes recent work focused on elucidating the molecular mechanisms of CRP-cAMP complex-mediated processes. For other bacteria, less detail is known. cAMP has been implicated in regulating antibiotic production, phototrophic growth, and pathogenesis. A role for cAMP has been suggested in nitrogen fixation. Often the only data that support cAMP involvement in these processes includes cAMP measurement, detection of the enzymes involved in cAMP metabolism, or observed effects of high concentrations of the nucleotide on cell growth. PMID:1315922

  17. The cyclic AMP receptor protein is the main activator of pectinolysis genes in Erwinia chrysanthemi.

    PubMed

    Reverchon, S; Expert, D; Robert-Baudouy, J; Nasser, W

    1997-06-01

    The main virulence factors of the phytopathogenic bacterium Erwinia chrysanthemi are pectinases that cleave pectin, a major constituent of the plant cell wall. Although physiological studies suggested that pectinase production in Erwinia species is subjected to catabolite repression, the direct implication of the cyclic AMP receptor protein (CRP) in this regulation has never been demonstrated. To investigate the role of CRP in pectin catabolism, we cloned the E. chrysanthemi crp gene by complementation of an Escherichia coli crp mutation and then constructed E. chrysanthemi crp mutants by reverse genetics. The carbohydrate fermentation phenotype of the E. chrysanthemi crp mutants is similar to that of an E. coli crp mutant. Furthermore, these mutants are unable to grow on pectin or polygalacturonate as the sole carbon source. Analysis of the nucleotide sequence of the E. chrysanthemi crp gene revealed the presence of a 630-bp open reading frame (ORF) that codes for a protein highly similar to the CRP of E. coli. Using a crp::uidA transcriptional fusion, we demonstrated that the E. chrysanthemi CRP represses its own expression, probably via a mechanism similar to that described for the E. coli crp gene. Moreover, in the E. chrysanthemi crp mutants, expression of pectinase genes (pemA, pelB, pelC, pelD, and pelE) and of genes of the intracellular part of the pectin degradation pathway (ogl, kduI, and kdgT), which are important for inducer formation and transport, is dramatically reduced in induced conditions. In contrast, expression of pelA, which encodes a pectate lyase important for E. chrysanthemi pathogenicity, seems to be negatively regulated by CRP. The E. chrysanthemi crp mutants have greatly decreased maceration capacity in potato tubers, chicory leaves, and celery petioles as well as highly diminished virulence on saintpaulia plants. These findings demonstrate that CRP plays a crucial role in expression of the pectinolysis genes and in the pathogenicity of E

  18. Different effect of prostaglandin E2 on B-cell activation by two distinct B-cell differentiation factors, B151-TRF1/IL-5 and B151-TRF2: selective inhibition of B151-TRF2-induced antibody response through increases in intracellular cyclic AMP levels

    PubMed Central

    Ishihara, K.; Ono, S.; Takahama, Y.; Hirayama, F.; Hirano, H.; Itoh, K.; Dobashi, K.; Murakami, S.; Katoh, Y.; Yamaguchi, M.; Hamaoka, T.

    1989-01-01

    Effects of prostaglandin E2 (PGE2) on murine B-cell activation induced by two distinct B-cell differentiation factors, B151-TRF1/IL-5 and B151-TRF2, were examined. A final differentiation of unprimed B cells into IgM-producing cells induced by B151-TRF2 was markedly inhibited by PGE2 at physiological concentrations (around 10-8 M), whereas B151-TRF1/IL-5-induced antibody responses of unprimed as well as activated B cells were not affected by PGE2, even at 10-6 M. B-cell responses induced by B151-TRF2-like factors from autoimmune-prone MRL/1pr mice were also inhibited by PGE2. Biphasic increases in intracellular cyclic AMP (cAMP) levels were induced by culturing B cells with 10-6 or 10-8 M PGE2: rapid increases within 8 min and delayed increases around 16 hr. The direct addition of dibutyryl cAMP to cultures of B cells resulted in marked inhibition of antibody responses when stimulated with B151-TRF2 but not with B151-TRF1/IL-5. The B151-TRF2-induced antibody responses were also inhibited by cAMP-elevating reagents such as forskolin, cholera toxin and theophyline. Furthermore, 2′, 5′-dideoxyadenosine, which is an inhibitor of adenylate cyclase, prevented the PGE2-mediated cAMP accumulation in unprimed B cells as well as the PGE2-mediated inhibition of B151-TRF2-induced B-cell responses when added at the initiation of culture. These results suggest that PGE2 inhibits B151-TRF2-induced antibody responses through the activation of adenylate cyclase and subsequent accumulation of intracellular cAMP, whereas B151-TRF1/IL-5-responsive B cells are resistant to the inhibitory effect of PGE2 and cAMP. PMID:2553585

  19. Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP-stimulated EAhy 926 endothelial cells.

    PubMed Central

    Graham, A; McLees, A; Malarkey, K; Gould, G W; Plevin, R

    1996-01-01

    We have investigated the mechanisms that bring about the termination of mitogen-activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re-application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1-2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein-synthesis step and by a mechanism that does not involve the inactivation of c-Raf-1 or MAP kinase kinase. PMID:8615830

  20. Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP-stimulated EAhy 926 endothelial cells.

    PubMed

    Graham, A; McLees, A; Malarkey, K; Gould, G W; Plevin, R

    1996-04-15

    We have investigated the mechanisms that bring about the termination of mitogen-activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re-application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1-2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein-synthesis step and by a mechanism that does not involve the inactivation of c-Raf-1 or MAP kinase kinase. PMID:8615830

  1. Regulation of cyclic AMP response element-binding protein during neuroglial interactions.

    PubMed

    Qin, LiMei; Bouchard, Ron; Pugazhenthi, Subbiah

    2016-03-01

    Communications between neurons and glial cells play an important role in regulating homeostasis in the central nervous system. cAMP response element-binding protein (CREB), a transcription factor, is down-regulated by neurotoxins, which are known to be released by activated glial cells. To determine the role of CREB signaling in neuroglial interactions, we used three neuroglial coculture models consisting of human neuroprogenitor cell (NPC)-derived neurons and human microglia. Conditioned medium from the Abeta (Aβ)-activated microglia decreased CREB phosphorylation and brain-derived neurotrophic factor promoter activity (47%), whereas the same medium induced (p < 0.01) the promoter of CXCL10, a chemokine, in NPC-derived neuron-rich cultures. These effects were reversed when microglia were exposed to Aβ in the presence of minocycline, an anti-inflammatory agent. The expression of CREB targets, including brain-derived neurotrophic factor, synapsin-1, and BIRC3 decreased by 50-65% (p < 0.01) in neurons isolated by laser capture microdissection in close proximity of microglia in neuroglial mixed cultures. Neuronal survival actively modulated microglial behavior when neurons and microglia were cocultured side-by-side on semicircles of ACLAR membrane. Neuronal injury, caused by the over-expression of dominant negative form of CREB, exacerbated Aβ-mediated microglial activation, whereas CREB over-expression resulted in decreased microglial activation. Decreases in the levels of neuronal markers were observed when NPCs were differentiated in the presence of proinflammatory cytokines IL-1β, tumor necrosis factor α, or IL-6. Instead, the NPCs differentiated into a glial phenotype, and these effects were more pronounced in the presence of tumor necrosis factor α. Our findings suggest that CREB down-regulation is an important component of defective neuroglial communications in the brain during neuroinflammation. Neuroglial interactions were examined using coculture

  2. SF-1 (Nuclear Receptor 5A1) Activity Is Activated by Cyclic AMP via p300-Mediated Recruitment to Active Foci, Acetylation, and Increased DNA Binding

    PubMed Central

    Chen, Wei-Yi; Juan, Li-Jung; Chung, Bon-chu

    2005-01-01

    Steroidogenic factor 1 (SF-1) is a nuclear receptor essential for steroidogenic gene expression, but how its activity is regulated is unclear. Here we demonstrate that p300 plays an important role in regulating SF-1 function. SF-1 was acetylated in vitro and in vivo by p300 at the KQQKK motif in the Ftz-F1 (Fushi-tarazu factor 1) box adjacent to its DNA-binding domain. Mutation of the KQQKK motif reduced the DNA-binding activity and p300-dependent activation of SF-1. When stimulated with cyclic AMP (cAMP), adrenocortical Y1 cells expressed more p300, leading to additional SF-1 association with p300 and increased SF-1 acetylation and DNA binding. It also increased SF-1 colocalization with p300 in nuclear foci. Collectively, these results indicate that SF-1 transcriptional activity is regulated by p300 in response to the cAMP signaling pathway by way of increased acetylation, DNA binding, and recruitment to nuclear foci. PMID:16287857

  3. A mouse model for the Carney complex tumor syndrome develops neoplasia in cyclic AMP-responsive tissues.

    PubMed

    Kirschner, Lawrence S; Kusewitt, Donna F; Matyakhina, Ludmila; Towns, William H; Carney, J Aidan; Westphal, Heiner; Stratakis, Constantine A

    2005-06-01

    Carney complex is an autosomal dominant neoplasia syndrome characterized by spotty skin pigmentation, myxomatosis, endocrine tumors, and schwannomas. This condition may be caused by inactivating mutations in PRKAR1A, the gene encoding the type 1A regulatory subunit of protein kinase A. To better understand the mechanism by which PRKAR1A mutations cause disease, we have developed conventional and conditional null alleles for Prkar1a in the mouse. Prkar1a(+/-) mice developed nonpigmented schwannomas and fibro-osseous bone lesions beginning at approximately 6 months of age. Although genotype-specific cardiac and adrenal lesions were not seen, benign and malignant thyroid neoplasias were observed in older mice. This spectrum of tumors overlaps that seen in Carney complex patients, confirming the validity of this mouse model. Genetic analysis indicated that allelic loss occurred in a subset of tumor cells, suggesting that complete loss of Prkar1a plays a key role in tumorigenesis. Similarly, tissue-specific ablation of Prkar1a from a subset of facial neural crest cells caused the formation of schwannomas with divergent differentiation. These observations confirm the identity of PRKAR1A as a tumor suppressor gene with specific importance to cyclic AMP-responsive tissues and suggest that these mice may be valuable tools not only for understanding endocrine tumorigenesis but also for understanding inherited predispositions for schwannoma formation. PMID:15930266

  4. Regulation of genotoxic stress response by homeodomain-interacting protein kinase 2 through phosphorylation of cyclic AMP response element-binding protein at serine 271.

    PubMed

    Sakamoto, Kensuke; Huang, Bo-Wen; Iwasaki, Kenta; Hailemariam, Kiros; Ninomiya-Tsuji, Jun; Tsuji, Yoshiaki

    2010-08-15

    CREB (cyclic AMP response element-binding protein) is a stimulus-induced transcription factor that plays pivotal roles in cell survival and proliferation. The transactivation function of CREB is primarily regulated through Ser-133 phosphorylation by cAMP-dependent protein kinase A (PKA) and related kinases. Here we found that homeodomain-interacting protein kinase 2 (HIPK2), a DNA-damage responsive nuclear kinase, is a new CREB kinase for phosphorylation at Ser-271 but not Ser-133, and activates CREB transactivation function including brain-derived neurotrophic factor (BDNF) mRNA expression. Ser-271 to Glu-271 substitution potentiated the CREB transactivation function. ChIP assays in SH-SY5Y neuroblastoma cells demonstrated that CREB Ser-271 phosphorylation by HIPK2 increased recruitment of a transcriptional coactivator CBP (CREB binding protein) without modulation of CREB binding to the BDNF CRE sequence. HIPK2-/- MEF cells were more susceptible to apoptosis induced by etoposide, a DNA-damaging agent, than HIPK2+/+ cells. Etoposide activated CRE-dependent transcription in HIPK2+/+ MEF cells but not in HIPK2-/- cells. HIPK2 knockdown in SH-SY5Y cells decreased etoposide-induced BDNF mRNA expression. These results demonstrate that HIPK2 is a new CREB kinase that regulates CREB-dependent transcription in genotoxic stress. PMID:20573984

  5. Phosphorylation and activation of calcineurin by glycogen synthase (casein) kinase-1 and cyclic AMP-dependent protein kinase

    SciTech Connect

    Singh, T.J.; Wang, J.H.

    1986-05-01

    Calcineurin is a phosphoprotein phosphatase that is activated by divalent cations and further stimulated by calmodulin. In this study calcineurin is shown to be a substrate for both glycogen synthase (casein) kinase-1 (CK-1) and cyclic AMP-dependent protein kinase (A-kinase). Either kinase can catalyze the incorporation of 1.0-1.4 mol /sup 32/P/mol calcineurin. Analysis by SDS-PAGE revealed that only the ..cap alpha.. subunit is phosphorylated. Phosphorylation of calcineurin by either kinase leads to its activation. Using p-nitrophenyl phosphate as a substrate the authors observed a 2-3 fold activation of calcineurin by either Mn/sup 2 +/ or Ni/sup 2 +/ (in the presence or absence of calmodulin) after phosphorylation of calcineurin by either CK-1 or A-kinase. In the absence of Mn/sup 2 +/ or Ni/sup 2 +/ phosphorylated calcineurin, like the nonphosphorylated enzyme, showed very little activity. Ni/sup 2 +/ was a more potent activator of phosphorylated calcineurin compared to Mn/sup 2 +/. Higher levels of activation (5-8 fold) of calcineurin by calmodulin was observed when phosphorylated calcineurin was pretreated with Ni/sup 2 +/ before measurement of phosphatase activity. These results indicate that phosphorylation may be an important mechanism by which calcineurin activity is regulated by Ca/sup 2 +/.

  6. AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

    PubMed Central

    Johanns, M.; Lai, Y.-C.; Hsu, M.-F.; Jacobs, R.; Vertommen, D.; Van Sande, J.; Dumont, J. E.; Woods, A.; Carling, D.; Hue, L.; Viollet, B.; Foretz, M; Rider, M H

    2016-01-01

    Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation. PMID:26952277

  7. Activation of portal-hepatic osmoreceptors in rats: role of calcium, acetylcholine and cyclic AMP.

    PubMed

    Stoppini, L; Baertschi, A J

    1984-11-01

    Osmoreceptors are sensory organs of paramount importance in water and electrolyte balance, yet the mechanisms for their activation are virtually unknown. Peripheral osmoreceptors have been localised in the hepatic portal vein area of rats. We thus superfused the portal adventitia with 0.2 ml of 4% NaCl before and after various pharmacological pretreatments (0.4 ml of 1 mM solutions) of the portal area, while monitoring the neural activity of the hypothalamo-neurohypophysial system. Portal superfusion with verapamil, to reduce Ca-influx, reversibly inhibited the response to osmotic stimuli by up to 50% (P less than 0.0005). Such inhibition (58%; P less than 0.0005) was also seen with portal superfusion by atropine. Atropine did not affect hypothalamo-neurohypophysial responses to stimulation of portal bradykinin receptors with 0.2 ml 1 muM bradykinin, and portal superfusion with acetylcholine activated the hypothalamo-neurohypophysial system. The results thus support the hypothesis of a cholinergic neurotransmission linking portal osmoreceptive structures and afferent nerve endings. Diamide, which inhibits water efflux in frog skin, also reversibly inhibited responses to osmotic stimuli by 38% (P less than 0.0005). Pretreatments with trifluoperazine, a calmodulin inhibitor, and cordycepin, an adenylate cyclase inhibitor, diminished responses to osmotic stimuli by 30-45% (P less than 0.005), while cAMP and theophilline potentiated them by 38% (P less than 0.0005). Responses to bradykinin superfusion were reduced 20-30% (P less than 0.05) by both cordycepin and cAMP. The results suggest that portal osmoreceptors release acetylcholine to excite afferent nerves when exposed to an osmotic gradient. The mechanism of this release may be mediated by an efflux of water and an increase of intracellular calcium activity and cAMP. PMID:6150955

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

    SciTech Connect

    Kim, Kyoung Mi; Kwon, Shi-Nae; Kang, Ju-Il; Lee, Song Hee; Jang, Sung Key; Ahn, Byung-Yoon; Kim, Yoon Ki . E-mail: yk-kim@korea.ac.kr

    2007-05-18

    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. Calspermin gene transcription is regulated by two cyclic AMP response elements contained in an alternative promoter in the calmodulin kinase IV gene.

    PubMed Central

    Sun, Z; Sassone-Corsi, P; Means, A R

    1995-01-01

    The transcript for the high-affinity Ca2+/calmodulin-binding protein calspermin is generated from the gene encoding Ca2+/calmodulin-dependent protein kinase IV only in postmeiotic germ cells during spermatogenesis. We demonstrate that this testis-specific calspermin transcript can be produced in heterologous cells by utilization of a promoter located in an intron of the calmodulin (CaM) kinase IV gene. Critical motifs within this promoter are two cyclic AMP response element (CRE)-like sequences located about -70 and -50 bp upstream of the transcriptional initiation site. Both CRE motifs are footprinted by the authentic testis-specific transcriptional activator CREM tau or by CREM tau present in adult testis nuclear extract. Whereas a 2.1-kb DNA fragment containing the calspermin promoter is inactive when transfected into NIH 3T3 cells, activity can be restored by cotransfection of CREM tau and protein kinase A or CaM kinase IV but not CaM kinase II alpha. Restoration of activity is greatly reduced by mutation of the two CRE motifs. Since CRE-like motifs have been identified in many genes uniquely expressed in postmeiotic germ cells, which contain abundant CREM tau protein, we suggest that CREM tau may function as one transcription factor responsible for the expression of postmeiotic germ cell-specific genes. PMID:7799965

  10. Mechanisms of tyrosine hydroxylase regulation in striatal synaptosomes: effects of activation of cyclic AMP-dependent protein kinase

    SciTech Connect

    Colby, K.A.

    1987-01-01

    The regulation of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis, was examined in synaptosomes prepared from rat corpus striatum. Exposure of striatal synaptosomes to dibutyryl-cyclic AMP (dbcAMP) causes an increase in the maximal velocity of TH, but does not change the K/sub m/ of the enzyme for the synthetic cofactor, 2-amino-4-hydroxy-6-methyl-tetrahydropterin. Activation of TH by synaptosomal exposure to dbcAMP also causes a decrease in the pH sensitivity and an increase in the thermolability of the enzyme. Striatal synaptosomes were used to examine the in vitro phosphorylation of TH. Under the protocol developed as part of this work, TH in synaptosomes can be labelled with /sup 32/P. This is the first report of in vitro labelling of TH in a biochemically intact CNS preparation. Under certain protocols, treatment of synaptosomes with dbcAMP causes an increase in the /sup 32/P labelling of TH. These results are consistent with the notion that dbcAMP produces changes in the physical properties of TH by activating cAMP-dependent protein kinase which subsequently phosphorylates TH. In vivo electrical stimulation of the rat medial forebrain bundle causes an activation of striatal TH as well as an decrease in the pH sensitivity of the enzyme. Since similar changes are produced upon activation of snaptosomal TH by dbcAMP, it is likely that phosphorylation of TH is involved in the increase in TH activity that is associated with neuronal depolarization.

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

    PubMed

    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. PMID:1445251

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

    PubMed Central

    Hoggett, J G; Brierley, I

    1992-01-01

    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. PMID:1445251

  13. The Role of Angiotensin II and Cyclic AMP in Alveolar Active Sodium Transport

    PubMed Central

    Ismael-Badarneh, Reem; Guetta, Julia; Klorin, Geula; Berger, Gidon; Abu-saleh, Niroz; Abassi, Zaid; Azzam, Zaher S.

    2015-01-01

    Active alveolar fluid clearance is important in keeping airspaces free of edema. Angiotensin II plays a role in the pathogenesis of hypertension, heart failure and others. However, little is known about its contribution to alveolar fluid clearance. Angiotensin II effects are mediated by two specific receptors; AT1 and AT2. The localization of these two receptors in the lung, specifically in alveolar epithelial cells type II, was recently reported. We hypothesize that Angiotensin II may have a role in the regulation of alveolar fluid clearance. We investigated the effect of Angiotensin II on alveolar fluid clearance in rats using the isolated perfused lung model and isolated rat alveolar epithelial cells. The rate of alveolar fluid clearance in control rats was 8.6% ± 0.1 clearance of the initial volume and decreased by 22.5%, 28.6%, 41.6%, 48.7% and 39% in rats treated with 10-10 M, 10-9 M, 10-8 M, 10-7 M or 10-6 M of Ang II respectively (P < 0.003). The inhibitory effect of Angiotensin II was restored in losartan, an AT1 specific antagonist, pretreated rats, indicating an AT1 mediated effect of Ang II on alveolar fluid clearance. The expression of Na,K-ATPase proteins and cAMP levels in alveolar epithelial cells were down-regulated following the administration of Angiotensin II; suggesting that cAMP may be involved in AngII-induced reduced Na,K-ATPase expression, though the contribution of additional factors could not be excluded. We herein suggest a novel mechanism of clinical relevance by which angiotensin adversely impairs the ability of the lungs to clear edema. PMID:26230832

  14. Role of Exchange Protein Directly Activated by Cyclic AMP Isoform 1 in Energy Homeostasis: Regulation of Leptin Expression and Secretion in White Adipose Tissue.

    PubMed

    Hu, Yaohua; Robichaux, William G; Mei, Fang C; Kim, Eun Ran; Wang, Hui; Tong, Qingchun; Jin, Jianping; Xu, Mingxuan; Chen, Ju; Cheng, Xiaodong

    2016-10-01

    Epacs (exchange proteins directly activated by cyclic AMP [cAMP]) act as downstream effectors of cAMP and play important roles in energy balance and glucose homeostasis. While global deletion of Epac1 in mice leads to heightened leptin sensitivity in the hypothalamus and partial protection against high-fat diet (HFD)-induced obesity, the physiological functions of Epac1 in white adipose tissue (WAT) has not been explored. Here, we report that adipose tissue-specific Epac1 knockout (AEKO) mice are more prone to HFD-induced obesity, with increased food intake, reduced energy expenditure, and impaired glucose tolerance. Despite the fact that AEKO mice on HFD display increased body weight, these mice have decreased circulating leptin levels compared to their wild-type littermates. In vivo and in vitro analyses further reveal that suppression of Epac1 in WAT decreases leptin mRNA expression and secretion by inhibiting cAMP response element binding (CREB) protein and AKT phosphorylation, respectively. Taken together, our results demonstrate that Epac1 plays an important role in regulating energy balance and glucose homeostasis by promoting leptin expression and secretion in WAT. PMID:27381457

  15. Modulation by atrial natriuretic factor of receptor-mediated cyclic AMP-dependent responses in canine pulmonary artery during heart failure.

    PubMed Central

    Mathew, R.; Omar, H. A.; Fayngersh, R.; Shen, W.; Wang, J.; Gewitz, M. H.; Hintze, T. H.; Wolin, M. S.

    1996-01-01

    1. Pacing-induced congestive heart failure (CHF) in dogs is associated with increased plasma levels of atrial natriuretic factor (ANF) and inhibition of receptor-mediated cyclic AMP-dependent relaxation in isolated pulmonary arteries (PA). Since ANF is known to be negatively coupled to adenylate cyclase, we studied cyclic AMP-mediated relaxation to isoprenaline (Iso) and arachidonic acid (AA) in PA from control dogs (C), dogs with pacing-induced CHF (CHF) and dogs with bilateral atrial appendectomy and CHF (ATR APP+CHF). 2. In CHF, plasma ANF levels increased from a baseline of 80 +/- 8 pg ml-1 to 283 +/- 64 pg ml-1 (P < 0.05), but the ATR APP+CHF group failed to show this increase (67 +/- 7 pg ml-1 vs 94 +/- 15 pg ml-1, P = NS). Plasma ANF levels, however, did not influence myocardial dysfunction in CHF. 3. The relaxation of 49 +/- 5% to 1 microM Iso in C was reduced to 23 +/- 4% in CHF (P < 0.05), but relaxation of 49 +/- 12% was observed in the ATR APP+CHF group (P = NS vs C). Relaxation responses to 10 microM AA were as follows: 77 +/- 5% (C, n = 8), 27 +/- 8% (CHF, n = 10, P < 0.05 vs C), and 93 +/- 5% (ATR APP+CHF, n = 5). The presence of CHF, or the plasma ANF levels, did not affect responses to cyclic GMP-mediated relaxing agents in PA. 4. These data indicate that the myocardial performance in CHF is not influenced by plasma ANF levels. However, altered cyclic AMP-mediated relaxation in PA during CHF is, in part, modulated by circulating ANF levels. PMID:8864519

  16. Inducing coproporphyria in rat hepatocyte cultures using cyclic AMP and cyclic AMP-releasing agents.

    PubMed

    De Matteis, Francesco; Harvey, Carolyn

    2005-07-01

    Cyclic AMP (c-AMP), added on its own to rat hepatocyte cultures, caused a marked accumulation of coproporphyrin III. The results obtained by comparing the effect of c-AMP to that of exogenous 5-aminolevulinate (ALA), and from adding c-AMP and ALA together, indicated that the coproporphyrinogen III metabolism was blocked, even though no inhibition of the relevant enzyme, coproporphyrinogen oxidase, could be demonstrated. Preferential accumulation of coproporphyrin could also be produced in cultures of rat hepatocytes by agents that raise the cellular levels of cyclic AMP, such as glucagon. The effect of supplementing the culture medium with triiodothyronine (T3) on the response of rat hepatocytes to c-AMP was also investigated. T3, which is known to stimulate mitochondrial respiration, uncoupling O2 consumption from ATP synthesis, produced a c-AMP-like effect when given on its own and potentiated the effect of c-AMP, with an apparent increase in the severity of the metabolic block. It is suggested that an oxidative mechanism may be activated in c-AMP and T3-induced coproporphyria, preferentially involving the mitochondrial compartment, leading to oxidation of porphyrinogen intermediates of haem biosynthesis, especially coproporphyrinogen. Coproporphyin, the fully oxidized aromatic derivative produced, cannot be metabolized and will therefore accumulate. PMID:15902420

  17. Adenylyl cyclase 6 mediates the action of cyclic AMP-dependent secretagogues in mouse pancreatic exocrine cells via protein kinase A pathway activation

    PubMed Central

    Sabbatini, Maria E; D’Alecy, Louis; Lentz, Stephen I; Tang, Tong; Williams, John A

    2013-01-01

    Both secretin and vasoactive intestinal polypeptide (VIP) receptors are responsible for the activation of adenylyl cyclases (ACs), which increase intracellular cyclic AMP (cAMP) levels in the exocrine pancreas. There are nine membrane-associated isoforms, each with its own pattern of expression and regulation. In this study we sought to establish which AC isoforms play a regulatory role in pancreatic exocrine cells. Using RT-PCR, AC3, AC4, AC6, AC7 and AC9 were found to be expressed in the pancreas. AC3, AC4, AC6 and AC9 were expressed in both pancreatic acini and ducts, whereas AC7 was expressed only in pancreatic ducts. Based on known regulation by intracellular signals, selective inhibitors and stimulators were used to suggest which isoforms play an important role in the induction of cAMP formation. AC6 appeared to be an important isoform because protein kinase A (PKA), PKC and calcium all inhibited VIP-induced cAMP formation, whereas calcineurin or calmodulin did not modify the response to VIP. Mice with genetically deleted AC6 were studied and showed reduced cAMP formation and PKA activation in both isolated pancreatic acini and duct fragments. The absence of AC6 reduced cAMP-dependent secretagogue-stimulated amylase secretion, and abolished fluid secretion in both in vivo and isolated duct fragments. In conclusion, several AC isoforms are expressed in pancreatic acini and ducts. AC6 mediates a significant part of pancreatic amylase and fluid secretion in response to secretin, VIP and forskolin through cAMP/PKA pathway activation. PMID:23753526

  18. Cyclic AMP response element-binding protein in post-mortem brain of teenage suicide victims: specific decrease in the prefrontal cortex but not the hippocampus.

    PubMed

    Pandey, Ghanshyam N; Dwivedi, Yogesh; Ren, Xinguo; Rizavi, Hooriyah S; Roberts, Rosalinda C; Conley, Robert R

    2007-10-01

    Abnormalities in both adenylyl cyclase (AC) and phosphoinositide (PI) signalling systems have been observed in the post-mortem brain of suicide victims. Cyclic AMP response element-binding protein (CREB) is a transcription factor that is activated by phosphorylating enzymes such as protein kinase A (PKA) and protein kinase C (PKC), which suggests that both AC and PI signalling systems converge at the level of CREB. CREB is involved in the transcription of many neuronally expressed genes that have been implicated in the pathophysiology of depression and suicide. Since we observed abnormalities of both PKA and PKC in the post-mortem brain of teenage suicide victims, we examined if these abnormalities are also associated with abnormalities of CREB, which is activated by these phosphorylating enzymes. We determined CRE-DNA binding using the gel shift assay, as well as protein expression of CREB using the Western blot technique, and the mRNA expression of CREB using a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) technique in the prefrontal cortex (PFC), and hippocampus obtained from 17 teenage suicide victims and 17 matched normal control subjects. We observed that the CRE-DNA binding and the protein expression of CREB were significantly decreased in the PFC of teenage suicide victims compared with controls. There was also a significant decrease in mRNA expression of CREB in the PFC of teenage suicide victims compared with control subjects. However, there were no significant differences in CRE-DNA binding or the protein and mRNA expression of CREB in the hippocampus of teenage suicide victims compared with control subjects. These results suggest that the abnormalities of PKA, and of PKC, observed in teenage suicide victims are also associated with abnormalities of the transcription factor CREB, and that this may also cause alterations of important neuronally expressed genes, and provide further support of the signal transduction of abnormalities

  19. Hormone-sensitive lipase in differentiated 3T3-L1 cells and its activation by cyclic AMP-dependent protein kinase.

    PubMed Central

    Kawamura, M; Jensen, D F; Wancewicz, E V; Joy, L L; Khoo, J C; Steinberg, D

    1981-01-01

    Differentiation of 3T3-L1 fibroblasts to adipocyte-like cells was accompanied by a 19-fold increase in neutral triglyceride lipase activity, a 12-fold increase in diglyceride lipase activity, a 10-fold increase in monoglyceride lipase activity, and a 280-fold increase in cholesterol esterase activity. In contrast, acid acylhydrolase activities did not increase during differentiation. The rate of glycerol release from unstimulated intact cells increased by more than 1 order of magnitude upon differentiation. Isoproterenol (1 microM) and 1-methyl-3-isobutylxanthine (0.1 mM) further stimulated this rate of glycerol release 3-fold. The neutral triglyceride lipase activity in cell-free preparations of differentiated cells was activated 105% by cyclic AMP-dependent protein kinase. Neutral cholesterol esterase, diglyceride lipase, and monoglyceride lipase were also activated (117%, 10%, and 37+, respectively) by cyclic AMP-dependent protein kinase. In contrast, protein kinase had no effect on any of the four lysosomal acid acylhydrolase activities. Thus, hormone-sensitive lipase, the most characteristic and functionally important enzyme of adipose tissue, has been characterized in differentiated 3T3-L1 cells. The 3T3-L1 cell should be a valuable model system in which to study regulation of hormone-sensitive lipase, particularly its long-term regulation. PMID:6262767

  20. Involvement of platelet cyclic GMP but not cyclic AMP suppression in leukocyte-dependent platelet adhesion to endothelial cells induced by platelet-activating factor in vitro.

    PubMed Central

    Hirafuji, M.; Nezu, A.; Shinoda, H.; Minami, M.

    1996-01-01

    1. Incubation of endothelial cells with platelets in the absence or the presence of PAF (10 nM) markedly increased platelet cyclic AMP levels, which were significantly decreased by indomethacin (3 microM). Co-incubation of endothelial cells and platelets with polymorphonuclear leukocytes (PMNs) did not change the platelet cyclic AMP levels. 2. Incubation of endothelial cells with platelets in the absence of PAF increased platelet cyclic GMP levels, which were increased 3.5 fold by PAF. These cyclic GMP levels were significantly decreased by NG-nitro-L-arginine (100 microM), and completely by methylene blue (10 microM). When endothelial cells and platelets were co-incubated with PMNs, the cyclic GMP level in the cell mixture was 42.5 and 65.3% lower than that in endothelial cells and platelets without and with PAF stimulation, respectively. 3. PAF induced platelet adhesion to endothelial cells only when PMNs were present. Methylene blue dose-dependently potentiated the PMN-dependent platelet adhesion induced by PAF, although it had no effect in the absence of PMNs. 4. Sodium nitroprusside and 8-bromo cyclic GMP but not dibutyryl cyclic AMP significantly, although partially, inhibited the platelet adhesion. Inhibition of cyclic GMP-specific phosphodiesterase by zaprinast slightly inhibited the PMN-induced platelet adhesion and potentiated the inhibitory effect of 8-bromo cyclic GMP, while these drugs markedly inhibited the adhesion of platelet aggregates induced by PMN sonicates. 5. These results suggest that the impairment by activated PMNs of EDRF-induced platelet cyclic GMP formation is involved in part in the mechanism of PMN-dependent platelet adhesion to endothelial cells induced by PAF in vitro. The precise mechanism still remains to be clarified. PMID:8789382

  1. A Ric8/Synembryn Homolog Promotes Gpa1 and Gpa2 Activation To Respectively Regulate Cyclic AMP and Pheromone Signaling in Cryptococcus neoformans

    PubMed Central

    Gong, Jinjun; Grodsky, Jacob D.; Zhang, Zhengguang

    2014-01-01

    The G protein α subunits Gpa1, Gpa2, and Gpa3 mediate signal transduction and are important in the growth and virulence of Cryptococcus neoformans. To understand how Gpa1 functions without a conventional Gβ subunit, we characterized a resistance to inhibitors of cholinesterase 8 (Ric8) homolog from C. neoformans, which shares amino acid sequence homology with other Ric8 proteins that exhibit guanine nucleotide exchange factor (GEF) activity toward Gα. We found that the ric8 mutant was reduced in capsule size and melanin formation, which could be suppressed by cyclic AMP (cAMP) supplementation or by introducing the activated GPA1Q284L allele. Consistent with the fact that Ric8 participates in cAMP signaling to regulate virulence, the ric8 mutant was attenuated in virulence toward mice. Interestingly, disruption of RIC8 also resulted in opposing effects on pheromone signaling, as the ric8 mutant showed reduced mating but an enhanced ability to induce the pheromone response in the mating partner. To identify Ric8 functional mechanisms, we examined the interactions between Ric8 and the three Gα proteins. Ric8 interacted with Gpa1 and Gpa2, but not Gpa3. The presence of Gpa1Q284L negatively affected its interaction with Ric8, whereas the activated Gpa2Q203L allele abolished the interaction. Collectively, these findings suggest that Ric8 functions as a GEF to facilitate the activation of Gpa1-cAMP signaling and to promote Gpa2, affecting mating efficiency. Our study highlights the distinct and conserved characteristics associated with G protein signaling and contributes to our overall understanding of how G protein α subunits function with or without a canonical Gβ partner in C. neoformans. PMID:25084863

  2. Differentiation-Coupled Induction of Human Cytomegalovirus Replication by Union of the Major Enhancer Retinoic Acid, Cyclic AMP, and NF-κB Response Elements

    PubMed Central

    Yuan, Jinxiang; Li, Ming; Torres, Yasaira Rodriguez; Galle, Courtney S.

    2015-01-01

    ABSTRACT Triggers and regulatory pathways that effectively link human cytomegalovirus (HCMV) major immediate early (MIE) latent-lytic switch activation with progeny production are incompletely understood. In the quiescently infected human NTera2 cell model of primitive neural stem cells, we found that costimulation with vasoactive intestinal peptide (V) and phorbol ester (P) synergistically activated viral infection, but this effect waned over time. Coupling retinoic acid (R), an inducer of neuronal differentiation, to VP pulse stimulation attenuated the decline in viral activity and promoted the spread of the active infection through concentric layers of neighboring cells as cellular differentiation progressed. R stimulation alone was unable to activate the infection. The MIE enhancer cis-regulatory mechanisms responsible for this result were characterized by a strategy of combinatorial mutagenesis of five cis-acting element types (retinoic acid receptor binding elements [RARE], cyclic AMP [cAMP] response elements [CRE], NF-κB binding sites [kB], serum response element, and ETS/ELK-1 binding site) and multiple methods of assessment. We found that the CRE and kB combination sets the preinduction enhancer tone, is the major initiator and amplifier of RVP-induced MIE gene expression, and cooperates with RARE during cellular differentiation to enhance viral spread. In predifferentiated NTera2, we also found that the CRE-kB combination functions as initiator and amplifier of unstimulated HCMV MIE gene expression and cooperatively interacts with RARE to enhance viral spread. We conclude that RVP-stimulated signaling cascades and cellular differentiation operate through the enhancer CRE-kB-RARE core in strengthening induction of HCMV MIE gene expression in linkage with viral propagation. IMPORTANCE Cytomegalovirus-seropositive persons commonly lack detectable levels of cytomegalovirus replication, even when profoundly immunocompromised. In a human NTera2 cell model of

  3. Cyclic AMP Receptor Protein-Aequorin Molecular Switch for Cyclic AMP

    PubMed Central

    Scott, Daniel; Hamorsky, Krystal Teasley; Ensor, C. Mark; Anderson, Kimberly W.; Daunert, Sylvia

    2011-01-01

    Molecular switches are designer molecules that combine the functionality of two individual proteins into one, capable of manifesting an “on/off” signal in response to a stimulus. These switches have unique properties and functionalities and thus, can be employed as nanosensors in a variety of applications. To that end, we have developed a bioluminescent molecular switch for cyclic AMP. Bioluminescence offers many advantages over fluorescence and other detection methods including the fact that there is essentially zero background signal in physiological fluids, allowing for more sensitive detection and monitoring. The switch was created by combining the properties of the cyclic AMP receptor protein (CRP), a transcriptional regulatory protein from E. coli that binds selectively to cAMP with those of aequorin, a bioluminescent photoprotein native of the jellyfish Aequorea victoria. Genetic manipulation to split the genetic coding sequence of aequorin in two and genetically attach the fragments to the N and C termini of CRP, resulted in a hybrid protein molecular switch. The conformational change experienced by CRP upon the binding of cyclic AMP is suspected to result in the observed loss of bioluminescent signal from aequorin. The “on/off” bioluminescence can be modulated by cyclic AMP over a range of several orders of magnitude in a linear fashion in addition to the capacity to detect changes in cellular cyclic AMP of intact cells exposed to different external stimuli without the need to lyse the cells. We envision that the molecular switch could find applications in vitro as well as in vivo cyclic AMP detection and/or imaging. PMID:21329338

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

    PubMed

    Mednieks, M I; Popova, I A; Grindeland, R E

    1991-10-01

    A frequent cellular response to organismal stress is the increase in ligand binding by beta-adrenergic receptors. The extracellular signal is amplified by intracellular increases in cyclic AMP and the ensuing activation of cyclic AMP-dependent protein kinase (cAPK). The molecular mechanisms involve the binding of cyclic AMP to regulatory (R) subunits of cAPK, thus freeing the catalytic subunit for protein phosphorylation. This study was carried out to determine the cellular compartmentalization of the cyclic AMP-receptor proteins in heart ventricular tissue obtained from rats flown on the Cosmos 1887 mission. Photoaffinity labeling of soluble and particulate cell fractions with an [32P]-8-azido analog of cyclic AMP was followed by electrophoretic separation of the proteins and by autoradiographic identification of the labeled isoforms of cAPK R subunits. The results showed 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 showed some variability in tissues of individual animals, but exhibited 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. These findings indicate that the cardiac cell integrity or its protein content is not compromised under flight conditions. There is, however, what appears to be an adaptive molecular response which can be detected using microanalytical methods, indicating that a major hormone regulated mechanism may be affected during some phase of travel in space. PMID:1662483

  5. Comparative Transcriptome Analysis Reveals Novel Roles of the Ras and Cyclic AMP Signaling Pathways in Environmental Stress Response and Antifungal Drug Sensitivity in Cryptococcus neoformans ▿ †

    PubMed Central

    Maeng, Shinae; Ko, Young-Joon; Kim, Gyu-Bum; Jung, Kwang-Woo; Floyd, Anna; Heitman, Joseph; Bahn, Yong-Sun

    2010-01-01

    The cyclic AMP (cAMP) pathway plays a central role in the growth, differentiation, and virulence of pathogenic fungi, including Cryptococcus neoformans. Three upstream signaling regulators of adenylyl cyclase (Cac1), Ras, Aca1, and Gpa1, have been demonstrated to control the cAMP pathway in C. neoformans, but their functional relationship remains elusive. We performed a genome-wide transcriptome analysis with a DNA microarray using the ras1Δ, gpa1Δ, cac1Δ, aca1Δ, and pka1Δ pka2Δ mutants. The aca1Δ, gpa1Δ, cac1Δ, and pka1Δ pka2Δ mutants displayed similar transcriptome patterns, whereas the ras1Δ mutant exhibited transcriptome patterns distinct from those of the wild type and the cAMP mutants. Interestingly, a number of environmental stress response genes are modulated differentially in the ras1Δ and cAMP mutants. In fact, the Ras signaling pathway was found to be involved in osmotic and genotoxic stress responses and the maintenance of cell wall integrity via the Cdc24-dependent signaling pathway. Notably, the Ras and cAMP mutants exhibited hypersensitivity to a polyene drug, amphotericin B, without showing effects on ergosterol biosynthesis, which suggested a novel method of antifungal combination therapy. Among the cAMP-dependent gene products that we characterized, two small heat shock proteins, Hsp12 and Hsp122, were found to be involved in the polyene antifungal drug susceptibility of C. neoformans. PMID:20097740

  6. Cyclic AMP responsive element-binding protein promotes renal cell carcinoma proliferation probably via the expression of spindle and kinetochore-associated protein 2

    PubMed Central

    Zhuang, Haihui; Meng, Xiangyu; Li, Yanyuan; Wang, Xue; Huang, Shuaishuai; Liu, Kaitai; Hehir, Michael; Fang, Rong; Jiang, Lei; Zhou, Jeff X.; Wang, Ping; Ren, Yu

    2016-01-01

    Emerging evidence shows that the aberrantly expressed cyclic AMP responsive element-binding protein (CREB) is associated with tumor development and progression in several cancers. Spindle and kinetochore-associated protein 2 (SKA2) is essential for regulating the progress of mitosis. In this study, we evaluate in vitro and in vivo the functional relationship between CREB and SKA2 in renal cell carcinoma (RCC). Suppressing and replenishing CREB levels were used to manipulate SKA2 expression, observing the effects on RCC cell lines. Computational prediction and ChIP assay identified that CREB targeted ska2 by binding its CRE sequence in the human genome. Overexpression of CREB reversed the inhibited cell growth following siSKA2 treatment, and reduced the number of cells holding in mitosis. Decreased expression of CREB suppressed RCC cell growth and xenograft tumor formation, accompanied by reduced expression of SKA2. In RCC tumor samples from patients, mRNA for SKA2 were plotted near those of CREB in each sample, with significantly increased immunohistochemical staining of higher SKA2 and CREB in the higher TNM stages. The study adds evidence that CREB, a tumor oncogene, promotes RCC proliferation. It probably achieves this by increasing SKA2 expression. PMID:26824422

  7. Involvement of a cyclic-AMP pathway in group I metabotropic glutamate receptor responses in neonatal rat cortex.

    PubMed

    Schaffhauser, H; de Barry, J; Muller, H; Heitz, M P; Gombos, G; Mutel, V

    1997-09-10

    3,5-Dihydroxyphenylglycine (DHPG), (S)-3-hydroxyphenylglycine and (S)-4-carboxy-3-hydroxyphenylglycine (S-4C3HPG) stimulated phosphoinositide hydrolysis in neonatal rat cortical slices, but with lower maximal effect, in comparison with 2S,1'S,2'S-2-(2'-carboxycyclopropyl)glycine (L-CCG I) or (1S,3R)-1-aminocyclo-pentane-1,3-dicarboxylic acid (1S,3R-ACPD). DHPG, 1S,3R-ACPD, and S-4C3HPG also evoked a rapidly desensitizing increase in [Ca2+]i in cortical layers of neonatal brain slices. (R,S)-alpha-methyl-4-tetrazolyl-phenylglycine (MTPG), and (R,S)-alpha-methyl-4-phosphono-phenylglycine (MPPG) inhibited the increase of phosphoinositide hydrolysis elicited by 1S,3R-ACPD but not that by R,S-DHPG. In contrast, the selective group II receptor agonist (1S,2S,5R,6S)-2-amino-bicyclo-[3.1.0]-hexane-2,6-dicarboxylate (LY 354740) potentiated the response of R,S-DHPG. Finally, 8-(4-chlorophenylthio)-cAMP, a membrane permeant analogue of cAMP, reversed the stimulatory effect of 1S,3R-ACPD and S-4C3HPG on phosphoinositide hydrolysis and [Ca2+]i mobilization, without affecting the response induced by R,S-DHPG. These data suggest that, in neonatal rat cortex, the activation of group II metabotropic glutamate receptors potentiates the phosphoinositide hydrolysis and [Ca2+]i responses mediated by group I metabotropic glutamate receptors. PMID:9369360

  8. Activation of Exchange Protein Activated by Cyclic-AMP Enhances Long-Lasting Synaptic Potentiation in the Hippocampus

    ERIC Educational Resources Information Center

    Gelinas, Jennifer N.; Banko, Jessica L.; Peters, Melinda M.; Klann, Eric; Weeber, Edwin J.; Nguyen, Peter V.

    2008-01-01

    cAMP is a critical second messenger implicated in synaptic plasticity and memory in the mammalian brain. Substantial evidence links increases in intracellular cAMP to activation of cAMP-dependent protein kinase (PKA) and subsequent phosphorylation of downstream effectors (transcription factors, receptors, protein kinases) necessary for long-term…

  9. Cyclic AMP-dependent activation of rhodopsin gene transcription in cultured retinal precursor cells of chicken embryo.

    PubMed

    Voisin, Pierre; Bernard, Marianne

    2009-07-01

    The present study describes a robust 50-fold increase in rhodopsin gene transcription by cAMP in cultured retinal precursor cells of chicken embryo. Retinal cells isolated at embryonic day 8 (E8) and cultured for 3 days in serum-supplemented medium differentiated mostly into red-sensitive cones and to a lesser degree into green-sensitive cones, as indicated by real-time RT-PCR quantification of each specific opsin mRNA. In contrast, both rhodopsin mRNA concentration and rhodopsin gene promoter activity required the presence of cAMP-increasing agents [forskolin and 3-isobutyl-1-methylxanthine (IBMX)] to reach significant levels. This response was rod-specific and was sufficient to activate rhodopsin gene transcription in serum-free medium. The increase in rhodopsin mRNA levels evoked by a series of cAMP analogs suggested the response was mediated by protein kinase A, not by EPAC. Membrane depolarization by high KCl concentration also increased rhodopsin mRNA levels and this response was strongly potentiated by IBMX. The rhodopsin gene response to cAMP-increasing agents was developmentally gated between E6 and E7. Rod-specific transducin alpha subunit mRNA levels also increased up to 50-fold in response to forskolin and IBMX, while rod-specific phosphodiesterase-VI and rod arrestin transcripts increased 3- to 10-fold. These results suggest a cAMP-mediated signaling pathway may play a role in rod differentiation. PMID:19457115

  10. The plasma cyclic-AMP response to noise in humans and rats—short-term exposure to various noise levels

    NASA Astrophysics Data System (ADS)

    Iwamoto, M.; Dodo, H.; Ishii, F.; Yoneda, J.; Yamazaki, S.; Goto, H.

    1988-12-01

    Rats were exposed to short-term noise which was found to activate the hypothalamohypophyseal-adrenal system and result in a decrease of adrenal ascorbic acid (AAA) and an increase of serum corticosterone (SCS). The threshold limit value lay between 60 and 70 dB(A). To characterize better the effect of noise on the human hypothalamo-hypophyseal-adrenal system, a large group of subjects was exposed to short-term noise at 85 dB(A) and higher, and tested for levels of adrenocortical steroid (cortisol) and anterior pituitary hormones such as ACTH, growth hormone (GH) and prolactin (PRL). Results in humans showed hyperfunction of the hypothalamo-pituitary system. However, as the responses in rats and humans differed, a further experiment was performed using C-AMP, a second messenger mediating many of the effects of a variety of hormones. Plasma C-AMP in humans and rats increased significantly after exposure to noise greater than 70 dB(A). We suggest that plasma C-AMP could be useful as a sensitive index for noise-related stress in the daily living environment of humans and rats.

  11. Age-related changes of cyclic AMP phosphodiesterase activity in rat brain regions and a new phosphodiesterase inhibitor--nootropic agent adafenoxate.

    PubMed

    Stancheva, S L; Alova, L G

    1991-01-01

    1. The low- and high-KM cyclic AMP phosphodiesterase (cAMP PDE) activity in cerebral cortex, striatum, hypothalamus and hippocampus of young (4-5-month-old) and aged (22-month-old) rats has been studied. 2. A significant rise in the high-KM cAMP PDE activity in the cerebral cortex, hypothalamus and hippocampus in aged rats has been found. 3. The activity of the low-KM cAMP PDE does not change during senescence in all the brain structures studied. 4. In a series of increased concentrations (from 5 x 10(-4) to 1 x 10(-5) M) adafenoxate inhibits low- and high-KM cAMP PDE in most of the brain structures studied in both age groups. 5. The present results provide evidence for realization of the CNS effects of adafenoxate through inhibition of cAMP PDE activity and regulation of the intracellular level of cAMP. PMID:1662175

  12. Ethanol-induced loss of brain cyclic AMP binding proteins: correlation with growth suppression

    SciTech Connect

    Pennington, S.; Kalmus, G.

    1987-05-01

    Brain hypoplasia secondary to maternal ethanol consumption is a common fetal defect observed in all models of fetal alcohol syndrome. The molecular mechanism by which ethanol inhibits growth is unknown but has been hypothesized to involve ethanol-induced changes in the activity of cyclic-AMP stimulated protein kinase. Acute and chronic alcohol exposure elevate cyclic AMP level in many tissues, including brain. This increase in cyclic AMP should increase the phosphorylating activity of kinase by increasing the amount of dissociated (active) kinase catalytic subunit. In 7-day embryonic chick brains, ethanol-induced growth suppression was correlated with increased brain cyclic AMP content but neither basal nor cyclic AMP stimulated kinase catalytic activity was increased. However, the levels of cyclic AMP binding protein (kinase regulatory subunit) were significantly lowered by ethanol exposure. Measured as either /sup 3/H cyclic AMP binding or as 8-azido cyclic AM/sup 32/P labeling, ethanol-exposed brains had significantly less cyclic AMP binding activity (51 +/- 14 versus 29 +/- 10 units/..mu..g protein for 8-azido cyclic AMP binding). These findings suggest that ethanol's effect on kinase activity may involve more than ethanol-induced activation of adenylate cyclase.

  13. Transcription activation at Escherichia coli promoters dependent on the cyclic AMP receptor protein: effects of binding sequences for the RNA polymerase alpha-subunit.

    PubMed Central

    Savery, N J; Rhodius, V A; Wing, H J; Busby, S J

    1995-01-01

    Transcription activation at two semi-synthetic Escherichia coli promoters, CC(-41.5) and CC(-72.5), is dependent on the cyclic AMP receptor protein (CRP) that binds to sites centred 41.5 and 72.5 bp upstream from the respective transcription startpoints. An UP-element that can bind the C-terminal domain of the RNA polymerase (RNAP) alpha-subunit was cloned upstream of the DNA site for CRP at CC(-41.5) and downstream of the DNA site for CRP at CC(-72.5). In both cases CRP-dependent promoter activity was increased by the UP-element, but CRP-independent activity was not increased. DNase I footprinting was exploited to investigate the juxtaposition of bound CRP and RNAP alpha-subunits. In both cases, CRP and RNAP alpha-subunits occupy their cognate binding sites in ternary CRP-RNAP promoter complexes. RNAP alpha-subunits can occupy the UP-element in the absence of CRP, but this is not sufficient for open complex formation. The positive effects of binding RNAP alpha-subunits upstream of the DNA site for CRP at -41.5 are suppressed if the UP-element is incorrectly positioned. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7619086

  14. Activation of Endogenous Anti-Inflammatory Mediator Cyclic AMP Attenuates Acute Pyelonephritis in Mice Induced by Uropathogenic Escherichia coli

    PubMed Central

    Wei, Yang; Li, Ke; Wang, Na; Cai, Gui-Dong; Zhang, Ting; Lin, Yan; Gui, Bao-Song; Liu, En-Qi; Li, Zong-Fang; Zhou, Wuding

    2015-01-01

    The pathogenesis of pyelonephritis caused by uropathogenic Escherichia coli (UPEC) is not well understood. Here, we show that besides UPEC virulence, the severity of the host innate immune response and invasion of renal epithelial cells are important pathogenic factors. Activation of endogenous anti-inflammatory mediator cAMP significantly attenuated acute pyelonephritis in mice induced by UPEC. Administration of forskolin (a potent elevator of intracellular cAMP) reduced kidney infection (ie, bacterial load, tissue destruction); this was associated with attenuated local inflammation, as evidenced by the reduction of renal production of proinflammatory mediators, renal infiltration of inflammatory cells, and renal myeloperoxidase activity. In primary cell culture systems, forskolin not only down-regulated UPEC-stimulated production of proinflammatory mediators by renal tubular epithelial cells and inflammatory cells (eg, monocyte/macrophages) but also reduced bacterial internalization by renal tubular epithelial cells. Our findings clearly indicate that activation of endogenous anti-inflammatory mediator cAMP is beneficial for controlling UPEC-mediated acute pyelonephritis in mice. The beneficial effect can be explained at least in part by limiting excessive inflammatory responses through acting on both renal tubular epithelial cells and inflammatory cells and by inhibiting bacteria invasion of renal tubular epithelial cells. PMID:25478807

  15. Activation of endogenous anti-inflammatory mediator cyclic AMP attenuates acute pyelonephritis in mice induced by uropathogenic Escherichia coli.

    PubMed

    Wei, Yang; Li, Ke; Wang, Na; Cai, Gui-Dong; Zhang, Ting; Lin, Yan; Gui, Bao-Song; Liu, En-Qi; Li, Zong-Fang; Zhou, Wuding

    2015-02-01

    The pathogenesis of pyelonephritis caused by uropathogenic Escherichia coli (UPEC) is not well understood. Here, we show that besides UPEC virulence, the severity of the host innate immune response and invasion of renal epithelial cells are important pathogenic factors. Activation of endogenous anti-inflammatory mediator cAMP significantly attenuated acute pyelonephritis in mice induced by UPEC. Administration of forskolin (a potent elevator of intracellular cAMP) reduced kidney infection (ie, bacterial load, tissue destruction); this was associated with attenuated local inflammation, as evidenced by the reduction of renal production of proinflammatory mediators, renal infiltration of inflammatory cells, and renal myeloperoxidase activity. In primary cell culture systems, forskolin not only down-regulated UPEC-stimulated production of proinflammatory mediators by renal tubular epithelial cells and inflammatory cells (eg, monocyte/macrophages) but also reduced bacterial internalization by renal tubular epithelial cells. Our findings clearly indicate that activation of endogenous anti-inflammatory mediator cAMP is beneficial for controlling UPEC-mediated acute pyelonephritis in mice. The beneficial effect can be explained at least in part by limiting excessive inflammatory responses through acting on both renal tubular epithelial cells and inflammatory cells and by inhibiting bacteria invasion of renal tubular epithelial cells. PMID:25478807

  16. TSH-induced cyclic AMP production in an ovine thyroid cell line: OVNIS 5H.

    PubMed

    Fayet, G; Aouani, A; Hovsépian, S

    1986-01-01

    The TSH-induced cyclic AMP response was studied using a 3-year-old ovine thyroid cell line TSH-independent for growth: OVNIS 5H. The kinetics of cyclic AMP production was followed both in cell layers and in cell culture media, with or without phosphodiesterase inhibitor. It is noteworthy that following the first wave in cyclic AMP obtained within minutes, we observed later a sustained exponential increase in cyclic AMP during the 5 days following TSH stimulation. A bioassay of TSH was derived allowing measurement of 1 microU/ml TSH from a crude bTSH preparation. PMID:3000830

  17. CRP-Cyclic AMP Dependent Inhibition of the Xylene-Responsive σ54-Promoter Pu in Escherichia coli

    PubMed Central

    Tian, Zhe-Xian; Huo, Yi-Xin; Sun, Yi-Cheng; Wang, Yi-Ping

    2014-01-01

    The expression of σ54-dependent Pseudomonas putida Pu promoter is activated by XylR activator when cells are exposed to a variety of aromatic inducers. In this study, the transcriptional activation of the P. putida Pu promoter was recreated in the heterologous host Escherichia coli. Here we show that the cAMP receptor protein (CRP), a well-known carbon utilization regulator, had an inhibitory effect on the expression of Pu promoter in a cAMP-dependent manner. The inhibitory effect was not activator specific. In vivo KMnO4 and DMS footprinting analysis indicated that CRP-cAMP poised the RNA polymerase at Pu promoter, inhibiting the isomerization step of the transcription initiation even in the presence of an activator. Therefore, the presence of PTS-sugar, which eliminates cAMP, could activate the poised RNA polymerase at Pu promoter to transcribe. Moreover, the activation region 1 (AR1) of CRP, which interacts directly with the αCTD (C-terminal domain of α-subunit) of RNA polymerase, was found essential for the CRP-mediated inhibition at Pu promoter. A model for the above observations is discussed. PMID:24466213

  18. Phosphorylation of CREB, a cyclic AMP responsive element binding protein, contributes partially to lysophosphatidic acid-induced fibroblast cell proliferation

    SciTech Connect

    Kwon, Yong-Jun; Sun, Yuanjie; Kim, Nam-Ho; Huh, Sung-Oh

    2009-03-13

    Lysophospholipids regulate a wide array of biological processes including cell survival and proliferation. In our previous studies, we found that in addition to SRE, CRE is required for maximal c-fos promoter activation triggered by lysophosphatidic acid (LPA). c-fos is an early indicator of various cells into the cell cycle after mitogenic stimulation. However, role of CREB activation in LPA-stimulated proliferation has not been elucidated yet. Here, we investigate how LPA induces proliferation in Rat-2 fibroblast cell via CREB activation. We found that total cell number and BrdU-positive cells were increased by LPA. Moreover, levels of c-fos mRNA and cyclin D1 protein were increased via LPA-induced CREB phosphorylation. Furthermore, LPA-induced Rat-2 cell proliferation was decreased markedly by ERK inhibitor (U0126) and partially by MSK inhibitor (H89). Taken together, these results suggest that CREB activation could partially up-regulate accumulation of cyclin D1 protein level and proliferation of LPA-stimulated Rat-2 fibroblast cells.

  19. 4-Phenylbutyrate Attenuates the ER Stress Response and Cyclic AMP Accumulation in DYT1 Dystonia Cell Models

    PubMed Central

    Cho, Jin A.; Zhang, Xuan; Miller, Gregory M.; Lencer, Wayne I.; Nery, Flavia C.

    2014-01-01

    Dystonia is a neurological disorder in which sustained muscle contractions induce twisting and repetitive movements or abnormal posturing. DYT1 early-onset primary dystonia is the most common form of hereditary dystonia and is caused by deletion of a glutamic acid residue (302/303) near the carboxyl-terminus of encoded torsinA. TorsinA is localized primarily within the contiguous lumen of the endoplasmic reticulum (ER) and nuclear envelope (NE), and is hypothesized to function as a molecular chaperone and an important regulator of the ER stress-signaling pathway, but how the mutation in torsinA causes disease remains unclear. Multiple lines of evidence suggest that the clinical symptoms of dystonia result from abnormalities in dopamine (DA) signaling, and possibly involving its down-stream effector adenylate cyclase that produces the second messenger cyclic adenosine-3′, 5′-monophosphate (cAMP). Here we find that mutation in torsinA induces ER stress, and inhibits the cyclic adenosine-3′, 5′-monophosphate (cAMP) response to the adenylate cyclase agonist forskolin. Both defective mechanins are corrected by the small molecule 4-phenylbutyrate (4-PBA) that alleviates ER stress. Our results link torsinA, the ER-stress-response, and cAMP-dependent signaling, and suggest 4-PBA could also be used in dystonia treatment. Other pharmacological agents known to modulate the cAMP cascade, and ER stress may also be therapeutic in dystonia patients and can be tested in the models described here, thus supplementing current efforts centered on the dopamine pathway. PMID:25379658

  20. Activation of Cyclic AMP Synthesis by Full and Partial Beta-Adrenergic Receptor Agonists in Chicken Skeletal Muscle Cells

    NASA Technical Reports Server (NTRS)

    Young, R. B.; Bridge, K. Y.; Cureri, Peter A. (Technical Monitor)

    2002-01-01

    Several beta-adrenergic receptor (bAR) agonists are known to cause hypertrophy of skeletal muscle tissue. Accordingly, five bAR agonists encompassing a range in activity from strong to weak were evaluated for their ability to stimulate cAMP accumulation in embryonic chicken skeletal muscle cells in culture. Two strong agonists (epinephrine and isoproterenol), one moderate agonist (albuterol), and two weak agonists known to cause hypertrophy in animals (clenbuterol and cimaterol) were studied. Dose response curves were determined over six orders of magnitude in concentration for each agonist, and values were determined for their maximum stimulation of cAMP synthesis rate (Bmax) and the agonist concentration at which 50% stimulation of cAMP synthesis (EC50) occurred. Bmax values decreased in the following order: isoproterenol, epinephrine, albuterol, cimaterol, clenbuterol. Cimaterol and clenbuterol at their Bmax concentrations were approximately 15-fold weaker than isoproterenol in stimulating the rate of cAMP synthesis. When cimaterol and clenbuterol were added to culture media at concentrations known to cause significant muscle hypertrophy in animals, there was no detectable effect on stimulation of cAMP synthesis. Finally, these same levels of cimaterol and clenbuterol did not antagonize the stimulation of cAMP by either epinephrine or isoproterenol.

  1. Activation of Cyclic AMP Synthesis by Full and Partial Beta-Adrenergic Receptor Agonists in Chicken Skeletal Muscle Cells

    NASA Technical Reports Server (NTRS)

    Young, R. B.; Bridge, K. Y.

    2003-01-01

    Several beta-adrenergic receptor (bAR) agonists are known to cause hypertrophy of skeletal muscle tissue. Accordingly, five bAR agonists encompassing a range in activity from strong to weak were evaluated for their ability to stimulate CAMP accumulation in embryonic chicken skeletal muscle cells in culture. Two strong agonists (epinephrine and isoproterenol), one moderate agonist (albuterol), and two weak agonists known to cause hypertrophy in animals (clenbuterol and cimaterol) were studied. Dose response curves were determined over six orders of magnitude in concentration for each agonist, and values were determined for their maximum stimulation of CAMP synthesis rate (Bmax) and the agonist concentration at which 50% stimulation of CAMP synthesis (EC50) occurred. Bmax values decreased in the following order: isoproterenol, epinephrine, albuterol, cimaterol, clenbuterol. Cimaterol and clenbuterol at their Bmax concentrations were approximately 15-fold weaker than isoproterenol in stimulating the rate of CAMP synthesis. When cimaterol and clenbuterol were added to culture media at concentrations known to cause significant muscle hypertrophy in animals, there was no detectable effect on stimulation of CAMP synthesis. Finally, these same levels of cimaterol and clenbuterol did not antagonize the stimulation of CAMP by either epinephrine or isoproterenol.

  2. The dependence of Escherichia coli asparaginase II formation on cyclic AMP and cyclic AMP receptor protein.

    PubMed

    Russell, L; Yamazaki, H

    1978-05-01

    The amount of asparaginase II in an Escherichia coli wild-type strain (cya+, crp+) markedly increased upon a shift from aerobic to anaerobic growth. However, no such increase occurred in a mutant (cya) lacking cyclic AMP synthesis unless supplemented with exogenous cyclic AMP. Since a mutant (crp) deficient in cyclic AMP receptor protein also did not support the anaerobic formation of this enzyme, it is concluded that the formation of E. coli asparaginase II depends on both cyclic AMP and cyclic AMP receptor protein. PMID:207402

  3. Prostaglandin E2 Inhibits NLRP3 Inflammasome Activation through EP4 Receptor and Intracellular Cyclic AMP in Human Macrophages.

    PubMed

    Sokolowska, Milena; Chen, Li-Yuan; Liu, Yueqin; Martinez-Anton, Asuncion; Qi, Hai-Yan; Logun, Carolea; Alsaaty, Sara; Park, Yong Hwan; Kastner, Daniel L; Chae, Jae Jin; Shelhamer, James H

    2015-06-01

    PGE2 is a potent lipid mediator involved in maintaining homeostasis but also promotion of acute inflammation or immune suppression in chronic inflammation and cancer. Nucleotide-binding domain, leucine-rich repeat-containing protein (NLR)P3 inflammasome plays an important role in host defense. Uncontrolled activation of the NLRP3 inflammasome, owing to mutations in the NLRP3 gene, causes cryopyrin-associated periodic syndromes. In this study, we showed that NLRP3 inflammasome activation is inhibited by PGE2 in human primary monocyte-derived macrophages. This effect was mediated through PGE2 receptor subtype 4 (EP4) and an increase in intracellular cAMP, independently of protein kinase A or exchange protein directly activated by cAMP. A specific agonist of EP4 mimicked, whereas its antagonist or EP4 knockdown reversed, PGE2-mediated NLRP3 inhibition. PGE2 caused an increase in intracellular cAMP. Blockade of adenylate cyclase by its inhibitor reversed PGE2-mediated NLRP3 inhibition. Increase of intracellular cAMP by an activator of adenylate cyclase or an analog of cAMP, or a blockade of cAMP degradation by phosphodiesterase inhibitor decreased NLRP3 activation. Protein kinase A or exchange protein directly activated by cAMP agonists did not mimic, and their antagonists did not reverse, PGE2-mediated NLRP3 inhibition. Additionally, constitutive IL-1β secretion from LPS-primed PBMCs of cryopyrin-associated periodic fever syndromes patients was substantially reduced by high doses of PGE2. Moreover, blocking cytosolic phospholipase A2α by its inhibitor or small interfering RNA or inhibiting cyclooxygenase 2, resulting in inhibition of endogenous PGE2 production, caused an increase in NLRP3 inflammasome activation. Our results suggest that PGE2 might play a role in maintaining homeostasis during the resolution phase of inflammation and might serve as an autocrine and paracrine regulator. PMID:25917098

  4. Long-term memory of visually cued fear conditioning: roles of the neuronal nitric oxide synthase gene and cyclic AMP response element-binding protein.

    PubMed

    Kelley, J B; Anderson, K L; Altmann, S L; Itzhak, Y

    2011-02-01

    Nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) has a role in late-phase long-term potentiation (LTP) and long-term memory (LTM) formation. Our recent studies implicated NO signaling in contextual and auditory cued fear conditioning. The present study investigated the role of NO signaling in visually cued fear conditioning. First, visually cued fear conditioning was investigated in wild-type (WT) and nNOS knockout (KO) mice. Second, the effects of pharmacological modulators of NO signaling on the acquisition of visually cued fear conditioning were investigated. Third, plasma levels of corticosterone were measured to determine a relationship between physiological and behavioral responses to fear conditioning. Fourth, levels of extracellular signal-related kinase (ERK1/2) and cyclic AMP response element binding protein (CREB) phosphorylation, downstream of NO signaling, were determined in the amygdala as potential correlates of fear learning. Mice underwent single or multiple (4) spaced trainings that consisted of a visual cue (blinking light) paired with footshock. WT mice acquired cued and contextual LTM following single and multiple trainings. nNOS KO mice acquired neither cued nor contextual LTM following a single training; however, multiple trainings improved contextual but not cued LTM. The selective nNOS inhibitor S-methyl-thiocitrulline (SMTC) impaired cued and contextual LTM in WT mice. The NO donor molsidomine recovered contextual LTM but had no effect on cued LTM in nNOS KO mice. Re-exposure to the visual cue 24 h posttraining elicited freezing response and a marked increase in plasma corticosterone levels in WT but not nNOS KO mice. The expression of CREB phosphorylation (Ser-133) was significantly higher in naive nNOS KO mice than in WT counterparts, and pharmacological modulators of NO had significant effects on levels of CREB phosphorylation and expression. These findings suggest that visual cue-dependent LTM is impaired in nNOS KO

  5. The cyclic AMP response element-binding protein antisense oligonucleotide induced anti-nociception and decreased the expression of KIF17 in spinal cord after peripheral nerve injury in mice

    PubMed Central

    Bo, Jinhua; Zhang, Wei; Sun, Xiaofeng; Yang, Yan; Liu, Xiaojie; Jiang, Ming; Ma, Zhengliang; Gu, Xiaoping

    2014-01-01

    Backgrounds: The cyclic AMP response element-binding protein (CREB) plays an important role in neuropathic pain. Kinesin superfamily motor protein 17 (KIF17) is involved in long-term memory formation. CREB could increase the level of KIF17 when activated by synaptic input. This study is to investigate the role and mechanism of CREB antisense oligonucleotide (ODN) in neuropathic pain induced by chronic constriction injury (CCI) in mice. Results: CCI surgery decreased thresholds of mechanical allodynia and thermal hyperalgesia whereas CREB antisense oligonucleotide ODN significantly attenuated these pain behaviors (P < 0.05). CCI significantly induced the protein expression of phosphorylated CREB (pCREB) and KIF17, but not KIF5B, in the spinal cord of CCI mice (P < 0.05). Additionally, the mRNA expression of CREB and KIF17 was significantly increased by CCI (P < 0.05). However, CREB antisense ODN significantly decreased the protein expression of pCREB and KIF17 (but not KIF5B), and the mRNA expression of CREB and KIF17 (P < 0.05). Conclusions: CREB antisense oligonucleotide ODN may reduce neuropathic pain through targeting CREB and decreasing the expression of pCREB and KIF17. PMID:25664020

  6. The possible role of cyclic AMP in the neurotrophic control of skeletal muscle.

    PubMed Central

    Carlsen, R C

    1975-01-01

    1. Motoneurones provide trophic control of some of the functional characteristics of skeletal muscle fibres. This study has been designed to test whether the adenylate cyclase: cyclic AMP system may offer one potential mechanism for the mediation of neurotrophic regulation. 2. The concentration of cyclic AMP was measured at various intervals after muscle denervation. Muscle cyclic AMP concentration increases for the first 2 days after nerve section. It reaches a maximum value at 48 h and subsequently returns to the control value at 7 days. 3. Cyclic AMP concentration is unchanged by muscle disuse for the first 3 days following limb immobilization. Four days after immobilization, however, cyclic AMP increases in both the disused and contralateral control muscles. This phenomenon has been tentatively ascribed to some aspect of the inflammatory response. 4. Changing the level of nerve section, and therefore the length of the residual nerve stump, changes the temporal pattern of the increase in muscle cyclic AMP concentration. 5. Reinnervation of a denervated muscle produces a decrease in muscle cyclic AMP concentration. 6. It is concluded from the results that some aspect of nerve function provides trophic regulation of the muscle adenylate cyclase: cyclic AMP system. The mechanisms by which this regulation may be applied are considered in the Discussion. PMID:168354

  7. Activation of cyclic AMP-dependent protein kinase inhibits the desensitization and internalization of metabotropic glutamate receptors 1a and 1b.

    PubMed

    Mundell, Stuart J; Pula, Giordano; More, Julia C A; Jane, David E; Roberts, Peter J; Kelly, Eamonn

    2004-06-01

    In this study, we characterized the effects of activation of cyclic AMP-dependent protein kinase (PKA) on the internalization and functional coupling of the metabotropic glutamate receptor (mGluR1) splice variants mGluR1a and mGluR1b. Using an enzyme-linked immunosorbent assay technique to assess receptor internalization, we found that the glutamate-induced internalization of mGluR1a or mGluR1b transiently expressed in human embryonic kidney (HEK) 293 cells was inhibited by coactivation of endogenous beta2-adrenoceptors with isoprenaline or by direct activation of adenylyl cyclase with forskolin. The PKA inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked the effects of both isoprenaline and forskolin. The heterologous internalization of the mGluR1 splice variants triggered by carbachol was also inhibited by isoprenaline and forskolin in a PKA-sensitive fashion, whereas the constitutive (agonist-independent) internalization of mGluR1a was inhibited only modestly by PKA activation. Using inositol phosphate (IP) accumulation in cells prelabeled with [3H]inositol to assess receptor coupling, PKA activation increased basal IP accumulation in mGluR1a receptor-expressing cells and also increased glutamate-stimulated IP accumulation in both mGluR1a- and mGluR1b-expressing cells, but only at short times of glutamate addition. Furthermore, PKA activation completely blocked the carbachol-induced heterologous desensitization of glutamate-stimulated IP accumulation in both mGluR1a- and mGluR1b-expressing cells. In coimmunoprecipitation experiments, the ability of glutamate to increase association of GRK2 and arrestin-2 with mGluR1a and mGluR1b was inhibited by PKA activation with forskolin. Together, these results indicate that PKA activation inhibits the agonist-induced internalization and desensitization of mGluR1a and mGluR1b, probably by reducing their interaction with GRK2 and nonvisual arrestins. PMID:15155843

  8. Functional cyclic AMP response element in the breast cancer resistance protein (BCRP/ABCG2) promoter modulates epidermal growth factor receptor pathway- or androgen withdrawal-mediated BCRP/ABCG2 transcription in human cancer cells.

    PubMed

    Xie, Yi; Nakanishi, Takeo; Natarajan, Karthika; Safren, Lowell; Hamburger, Anne W; Hussain, Arif; Ross, Douglas D

    2015-03-01

    Phosphorylated cyclic-AMP (cAMP) response element binding protein (p-CREB) is a downstream effector of a variety of important signaling pathways. We investigated whether the human BCRP promoter contains a functional cAMP response element (CRE). 8Br-cAMP, a cAMP analogue, increased the activity of a BCRP promoter reporter construct and BCRP mRNA in human carcinoma cells. Epidermal growth factor receptor (EGFR) pathway activation also led to an increase in p-CREB and in BCRP promoter reporter activity via two major downstream EGFR signaling pathways: the phosphotidylinositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase (MAPK) pathway. EGF treatment increased the phosphorylation of EGFR, AKT, ERK and CREB, while simultaneously enhancing BCRP mRNA and functional protein expression. EGF-stimulated CREB phosphorylation and BCRP induction were diminished by inhibition of EGFR, PI3K/AKT or RAS/MAPK signaling. CREB silencing using RNA interference reduced basal levels of BCRP mRNA and diminished the induction of BCRP by EGF. Chromatin immunoprecipitation assays confirmed that a putative CRE site on the BCRP promoter bound p-CREB by a point mutation of the CRE site abolished EGF-induced stimulation of BCRP promoter reporter activity. Furthermore, the CREB co-activator, cAMP-regulated transcriptional co-activator (CRTC2), is involved in CREB-mediated BCRP transcription: androgen depletion of LNCaP human prostate cancer cells increased both CREB phosphorylation and CRTC2 nuclear translocation, and enhanced BCRP expression. Silencing CREB or CRTC2 reduced basal BCRP expression and BCRP induction under androgen-depletion conditions. This novel CRE site plays a central role in mediating BCRP gene expression in several human cancer cell lines following activation of multiple cancer-relevant signaling pathways. PMID:25615818

  9. Control of calcium transport in the myocardium by the cyclic AMP-Protein kinase system.

    PubMed

    Katz, A M; Tada, M; Kirchberger, M A

    1975-01-01

    At least three mechanical changes characterize the response of cardiac muscle to agents that enhance cyclic AMP production. In common with other inotropic interventions, tension is augmented and the rate of tension rise is increased. The third response, acceleration of the rate of relaxation, is characteristic of the actions of beta-adrenergic agonists. These mechanical effects can be attributed to changes in (1) the amount of Ca2+ released during systole, (2) the rate of Ca2+ release at the onset of systole, and (3) the rate at which Ca2+ is reaccumulated by the sarcoplasmic reticulum at the end of systole. The ability of cyclic AMP-dependent protein kinases to phosphorylate the cardiac sarcoplasmic reticulum in vitro parallels stimulation of both Ca2+ transport and Ca2+-activated ATPase. The phosphoprotein formed in the presence of cyclic AMP and protein kinase has the chemical characteristics of a phosphoester, contains mostly phosphoserine, and has an electrophoretic mobility in SDS polyacrylamide gels that corresponds to a protein of 22,000 daltons. This 22,000-dalton protein, tentatively named phospholamban, thus differs from the acyl phosphooprotein formed by the Ca2+-transport ATPase, which as an apparent molecular weight of 90,000 to 100,000 daltons. Phospholamban has not been found in fast skeletal muscle, nor is Ca2+ transport accelerated by cyclic AMP and protein kinase in sarcoplasmic reticulum from these muslces which do not respond to beta-adrenergic agonists with accelerated relaxation. It thus appears likely that phosphorylation of phospholamban correlates both with an increased rate of Ca2+ transport by cardiac sarcoplasmic reticulum in vitro and accelerated relaxation in the intact myocardium. Preliminary findings are consistent with the view that phosphorylation of phospholamban may be related to other actions on Ca2+ fluxes brought about by agents which activate adenylate cyclase in the myocardium, but these interpretations must remain

  10. Copper regulates cyclic-AMP-dependent lipolysis.

    PubMed

    Krishnamoorthy, Lakshmi; Cotruvo, Joseph A; Chan, Jefferson; Kaluarachchi, Harini; Muchenditsi, Abigael; Pendyala, Venkata S; Jia, Shang; Aron, Allegra T; Ackerman, Cheri M; Wal, Mark N Vander; Guan, Timothy; Smaga, Lukas P; Farhi, Samouil L; New, Elizabeth J; Lutsenko, Svetlana; Chang, Christopher J

    2016-08-01

    Cell signaling relies extensively on dynamic pools of redox-inactive metal ions such as sodium, potassium, calcium and zinc, but their redox-active transition metal counterparts such as copper and iron have been studied primarily as static enzyme cofactors. Here we report that copper is an endogenous regulator of lipolysis, the breakdown of fat, which is an essential process in maintaining body weight and energy stores. Using a mouse model of genetic copper misregulation, in combination with pharmacological alterations in copper status and imaging studies in a 3T3-L1 white adipocyte model, we found that copper regulates lipolysis at the level of the second messenger, cyclic AMP (cAMP), by altering the activity of the cAMP-degrading phosphodiesterase PDE3B. Biochemical studies of the copper-PDE3B interaction establish copper-dependent inhibition of enzyme activity and identify a key conserved cysteine residue in a PDE3-specific loop that is essential for the observed copper-dependent lipolytic phenotype. PMID:27272565

  11. Cyclic AMP induces maturation of trout sperm axoneme to initiate motility

    NASA Astrophysics Data System (ADS)

    Morisawa, Masaaki

    1982-02-01

    Cyclic AMP has long been implicated as an activator of sperm motility1-5. From more recent experiments using demembranated mammalian and sea urchin spermatozoa6,7, it was concluded that cyclic AMP only increases the motility of the axoneme after it has been initiated by MgATP2-. We have now carried out similar experiments using spermatozoa collected from the rainbow trout and demembranated by treatment with the detergent Triton X-100. Our results suggest that in this species, cyclic AMP is required before MgATP2- to trigger maturation of the nonmotile axoneme. Subsequent addition of an energy source then induces motility.

  12. Rapid regulation of PDE-2 and PDE-4 cyclic AMP phosphodiesterase activity following ligation of the T cell antigen receptor on thymocytes: analysis using the selective inhibitors erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) and rolipram.

    PubMed

    Michie, A M; Lobban, M; Müller, T; Harnett, M M; Houslay, M D

    1996-02-01

    The PDE2, cyclic GMP-stimulated, and the PDE4, cyclic AMP-specific enzymes provide the major, detectable cyclic AMP phosphodiesterase activities in murine thymocytes. In the absence of the cyclic GMP, PDE4 activity predominated (approximately 80% total) but in the presence of low (10 microM) cyclic GMP concentrations, PDE2 activity constituted the major PDE activity in thymocytes (approximately 80% total). The PDE4 selective inhibitor rolipram dose-dependently inhibited thymocyte PDE4 activity (IC50 approximately 65 nM). PDE2 was dose-dependently activated (EC50 approximately 1 microM) by cyclic GMP and inhibited by erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) (IC50 approximately 4 microM). EHNA was shown to serve as a selective inhibitor of PDE-2 activity as assessed from studies using separated PDE1, PDE2, PDE3 and PDE4 species from hepatocytes as well as human PDE2 and PDE4 enzymes. EHNA completely ablated the ability of cyclic GMP to activate PDE2 activity, whilst having a much smaller inhibitory effect on the unstimulated PDE2 activity. EHNA exhibited normal Michaelian kinetics of inhibition for the cyclic GMP-stimulated PDE2 activity with Hill plots near unity. Apparent negative co-operative effect were seen in the absence of cyclic GMP with Hill coefficients of approximately 0.3 for inhibition of PDE2 activity. Within 5 min of challenge of thymocytes with the lectin phytohaemagglutinin (PHA) there was a transient decrease (approximately 83%) in PDE-4 activity and in PDE2 activity (approximately 40%). Both anti-TCR antibodies also caused an initial reduction in the PDE4 activity which was followed by a sustained and profound increase in activity. In contrast to that observed with PHA, anti-TCR/CD3 antisera had little effect on PDE2 activity. It is suggested that, dependent upon the intracellular concentrations of cyclic GMP, thymocyte cyclic AMP metabolism can be expected to switch from being under the predominant control of PDE4 activity to that determined

  13. Is a decrease in cyclic AMP a necessary and sufficient signal for maturation of amphibian oocytes

    SciTech Connect

    Gelerstein, S.; Shapira, H.; Dascal, N.; Yekuel, R.; Oron, Y.

    1988-05-01

    Acetylcholine rapidly lowered the intracellular levels of cyclic AMP in stage 5 and 6 Xenopus laevis oocytes. Acetylcholine alone did not induce oocyte maturation, though it did accelerate maturation induced by progesterone. The effect of acetylcholine on oocyte maturation was independent of extracellular calcium concentration. Adenosine increased cyclic AMP and abolished the progesterone-induced decrease in cyclic AMP levels in follicles and in denuded oocytes. This effect of adenosine was blocked by the Ra purinergic receptor antagonist, theophylline. Despite those effects, adenosine alone induced maturation in stage 6 oocytes and accelerated progesterone-induced maturation in both stage 5 and 6 cells. Adenosine also induced a significant increase in the rate of /sup 45/Ca efflux from oocytes in the presence and the absence of external calcium. We suggest that the activation of cell surface receptors involved in the release of calcium from cellular stores may induce or accelerate oocyte maturation independently of small changes in intracellular cyclic AMP concentration.

  14. Dual bradykinin B2 receptor signalling in A431 human epidermoid carcinoma cells: activation of protein kinase C is counteracted by a GS-mediated stimulation of the cyclic AMP pathway.

    PubMed Central

    Liebmann, C; Graness, A; Ludwig, B; Adomeit, A; Boehmer, A; Boehmer, F D; Nürnberg, B; Wetzker, R

    1996-01-01

    Cell membranes of the human epidermoid cell line A431 express classical bradykinin (BK) B2 receptors, as assessed by [3H]BK binding studies. Furthermore, stimulation by BK induced a time-dependent modulation of protein kinase C (PKC) activity in A431 cells: a rapid activation (t1/2 approximately 1 min) is followed by a slow inhibition (t1/2 approximately 20 min) of PKC translocation measured by [3H]phorbol 12,13-dibutyrate binding. In addition, BK stimulated both adenylate cyclase activity in A431 membranes and accumulation of intracellular cyclic AMP (cAMP) in intact cells in a retarded manner. A possible BK-induced activation of the cAMP pathway mediated via PKC, phospholipase D, prostaglandins or Ca2+/calmodulin was excluded. A 35 kDa protein was found in A431 membranes to be specifically phosphorylated in the presence of both BK and protein kinase A (PKA). An anti-alpha s-antibody, AS 348, abolished stimulation of adenylate cyclase activity in response to BK, cholera toxin and isoprenaline, strongly suggesting the involvement of Gs proteins in the BK action. The BK-activated cAMP signalling system might be important for the observed inactivation of PKC slowly evoked by BK: the BK-induced rapid activation of PKC is decreased by dibutyryl cAMP, and the slow inhibition of PKC is prevented by an inhibitor of PKA, adenosine 3':5'-monophosphothioate (cyclic, Rp isomer). The inhibition of PKC translocation might be exerted directly at the level of PKC activation, since stimulation of phosphoinositide hydrolysis by BK was affected by neither dibutyryl cAMP nor forskolin. Thus our results provide the first evidence that A431 cells BK is able to activate two independent signal-transduction pathways via a single class of B2 receptors but two different G proteins. The lagging stimulation of the cAMP signalling pathway via Gs might serve to switch off PKC, which is rapidly activated via Gq-mediated stimulation of phosphoinositide hydrolysis. PMID:8546671

  15. Expression of the glycoprotein hormone alpha-subunit gene in the placenta requires a functional cyclic AMP response element, whereas a different cis-acting element mediates pituitary-specific expression.

    PubMed Central

    Bokar, J A; Keri, R A; Farmerie, T A; Fenstermaker, R A; Andersen, B; Hamernik, D L; Yun, J; Wagner, T; Nilson, J H

    1989-01-01

    The single-copy gene encoding the alpha subunit of glycoprotein hormones is expressed in the pituitaries of all mammals and in the placentas of only primates and horses. We have systematically analyzed the promoter-regulatory elements of the human and bovine alpha-subunit genes to elucidate the molecular mechanisms underlying their divergent patterns of tissue-specific expression. This analysis entailed the use of transient expression assays in a chorionic gonadotropin-secreting human choriocarcinoma cell line, protein-DNA binding assays, and expression of chimeric forms of human or bovine alpha subunit genes in transgenic mice. From the results, we conclude that placental expression of the human alpha-subunit gene requires a functional cyclic AMP response element (CRE) that is present as a tandem repeat in the promoter-regulatory region. In contrast, the promoter-regulatory region of the bovine alpha-subunit gene, as well as of the rat and mouse genes, was found to contain a single CRE homolog that differed from its human counterpart by a single nucleotide. This difference substantially reduced the binding affinity of the bovine CRE homolog for the nuclear protein that bound to the human alpha CRE and thereby rendered the bovine alpha-subunit promoter inactive in human choriocarcinoma cells. However, conversion of the bovine alpha CRE homolog to an authentic alpha CRE restored activity to the bovine alpha-subunit promoter in choriocarcinoma cells. Similarly, a human but not a bovine alpha transgene was expressed in placenta in transgenic mice. Thus, placenta-specific expression of the human alpha-subunit gene may be the consequence of the recent evolution of a functional CRE. Expression of the human alpha transgene in mouse placenta further suggests that evolution of placenta-specific trans-acting factors preceded the appearance of this element. Finally, in contrast to their divergent patterns of placental expression, both the human and bovine alpha

  16. Deficient guanine nucleotide regulatory unit activity in cultured fibroblast membranes from patients with pseudohypoparathyroidism type I. A cause of impaired synthesis of 3',5'-cyclic AMP by intact and broken cells

    PubMed Central

    Levine, Michael A.; Eil, Charles; Downs, Robert W.; Spiegel, Allen M.

    1983-01-01

    Deficient activity of the guanine nucleotide regulatory protein (G unit), an integral component of the membrane-bound adenylate cyclase complex, has been implicated as the biochemical lesion in many patients with pseudohypoparathyroidism (PHP) type I. In addition to renal resistance to parathyroid hormone in this disorder, there is decreased responsiveness of diverse tissues to hormones that act via 3',5'-cyclic AMP (cAMP). To assess whether a deficiency of G units could account for impaired adenylate cyclase activity, we studied cAMP production in intact cultured fibroblasts and fibroblast plasma membranes from five patients with PHP in response to several activators of adenylate cyclase. The number of G units in PHP fibroblast membranes, measured by cholera toxin-dependent [32P]ADP ribosylation of G-unit peptides, as well as the G-unit activity, determined by the ability of detergent extracts to reconstitute adenylate cyclase activity in G-unit-deficient S49 CYC- membranes, were found to be markedly reduced compared with control membranes (43 and 40%, respectively), The activation of fibroblast membrane adenylate cyclase by effectors that act directly through the G unit (guanosine triphosphate, guanosine 5'-0-[3-thiotriphosphate] [GTP-γ-S], NaF) was significantly greater in control membranes than in membranes from patients with PHP. Moreover, we found that hormone (prostaglandin E1) stimulated adenylate cyclase activity was also greater in control membranes than in PHP membranes. Neither the apparent affinity of membrane adenylate cyclase for GTP-γ-S (apparent Km =5 X 10-8 M) nor the rate of enzyme activation by GTP-γ-S was significantly different in fibroblast membranes from control subjects and patients with PHP. In contrast to the notable differences in hormone and G-unit-activated adenylate cyclase shown in fibroblast membranes from PHP patients and control subjects, the intrinsic catalytic activity of membranes, as determined by forskolin

  17. Looking downstream: the role of cyclic AMP-regulated genes in axonal regeneration

    PubMed Central

    Siddiq, Mustafa M.; Hannila, Sari S.

    2015-01-01

    Elevation of intracellular cyclic AMP (cAMP) levels has proven to be one of the most effective means of overcoming inhibition of axonal regeneration by myelin-associated inhibitors such as myelin-associated glycoprotein (MAG), Nogo, and oligodendrocyte myelin glycoprotein. Pharmacological manipulation of cAMP through the administration of dibutyryl cAMP or rolipram leads to enhanced axonal growth both in vivo and in vitro, and importantly, upregulation of cAMP within dorsal root ganglion neurons is responsible for the conditioning lesion effect, which indicates that cAMP plays a significant role in the endogenous mechanisms that promote axonal regeneration. The effects of cAMP are transcription-dependent and are mediated through the activation of protein kinase A (PKA) and the transcription factor cyclic AMP response element binding protein (CREB). This leads to the induction of a variety of genes, several of which have been shown to overcome myelin-mediated inhibition in their own right. In this review, we will highlight the pro-regenerative effects of arginase I (ArgI), interleukin (IL)-6, secretory leukocyte protease inhibitor (SLPI), and metallothionein (MT)-I/II, and discuss their potential for therapeutic use in spinal cord injury. PMID:26150769

  18. Looking downstream: the role of cyclic AMP-regulated genes in axonal regeneration.

    PubMed

    Siddiq, Mustafa M; Hannila, Sari S

    2015-01-01

    Elevation of intracellular cyclic AMP (cAMP) levels has proven to be one of the most effective means of overcoming inhibition of axonal regeneration by myelin-associated inhibitors such as myelin-associated glycoprotein (MAG), Nogo, and oligodendrocyte myelin glycoprotein. Pharmacological manipulation of cAMP through the administration of dibutyryl cAMP or rolipram leads to enhanced axonal growth both in vivo and in vitro, and importantly, upregulation of cAMP within dorsal root ganglion neurons is responsible for the conditioning lesion effect, which indicates that cAMP plays a significant role in the endogenous mechanisms that promote axonal regeneration. The effects of cAMP are transcription-dependent and are mediated through the activation of protein kinase A (PKA) and the transcription factor cyclic AMP response element binding protein (CREB). This leads to the induction of a variety of genes, several of which have been shown to overcome myelin-mediated inhibition in their own right. In this review, we will highlight the pro-regenerative effects of arginase I (ArgI), interleukin (IL)-6, secretory leukocyte protease inhibitor (SLPI), and metallothionein (MT)-I/II, and discuss their potential for therapeutic use in spinal cord injury. PMID:26150769

  19. [The effect of some pharmacological agents and electroshock on the level of cyclic AMP of the total mouse brain].

    PubMed

    Joanny, P; Devolx, B C; Garron, J; Giannellini, F

    1976-01-01

    Amphetamin, pentobarbital, pargyline, parachlorophenylalanine, pentetrasol and maximal electroshock all increased significantly cyclic AMP in mice whole brain conversely reserpine induced a decrease of cyclic nucleotide. All those changes were tentatively correlated toward central monoaminergic systems activation. PMID:192423

  20. Minocycline upregulates cyclic AMP response element binding protein and brain-derived neurotrophic factor in the hippocampus of cerebral ischemia rats and improves behavioral deficits

    PubMed Central

    Zhao, Yu; Xiao, Ming; He, Wenbo; Cai, Zhiyou

    2015-01-01

    Background and purpose The cAMP response element binding protein (CREB) plays an important role in the mechanism of cognitive impairment and is also pivotal in the switch from short-term to long-term memory. Brain-derived neurotrophic factor (BDNF) seems a promising avenue in the treatment of cerebral ischemia injury since this neurotrophin could stimulate structural plasticity and repair cognitive impairment. Several findings have displayed that the dysregulation of the CREB–BDNF cascade has been involved in cognitive impairment. The aim of this study was to investigate the effect of cerebral ischemia on learning and memory as well as on the levels of CREB, phosphorylated CREB (pCREB), and BDNF, and to determine the effect of minocycline on CREB, pCREB, BDNF, and behavioral functional recovery after cerebral ischemia. Methods The animal model was established by permanent bilateral occlusion of both common carotid arteries. Behavior was evaluated 5 days before decapitation with Morris water maze and open-field task. Four days after permanent bilateral occlusion of both common carotid arteries, minocycline was administered by douche via the stomach for 4 weeks. CREB and pCREB were examined by Western blotting, reverse transcription polymerase chain reaction, and immunohistochemistry. BDNF was measured by immunohistochemistry and Western blotting. Results The model rats after minocycline treatment swam shorter distances than control rats before finding the platform (P=0.0007). The number of times the platform position was crossed for sham-operation rats was more than that of the model groups in the corresponding platform location (P=0.0021). The number of times the platform position was crossed for minocycline treatment animals was significantly increased compared to the model groups in the corresponding platform position (P=0.0016). CREB, pCREB, and BDNF were downregulated after permanent bilateral occlusion of both common carotid arteries in the model group

  1. 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

    NASA Technical Reports Server (NTRS)

    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

  2. Cyclic AMP system in muscle tissue during prolonged hypokinesia

    NASA Technical Reports Server (NTRS)

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

    1980-01-01

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

  3. Pro-inflammatory cytokine regulation of cyclic AMP-phosphodiesterase 4 signaling in microglia in vitro and following CNS injury

    PubMed Central

    Ghosh, Mousumi; Garcia-Castillo, Daniela; Aguirre, Vladimir; Golshani, Roozbeh; Atkins, Coleen M.; Bramlett, Helen M.; Dietrich, W. Dalton; Pearse, Damien D.

    2015-01-01

    Cyclic AMP suppresses immune cell activation and inflammation. The positive feedback loop of pro-inflammatory cytokine production and immune activation implies that cytokines may not only be regulated by cyclic AMP but conversely regulate cyclic AMP. This study examined the effects of TNF-α and IL-1β on cyclic AMP-phosphodiesterase (PDE) signaling in microglia in vitro and after spinal cord or traumatic brain injury (SCI, TBI). TNF-α or IL-1β stimulation produced a profound reduction (>90%) of cyclic AMP within EOC2 microglia from 30min that then recovered after IL-1β but remained suppressed with TNF-α through 24h. Cyclic AMP was also reduced in TNF-α-stimulated primary microglia, albeit to a lesser extent. Accompanying TNF-α-induced cyclic AMP reductions, but not IL-1β, was increased cyclic AMP-PDE activity. The role of PDE4 activity in cyclic AMP reductions was confirmed by using Rolipram. Examination of pde4 mRNA revealed an immediate, persistent increase in pde4b with TNF-α; IL-1β increased all pde4 mRNAs. Immunoblotting for PDE4 showed that both cytokines increased PDE4A1, but only TNF-α increased PDE4B2. Immunocytochemistry revealed PDE4B nuclear translocation with TNF-α but not IL-1β. Acutely after SCI/TBI, where cyclic AMP levels are reduced, PDE4B was localized to activated OX-42+ microglia; PDE4B was absent in OX-42+ cells in uninjured spinal cord/cortex or inactive microglia. Immunoblotting showed PDE4B2 up-regulation from 24h to 1wk post-SCI, the peak of microglia activation. These studies show that TNF-α and IL-1β differentially affect cyclic AMP-PDE signaling in microglia. Targeting PDE4B2 may be a putative therapeutic direction for reducing microglia activation in CNS injury and neurodegenerative diseases. PMID:22865690

  4. Cyclic AMP infusion and blood sugar, serum insulin and serum nonesterified fatty acid responses to glucose in recent experimental hyperthyroid dogs.

    PubMed

    Renauld, A; Garrido, D

    1992-01-01

    Recent experimental hyperthyroid (REH) dogs exhibit poor "in vivo" insulin responses to glucose probably due to a failure somewhere in cAMP-adenylate cyclase system. The actions of exogenous cAMP on these responses and on the regulation of blood sugar (BS) and serum nonesterified fatty acids (NEFA) during glucose infusion tests (GIT) in REH and normal dogs were studied here. Hyperthyroidism was induced by 1-thyroxine administration (100 micrograms/kg body wt./die, 10 days). GIT consisted of i.v. glucose-priming followed by glucose i.v. continuous infusion (60 min). cAMP (0, 33 or 66 mg/kg body wt./min) was infused alone (30 min) and then overlapped to gluco-se infusion (60 min). Peripheral veins were used for infusions and blood sample withdrawal. BS, serum inmunoreactive insulin (IRI) and serum NEFA concentrations, basally and throughout the test, were measured. Basally, there was neither action nor interaction of hyperthyroidism and exogenous cAMP on these variables. During the GIT, the BS levels remained unaffected by hyperthyroidism; cAMP increased them, but failed to interact with hyperthyroidism. cAMP noninfused normal dogs responded to hyperglycemia with hyperinsulinemia, whereas REH dogs noninfused the nucleotide did not. cAMP administration at a high dose promoted their response in normal and REH dogs, particularly in the former; in the latter, the response was still lower than in cAMP noninfused normal controls. Although recent hyperthyroidism increased serum NEFA basal level, it exerted neither action nor interaction with the infused cAMP on serum NEFA during GIT. Results are discussed on the basis that the abolished insulin secretion "in vivo" characterizing the REH dogs, related to beta-adrenergic deficiency, can be for the most part restored by exogenous cAMP administration, despite which some glucose and triglyceride metabolism impairments are developed. PMID:1343982

  5. Sweet Taste Receptor Expressed in Pancreatic β-Cells Activates the Calcium and Cyclic AMP Signaling Systems and Stimulates Insulin Secretion

    PubMed Central

    Nakagawa, Yuko; Nagasawa, Masahiro; Yamada, Satoko; Hara, Akemi; Mogami, Hideo; Nikolaev, Viacheslav O.; Lohse, Martin J.; Shigemura, Noriatsu; Ninomiya, Yuzo; Kojima, Itaru

    2009-01-01

    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 Ca2+ ([Ca2+]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 [Ca2+]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 [Ca2+]c response. The effect of sucralose on [Ca2+]c was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a Gq 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 β-cells, and activation of this receptor induces insulin secretion by Ca2+ and cAMP-dependent mechanisms. PMID:19352508

  6. Antidepressant- and anxiolytic-like effects of the phosphodiesterase-4 (PDE4) inhibitor rolipram on behavior depend on cyclic AMP-response element binding protein (CREB)-mediated neurogenesis in the hippocampus

    PubMed Central

    Li, Yun-Feng; Huang, Ying; Amsdell, Simon L.; Xiao, Lan; O'Donnell, James M.; Zhang, Han-Ting

    2009-01-01

    Inhibition of phosphodiesterase-4 (PDE4), an enzyme that catalyzes the hydrolysis of cyclic AMP (cAMP), increases phosphorylation of cAMP-response element binding protein (pCREB) and hippocampal neurogenesis, and produces antidepressant-like effects on behavior; however, causal links among these have not been established. In the present study, chronic administration of rolipram produced antidepressant- and anxiolytic-like effects on behavior in mice. It also increased cAMP and pCREB levels in the hippocampus and prefrontal cortex, but increased Sox2, a marker for mitotic progenitor cells, only in the hippocampus. Chronic rolipram treatment also increased hippocampal neurogenesis, as evidenced by increased bromodeoxyuridine (BrdU)-positive cells in the hippocampal dentate gyrus. Methylazoxymethanol (MAM), which is toxic to proliferating cells, reversed rolipram-induced increases in BrdU-positive cells and pCREB in the hippocampus and partially blocked its behavioral effects. Approximately 84% of BrdU-positive cells became newborn neurons, 93% of which co-expressed pCREB; these proportions were not altered by rolipram or MAM, either alone or in combination. Finally, three weeks following the end of MAM treatment, when neurogenesis was no longer inhibited, rolipram again increased hippocampal pCREB, with its antidepressant- and anxiolytic-like effects resumed. Overall, the present results suggest that rolipram produces its effects on behavior in a manner that at least partially depends on its neurogenic action in the hippocampus, targeting mitotic progenitor cells rather than newborn or mature neurons; cAMP/CREB signaling in hippocampal newborn neurons is critical for neurogenesis and contributes to the behavioral effects of rolipram. PMID:19516250

  7. Mlc is a transcriptional activator with a key role in integrating cyclic AMP receptor protein and integration host factor regulation of leukotoxin RNA synthesis in Aggregatibacter actinomycetemcomitans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aggregatibacter actinomycetemcomitans, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the ltxA operon is induced during anaerobic growth. The cAMP receptor protein (CRP) indirectly increases ltxA expression...

  8. Glucose Evokes Rapid Ca2+ and Cyclic AMP Signals by Activating the Cell-Surface Glucose-Sensing Receptor in Pancreatic β-Cells

    PubMed Central

    Nakagawa, Yuko; Nagasawa, Masahiro; Medina, Johan; Kojima, Itaru

    2015-01-01

    Glucose is a primary stimulator of insulin secretion in pancreatic β-cells. High concentration of glucose has been thought to exert its action solely through its metabolism. In this regard, we have recently reported that glucose also activates a cell-surface glucose-sensing receptor and facilitates its own metabolism. In the present study, we investigated whether glucose activates the glucose-sensing receptor and elicits receptor-mediated rapid actions. In MIN6 cells and isolated mouse β-cells, glucose induced triphasic changes in cytoplasmic Ca2+ concentration ([Ca2+]c); glucose evoked an immediate elevation of [Ca2+]c, which was followed by a decrease in [Ca2+]c, and after a certain lag period it induced large oscillatory elevations of [Ca2+]c. Initial rapid peak and subsequent reduction of [Ca2+]c were independent of glucose metabolism and reproduced by a nonmetabolizable glucose analogue. These signals were also blocked by an inhibitor of T1R3, a subunit of the glucose-sensing receptor, and by deletion of the T1R3 gene. Besides Ca2+, glucose also induced an immediate and sustained elevation of intracellular cAMP ([cAMP]c). The elevation of [cAMP]c was blocked by transduction of the dominant-negative Gs, and deletion of the T1R3 gene. These results indicate that glucose induces rapid changes in [Ca2+]c and [cAMP]c by activating the cell-surface glucose-sensing receptor. Hence, glucose generates rapid intracellular signals by activating the cell-surface receptor. PMID:26630567

  9. Muscarinic receptor stimulation and cyclic AMP-dependent effects in guinea-pig ventricular myocardium.

    PubMed Central

    Schmied, R.; Korth, M.

    1990-01-01

    1. The effect of carbachol on force of contraction, contraction duration, intracellular Na+ activity and cyclic AMP content was studied in papillary muscles of the guinea-pig exposed to isoprenaline or the phosphodiesterase inhibitor 3-isobutyl, 1-methyl xanthine (IBMX). The preparations were obtained from reserpine-pretreated animals and were electrically driven at a frequency of 0.2 Hz. 2. Isoprenaline (10 nM) and IBMX (100 microM) produced comparable positive inotropic effects of 9.8 and 9.7 mN, respectively. Carbachol (3 microM) attenuated the inotropic effects by 82% (isoprenaline) and by 79% (IBMX). The shortening of contraction duration which accompanied the positive inotropic effect of isoprenaline (by 14.9%) and of IBMX (by 22.4%) was not significantly affected by 3 microM carbachol. 3. The positive inotropic effect of 10 nM isoprenaline and of 100 microM IBMX was accompanied by an increase in cellular cyclic AMP content of 58 and 114%, respectively. Carbachol (3 microM) failed to reduce significantly the elevated cyclic AMP content of muscles exposed to either isoprenaline or IBMX. 4. In the quiescent papillary muscle, isoprenaline (10 nM) and IBMX (100 microM) reduced the intracellular Na+ activity by 28 and 17%, respectively. This decline was not influenced by the additional application of 3 microM carbachol. 5. The results demonstrate that muscarinic antagonism in guinea-pig ventricular myocardium exposed to cyclic AMP-elevating drugs is restricted to force of contraction. The underlying mechanism does not apparently involve the cytosolic signal molecule cyclic AMP. PMID:1691677

  10. D1 dopamine receptor-induced cyclic AMP-dependent protein kinase phosphorylation and potentiation of striatal glutamate receptors.

    PubMed

    Price, C J; Kim, P; Raymond, L A

    1999-12-01

    Dopamine receptor activation regulates cyclic AMP levels and is critically involved in modulating neurotransmission in the striatum. Others have shown that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptor-mediated current is potentiated by cyclic AMP-dependent protein kinase (PKA) activation. We made whole-cell patch clamp recordings from cultured striatal neurons and tested whether D1-type dopamine receptor activation affected AMPA receptor-mediated currents. After a 5-min exposure to the D1 agonist SKF 81297 (1 microM), kainate-evoked current amplitude was enhanced in approximately 75% of cells to 121+/-2.5% of that recorded prior to addition of drug. This response was inhibited by the D1 antagonist SCH 23390 and mimicked by activators of PKA. Moreover, by western blot analysis using an antibody specific for the phosphorylated PKA site Ser845 of GluR1, we observed a marked increase in phosphorylated GluR1 following a 10-min exposure of striatal neurons to 1 microM SKF 81297. Our data demonstrate that activation of D1-type dopamine receptors on striatal neurons promotes phosphorylation of AMPA receptors by PKA as well as potentiation of current amplitude. These results elucidate one mechanism by which dopamine can modulate neurotransmission in the striatum. PMID:10582604

  11. Dibutyryl cyclic AMP reduces the radiosensitivity of cultured endothelial cells

    SciTech Connect

    Ward, W.; Molteni, A.; Ts'ao, C.; Hinz, J. )

    1991-03-11

    The purpose of this study was to determine whether dibutyryl cyclic AMP modifies the radiosensitivity of confluent monolayers of bovine aortic endothelial cells (BAEC). Three indices of BAEC function were monitored from 4-24 hrs after exposure to 1-10 Gy of {sup 60}Co gamma rays: the release of {sup 51}Cr from prelabeled cells, and release of lactate dehydrogenase (LDH) and plasminogen activator (PLA) into the culture medium. There was a time- and radiation dose-dependent increase in {sup 51}Cr, LDH and PLA release from the BAEC, detectable within 12 hrs after 5 Gy or higher, and by 24 hrs after 1 Gy or higher. This increased release was accompanied by a radiation dose-dependent decrease in {sup 51}Cr and LDH, and an increase in PLA activity in the lysate of cells adherent to the monolayer at 24 hrs. The continuous presence of cAMP from 1 hr before to 24 hrs after irradiation reduced all of these radiation reactions, although mM concentrations of cAMP were required for significant sparing. The presence of cAMP from 1 hr before to 10 min after irradiation had no effect on BAEC sensitivity, whereas cAMP added 10 min after irradiation was fully as effective as continuously administered drug. Thus, cultured BAEC exhibit membrane dysfunction within 24 hrs after clinically relevant radiation doses, and this dysfunction is ameliorated by cAMP present after irradiation.

  12. Cyclic AMP Regulates Social Behavior in African Trypanosomes

    PubMed Central

    Oberholzer, Michael; Saada, Edwin A.

    2015-01-01

    ABSTRACT The protozoan parasite Trypanosoma brucei engages in surface-induced social behavior, termed social motility, characterized by single cells assembling into multicellular groups that coordinate their movements in response to extracellular signals. Social motility requires sensing and responding to extracellular signals, but the underlying mechanisms are unknown. Here we report that T. brucei social motility depends on cyclic AMP (cAMP) signaling systems in the parasite’s flagellum (synonymous with cilium). Pharmacological inhibition of cAMP-specific phosphodiesterase (PDE) completely blocks social motility without impacting the viability or motility of individual cells. Using a fluorescence resonance energy transfer (FRET)-based sensor to monitor cAMP dynamics in live cells, we demonstrate that this block in social motility correlates with an increase in intracellular cAMP levels. RNA interference (RNAi) knockdown of the flagellar PDEB1 phenocopies pharmacological PDE inhibition, demonstrating that PDEB1 is required for social motility. Using parasites expressing distinct fluorescent proteins to monitor individuals in a genetically heterogeneous community, we found that the social motility defect of PDEB1 knockdowns is complemented by wild-type parasites in trans. Therefore, PDEB1 knockdown cells are competent for social motility but appear to lack a necessary factor that can be provided by wild-type cells. The combined data demonstrate that the role of cyclic nucleotides in regulating microbial social behavior extends to African trypanosomes and provide an example of transcomplementation in parasitic protozoa. PMID:25922395

  13. The Cyclic AMP Phenotype of Fragile X and Autism

    PubMed Central

    Kelley, Daniel J; Bhattacharyya, Anita; Lahvis, Garet P; Yin, Jerry CP; Malter, Jim; Davidson, Richard J

    2008-01-01

    Cyclic AMP (cAMP) is a second messenger involved in many processes including mnemonic processing and anxiety. Memory deficits and anxiety are noted in the phenotype of fragile X (FX), the most common heritable cause of mental retardation and autism. Here we review reported observations of altered cAMP cascade function in FX and autism. Cyclic AMP is a potentially useful biochemical marker to distinguish autism comorbid with FX from autism per se and the cAMP cascade may be a viable therapeutic target for both FX and autism. PMID:18601949

  14. Sodium pump stimulation by oxytocin and cyclic AMP in the isolated epithelium of the frog skin.

    PubMed

    Aceves, J

    1977-11-23

    Activity of the Na pump was judged by Na extrusion in epithelial cells loaded with Na by a previous incubation in K-free solutions in the cold. Oxytocin significantly stimulated Na extrusion either at normal (3.5 mM) or low (0.25 mM) K in the medium. It was stimulated as well by cyclic AMP. Maximal concentrations of either agent caused about the same degree of stimulation. Addition of ouabain or removal of K prevented the action of both agents, but amiloride showed no effect at all. These results strongly suggest that, a) neurohypophyseal hormones not only increase Na entry across the mucosal barrier of the epithelium but they also stimulate the serosal Na pump, b) cyclic AMP not only mediates the action of neurohypophyseal hormones on Na and water permeability of the mucosal barrier, but it also mediates the action of the hormones on the Na pump of the serosal barrier. PMID:202919

  15. Inhibition of carbonic anhydrase by parathyroid hormone and cyclic AMP in rat renal cortex in vitro.

    PubMed Central

    Beck, N; Kim, K S; Wolak, M; Davis, B B

    1975-01-01

    It has been demonstrated that parathyroid hormone (PTH) inhibits the proximal tubular reabsorption of bicarbonate, and increases the urinary excretion of that ion. There is also a qualitative similarity between the alterations of the proximal tubular reabsorption of phosphate, sodium, and water after PTH administration and after acetazolamide administration. These findings suggest that the renal effect of PTH is possibly mediated through the inhibition of carbonic anhydrase in proximal tubules. Therefore, a possible inhibitory effect of PTH on carbonic anhydrase was evaluated in the homogenate of rat renal cortex by an indicator titration method. Incubation of cortical homogenates with PTH for 10 min at 37degreesC inhibited carbonic anhydrase activity. The inhibitory effect of PTH was ATP-, Mg++-, and K+-dependent and temperature-dependent; inactivation of PTH by heating at 100degreesC abolished the effect of PTH both to activate adenylate cyclase and to inhibit carbonic anhydrase. Calcium 5 mM also partially abolished effects of PTH to activate adenylate cyclase and to inhibit carbonic anhydrase. The inhibitory effect of PTH on carbonic anhydrase was specific to renal cortex. Cyclic AMP, the intracellular messenger substance for PTH, also inhibited carbonic anhydrase in renal cortex. The cyclic AMP-induced inhibition was also Mg++ dependent and temperature dependent, and required preincubation at 37degreesC. But 5'-AMP, a metabolic derivative of cyclic AMP without its biological effect, had no inhibitory effect on carbonic anhydrase. All the above results are consistent with the hypothesis that PTH inhibits proximal tubular reabsorption of bicarbonate and phosphate through the inhibition of carbonic anhydrase, and that inhibitory effect is mediated through the cyclic AMP system. PMID:233968

  16. 21 CFR 862.1230 - Cyclic AMP test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Cyclic AMP test system. 862.1230 Section 862.1230 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  17. 21 CFR 862.1230 - Cyclic AMP test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Cyclic AMP test system. 862.1230 Section 862.1230 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  18. 21 CFR 862.1230 - Cyclic AMP test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Cyclic AMP test system. 862.1230 Section 862.1230 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  19. 21 CFR 862.1230 - Cyclic AMP test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cyclic AMP test system. 862.1230 Section 862.1230 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  20. 21 CFR 862.1230 - Cyclic AMP test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Cyclic AMP test system. 862.1230 Section 862.1230 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems §...

  1. Characteristics of cyclic AMP transport by marine bacteria

    SciTech Connect

    Ammerman, J.W.; Azam, F.

    1987-12-01

    Uptake and autoradiography experiments with natural populations of marine bacteria, sea water cultures, and cultured isolates showed that the high-affinity cyclic AMP transport system in marine bacteria has stringent structural requirements, is found in a minority of cells in mixed bacterial assemblages, and appears to be related to the culture growth state.

  2. Cyclic AMP phosphodiesterase in Salmonella typhimurium: characteristics and physiological function.

    PubMed

    Botsford, J L

    1984-11-01

    The physiological function of cyclic AMP (cAMP) phosphodiesterase in Salmonella typhimurium was investigated with strains which were isogenic except for the cpd locus. In crude broken-cell extracts the properties of the enzyme were found to be similar to those reported for Escherichia coli. The specific activity in the mutant was less than 1% that in the wild type. Rates of cAMP production in the mutant were as much as twice those observed in the wild type. The amount of cAMP accumulated when cells grew overnight with limiting glucose was 4.5-fold greater in the mutant than in the wild type. The intracellular concentration of cAMP in the two strains was measured directly, using four different techniques to wash the cells to remove extracellular cAMP. The cAMP level in the cpd strain was only 25% greater than in the wild type. The functional concentration of the cAMP receptor protein-cAMP complex was estimated indirectly from the specific activity of beta-galactosidase in the two strains after introducing F'lac. When cells were grown with carbon sources permitting synthesis of different levels of cAMP, the specific activity of the enzyme was at most 25% greater in the cpd strain. The cpd strain was more sensitive to the effects of exogenous cAMP. Exogenous cAMP relieved both permanent and transient catabolite repression of the lac operon at lower concentrations in the cpd strain than in the wild type. When cells grew with glucose, glycerol, or ribose, exogenous cAMP inhibited growth of the mutant strain more than the wild type. PMID:6094495

  3. Farnesol and Cyclic AMP Signaling Effects on the Hypha-to-Yeast Transition in Candida albicans

    PubMed Central

    Lindsay, Allia K.; Deveau, Aurélie; Piispanen, Amy E.

    2012-01-01

    Candida albicans, a fungal pathogen of humans, regulates its morphology in response to many environmental cues and this morphological plasticity contributes to virulence. Farnesol, an autoregulatory molecule produced by C. albicans, inhibits the induction of hyphal growth by inhibiting adenylate cyclase (Cyr1). The role of farnesol and Cyr1 in controlling the maintenance of hyphal growth has been less clear. Here, we demonstrate that preformed hyphae transition to growth as yeast in response to farnesol and that strains with increased cyclic AMP (cAMP) signaling exhibit more resistance to farnesol. Exogenous farnesol did not induce the hypha-to-yeast transition in mutants lacking the Tup1 or Nrg1 transcriptional repressors in embedded conditions. Although body temperature is not required for embedded hyphal growth, we found that the effect of farnesol on the hypha-to-yeast transition varies inversely with temperature. Our model of Cyr1 activity being required for filamentation is also supported by our liquid assay data, which show increased yeast formation when preformed filaments are treated with farnesol. Together, these data suggest that farnesol can modulate morphology in preformed hyphal cells and that the repression of hyphal growth maintenance likely occurs through the inhibition of cAMP signaling. PMID:22886999

  4. Posttranscriptional Regulation of the Yersinia pestis Cyclic AMP Receptor Protein Crp and Impact on Virulence

    PubMed Central

    Lathem, Wyndham W.; Schroeder, Jay A.; Bellows, Lauren E.; Ritzert, Jeremy T.; Koo, Jovanka T.; Price, Paul A.; Caulfield, Adam J.; Goldman, William E.

    2014-01-01

    ABSTRACT The cyclic AMP receptor protein (Crp) is a transcriptional regulator that controls the expression of numerous bacterial genes, usually in response to environmental conditions and particularly by sensing the availability of carbon. In the plague pathogen Yersinia pestis, Crp regulates the expression of multiple virulence factors, including components of the type III secretion system and the plasminogen activator protease Pla. The regulation of Crp itself, however, is distinctly different from that found in the well-studied Escherichia coli system. Here, we show that at physiological temperatures, the synthesis of Crp in Y. pestis is positively regulated at the posttranscriptional level. The loss of the small RNA chaperone Hfq results in decreased Crp protein levels but not in steady-state Crp transcript levels, and this regulatory effect occurs within the 5′ untranslated region (UTR) of the Crp mRNA. The posttranscriptional activation of Crp synthesis is required for the expression of pla, and decoupling crp from Hfq through the use of an exogenously controlled promoter and 5′ UTR increases Pla protein levels as well as partially rescues the growth defect associated with the loss of Hfq. Finally, we show that both Hfq and the posttranscriptional regulation of Crp contribute to the virulence of Y. pestis during pneumonic plague. The Hfq-dependent, posttranscriptional regulation of Crp may be specific to Yersinia species, and thus our data help explain the dramatic growth and virulence defects associated with the loss of Hfq in Y. pestis. PMID:24520064

  5. Stimulation of Ca2+ uptake by cyclic AMP and protein kinase in sarcoplasmic reticulum-rich and sarcolemma-rich microsomal fractions from rabbit heart.

    PubMed

    Will, H; Schirpke, B; Wollenberger, A

    1976-01-01

    The effect of cyclic AMP on Ca2+ uptake by rabbit heart microsomal vesicular fractions representing mainly fragments of either sarcoplasmic reticulum or sarcolemma was investigated in the presence and absence of soluble cardiac protein kinase and with microsomes prephosphorylated by cyclic AMP-dependent protein kinase. The acceleration of oxalate-promoted Ca2+ uptake by fragmented sarcoplasmic reticulum following cyclic AMP-dependent membrane protein phosphorylation, observed by other authors, was confirmed. In addition it was found that the acceleration was greatest at pH 7.2 and almost negligible at pH 6.0 and pH 7.8. A very marked increase in Ca2+ uptake by cyclic AMP-dependent membrane protein phosphorylation was observed in the presence of boric acid, a reversible inhibitor of Ca2+ uptake. In addition to the microsomal fraction thought to represent mainly fragments of the sarcoplasmic reticulum, the effect of protein kinase and cyclic AMP on Ca2+ uptake was investigated in a cardiac sarcolemma-enriched membrane fraction. Ca2+ uptake by sarcolemmal vesicles, unlike Ca2+ uptake by sarcoplasmic reticulum vesicles, was inhibited by low doses of digitoxin. The acceleration of oxalate-promoted Ca2+ uptake by cyclic AMP and soluble cardiac protein kinase, however, was quite similar to what was seen in preparations of fragmented sarcoplasmic reticulum, which suggests that it may reflect an acceleration of active Ca2+ transport across the myocardial cell surface membrane. PMID:185862

  6. Electrical Stimulation Decreases Coupling Efficiency Between Beta-Adrenergic Receptors and Cyclic AMP Production in Cultured Muscle Cells

    NASA Technical Reports Server (NTRS)

    Young, R. B.; Bridge, K. Y.

    1999-01-01

    Electrical stimulation of skeletal muscle cells in culture is an effective way to simulate the effects of muscle contraction and its effects on gene expression in muscle cells. Expression of the beta-adrenergic receptor and its coupling to cyclic AMP synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy, and the goal of this project was to determine if electrical stimulation altered the beta-adrenergic response in muscle cells. Chicken skeletal muscle cells that had been grown for seven days in culture were subjected to electrical stimulation for an additional two days at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. At the end of this two-day stimulation period, beta-adrenergic receptor population was measured by the binding of tritium-labeled CGP-12177 to muscle cells, and coupling to cAMP synthesis was measured by Radioimmunoassay (RIA) after treating the cells for 10 min with the potent (beta)AR agonist, isoproterenol. The number of beta adrenergic receptors and the basal levels of intracellular cyclic AMP were not affected by electrical stimulation. However, the ability of these cells to synthesize cyclic AMP was reduced by approximately 50%. Thus, an enhanced level of contraction reduces the coupling efficiency of beta-adrenergic receptors for cyclic AMP production.

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

    NASA Technical Reports Server (NTRS)

    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.

  8. Cyclic AMP can promote APL progression and protect myeloid leukemia cells against anthracycline-induced apoptosis

    PubMed Central

    Gausdal, G; Wergeland, A; Skavland, J; Nguyen, E; Pendino, F; Rouhee, N; McCormack, E; Herfindal, L; Kleppe, R; Havemann, U; Schwede, F; Bruserud, Ø; Gjertsen, B T; Lanotte, M; Ségal-Bendirdjian, E; Døskeland, S O

    2013-01-01

    We show that cyclic AMP (cAMP) elevating agents protect blasts from patients with acute promyelocytic leukemia (APL) against death induced by first-line anti-leukemic anthracyclines like daunorubicin (DNR). The cAMP effect was reproduced in NB4 APL cells, and shown to depend on activation of the generally cytoplasmic cAMP-kinase type I (PKA-I) rather than the perinuclear PKA-II. The protection of both NB4 cells and APL blasts was associated with (inactivating) phosphorylation of PKA site Ser118 of pro-apoptotic Bad and (activating) phosphorylation of PKA site Ser133 of the AML oncogene CREB. Either event would be expected to protect broadly against cell death, and we found cAMP elevation to protect also against 2-deoxyglucose, rotenone, proteasome inhibitor and a BH3-only mimetic. The in vitro findings were mirrored by the findings in NSG mice with orthotopic NB4 cell leukemia. The mice showed more rapid disease progression when given cAMP-increasing agents (prostaglandin E2 analog and theophylline), both with and without DNR chemotherapy. The all-trans retinoic acid (ATRA)-induced terminal APL cell differentiation is a cornerstone in current APL treatment and is enhanced by cAMP. We show also that ATRA-resistant APL cells, believed to be responsible for treatment failure with current ATRA-based treatment protocols, were protected by cAMP against death. This suggests that the beneficial pro-differentiating and non-beneficial pro-survival APL cell effects of cAMP should be weighed against each other. The results suggest also general awareness toward drugs that can affect bone marrow cAMP levels in leukemia patients. PMID:23449452

  9. Diazepam and rolipram differentially inhibit cyclic AMP-specific phosphodiesterases PDE4A1 and PDE4B3 in the mouse.

    PubMed

    Cherry, J A; Thompson, B E; Pho, V

    2001-03-19

    Cyclic AMP is hydrolyzed by members of at least eight classes of cyclic nucleotide phosphodiesterases (PDEs). Although it has been reported that cyclic AMP PDE activity in mammalian tissues can be inhibited by benzodiazepines, it has not been conclusively demonstrated that members of the class of cyclic AMP-specific, rolipram-inhibitable PDEs (PDE4s) are targets for these drugs. Moreover, no PDE4s expressed in mice have been characterized. To address these issues, we isolated two cDNAs representing homologues of PDE4A1 and PDE4B3 from a mouse brain library. After transient transfection in human embryonic kidney (HEK) 293 cells, the mouse PDEs hydrolyzed cyclic AMP with a low K(m) and were inhibited by rolipram; both are properties typical of other mammalian PDE4 enzymes. In addition, we found that diazepam inhibited cyclic AMP hydrolysis by the mouse PDE4 subtypes. Interestingly, PDE4B was significantly more sensitive to inhibition by both rolipram and diazepam than the PDE4A subtype. This is the first demonstration that recombinantly expressed PDE4s are inhibited by diazepam, and should facilitate future studies with mouse models of depression and anxiety. PMID:11267656

  10. Cholera toxin, and the related nontoxic adjuvants mmCT and dmLT, promote human Th17 responses via cyclic AMP-protein kinase A and inflammasome-dependent IL-1 signaling.

    PubMed

    Larena, Maximilian; Holmgren, Jan; Lebens, Michael; Terrinoni, Manuela; Lundgren, Anna

    2015-04-15

    We have examined the molecular pathways involved in the adjuvant action of cholera toxin (CT) and two novel nontoxic molecules, multiple-mutated CT (mmCT) and double-mutant heat-labile toxin (dmLT) on human T cell responses. Human PBMCs or isolated monocytes were stimulated in vitro with CT, mmCT, or dmLT plus a polyclonal stimulus (staphylococcal enterotoxin B) or specific bacterial Ags, and effects on expression of cytokines and signaling molecules were determined. CT, mmCT, and dmLT strongly enhanced IL-17A and to a lesser extent IL-13 responses, but had little effect on IFN-γ production or cell proliferation. Intracellular cytokine staining revealed that the enhanced IL-17A production was largely confined to CD4(+) T cells and coculture experiments showed that the IL-17A promotion was effectively induced by adjuvant-treated monocytes. Relative to CT, mmCT and dmLT induced at least 100-fold lower levels of cAMP, yet this cAMP was enough and essential for the promotion of Th17 responses. Thus, inhibition of cAMP-dependent protein kinase A was abolished, and stimulation with a cAMP analog mimicked the adjuvant effect. Furthermore, CT, mmCT, and dmLT induced IL-1β production and caspase-1 activation in monocytes, which was associated with increased expression of key proinflammatory and inflammasome-related genes, including NLRP1, NLRP3, and NLRC4. Inflammasome inhibition with a specific caspase-1 inhibitor, or blocking of IL-1 signaling by IL-1 receptor antagonist, abrogated the Th17-promoting effect. We conclude that CT, mmCT, and dmLT promote human Th17 responses via cAMP-dependent protein kinase A and caspase-1/inflammasome-dependent IL-1 signaling. PMID:25786687

  11. Involvement of calyculin A inhibitable protein phosphatases in the cyclic AMP signal transduction pathway of mouse corticotroph tumour (AtT20) cells

    PubMed Central

    Antaraki, A; Ang, K L; Antoni, F A

    1997-01-01

    The role of non-calcineurin protein phosphatases in the cyclic AMP signal transduction pathway was examined in mouse pituitary corticotroph tumour (AtT20) cells. Blockers of protein phosphatases, calyculin A and okadaic acid, were applied in AtT20 cells depleted of rapidly mobilizable pools of intracellular calcium and activated by various cyclic AMP generating agonists. Inhibitors of cyclic nucleotide phosphodiesterases were present throughout. The accumulation of cyclic AMP was monitored by radioimmunoassay, phosphodiesterase activity in cell homogenates was measured by radiometric assay. Neither calyculin A nor okadaic acid altered basal cyclic AMP levels but cyclic AMP formation induced by 41 amino acid residue corticotrophin releasing-factor (CRF) was strongly inhibited (up to 80%). 1-Norokadaone was inactive. Similar data were also obtained when isoprenaline or pituitary adenylate cyclase activating peptide1–38 were used as agonists. Pertussis toxin did not modify the inhibition of CRF-induced cyclic AMP production by calyculin A. Pretreatment with calyculin A completely prevented the stimulation of cyclic AMP formation by cholera toxin even in the presence of 0.5 mM isobutylmethylxanthine (IBMX) and 0.1 mM rolipram. Cholera toxin mediated ADP-ribosylation of the 45K and 52K molecular weight Gsα isoforms in membranes from calyculin A-pretreated cells was enhanced to 150–200% when compared with controls. Cholera toxin-induced cyclic AMP was reduced by calyculin A within 10 min when calyculin A was applied after a 90 min pretreatment with cholera toxin. Under these conditions the effect of calyculin A could be blocked by the combination of 0.5 mM IBMX and 0.1 mM rolipram, but not by 0.5 mM IBMX alone. Phosphodiesterase activity in AtT20 cell homogenates showed a significant, 2.7 fold increase after treatment with calyculin A. In control cells phosphodiesterase activity was blocked by 80% in the presence of IBMX (0.5 mM), or IBMX plus

  12. Effect of electrical stimulation on beta-adrenergic receptor population and cyclic amp production in chicken and rat skeletal muscle cell cultures

    NASA Technical Reports Server (NTRS)

    Young, R. B.; Bridge, K. Y.; Strietzel, C. J.

    2000-01-01

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

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

    PubMed Central

    Pinkney, M; Hoggett, J G

    1988-01-01

    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. PMID:2839152

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

    PubMed

    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. PMID:2839152

  15. Tumour necrosis factor alpha-induced oxidative burst in neutrophils adherent to fibronectin: effects of cyclic AMP-elevating agents.

    PubMed

    Ottonello, L; Morone, M P; Dapino, P; Dallegri, F

    1995-11-01

    Human neutrophils, plated on fibronectin-coated polystyrene wells, were found to exhibit a prolonged production of superoxide anion (O2-) in response to tumour necrosis factor-alpha (TNF). The TNF-triggered O2- production was significantly reduced by 10 microM prostaglandin E2 (PGE2), which was ineffective at lower doses. Moreover, the O2- production was slightly reduced by the phosphodiesterase type IV (PDE IV) inhibitor RO 20-1724. When PGE2 and RO 20-1724 were added together to TNF-triggered neutrophils they caused a marked synergistic inhibition of O2- production. The action of PGE2 could be mimicked by forskolin (FK), a well-known direct activator of adenylate cyclase. These results suggest that cyclic AMP (cAMP)-elevating agents (PGE2, FK, RO 20-1724) down-regulate the capacity of adherent neutrophils to mount the respiratory burst in response to TNF. Consistent with this interpretation, PGE2 and RO 20-1724 increased the intracellular levels of cAMP displaying synergistic activity. Moreover, the membrane-permeable analogue of cAMP, dibutyryl cAMP, was found to inhibit the TNF-induced O2- production in a dose-dependent manner. As all the aforementioned cAMP-elevating agents did not affect the O2- production in response to phorbol myristate acetate, they appear to act by interfering with the assembly of the O2(-)-generating NADPH oxidase complex rather than by directly inhibiting the activity of already working oxidase complex. In conclusion, taking into account the TNF capacity to promote PGE2 formation at sites of inflammation, our observations suggest the existence of a negative PGE2-dependent feed-back, potentially capable of controlling the neutrophil response to TNF and susceptible to amplification by PDE IV-inhibiting compounds. PMID:8555055

  16. Cyclic AMP and Polyamines Overcome Inhibition by Myelin-Associated Glycoprotein through eIF5A-Mediated Increases in p35 Expression and Activation of Cdk5.

    PubMed

    He, Huifang; Deng, Kangwen; Siddiq, Mustafa M; Pyie, Aung; Mellado, Wilfredo; Hannila, Sari S; Filbin, Marie T

    2016-03-01

    Inhibitory molecules associated with CNS myelin, such as myelin-associated glycoprotein (MAG), represent major obstacles to axonal regeneration following CNS injury. Our laboratory has shown that elevating levels of intracellular cAMP, via application of the nonhydrolyzable analog dibutyryl cAMP (dbcAMP), can block the inhibitory effects of MAG and myelin. We have also shown that elevation of cAMP results in upregulation of arginase I and increased polyamine synthesis. Treatment with putrescine or spermidine blocks myelin-mediated inhibition of neurite outgrowth, but the mechanism underlying this effect has not yet been elucidated. Here we show that cyclin-dependent kinase 5 (Cdk5) is required for dbcAMP and putrescine to overcome MAG-mediated inhibition. The ability of dbcAMP and putrescine to overcome inhibition by MAG is abolished in the presence of roscovitine, a Cdk inhibitor that has greater selectivity for Cdk5, and expression of dominant negative Cdk5 abolishes the ability of dbcAMP or putrescine to enhance neurite outgrowth in the presence of MAG. Importantly, dbcAMP and putrescine increase expression of p35, the neuron-specific activator of Cdk5, and rat DRG neurons transduced with HSV overexpressing p35 can overcome inhibition by MAG. The upregulation of p35 by putrescine is also reflected in increased localization of p35 to neurites and growth cones. Last, we show that putrescine upregulates p35 expression by serving as a substrate for hypusine modification of eIF5A, and that this hypusination is necessary for putrescine's ability to overcome inhibition by MAG. Our findings reveal a previously unknown mechanism by which polyamines may encourage regeneration after CNS injury. PMID:26961960

  17. Modulation of 3',5'-cyclic AMP homeostasis in human platelets by coffee and individual coffee constituents.

    PubMed

    Montoya, Gina A; Bakuradze, Tamara; Eirich, Marion; Erk, Thomas; Baum, Matthias; Habermeyer, Michael; Eisenbrand, Gerhard; Richling, Elke

    2014-11-14

    3',5'-Cyclic AMP (cAMP) is one of the most important second messengers in mammalian cells, mediating a multitude of diverse cellular signalling responses. Its homeostasis is primarily regulated by adenylate cyclases and phosphodiesterases (PDE), the activities of which are partially dependent on the downstream events of adenosine receptor signalling. The present study was conducted to determine whether coffee constituents other than caffeine can influence the homeostasis of intracellular cAMP in vitro and in vivo by evaluating the effects of selected constituents present in coffee, coffee brews and coffee extracts on platelet PDE activity. In addition, to evaluate the potential effects of these constituents on platelet cAMP concentrations and PDE activity in humans, a 7-week pilot intervention study with eight subjects was conducted. The subjects consumed a regular commercial coffee and a low-caffeine coffee at a rate of 750 ml/d for 2 weeks each. The in vivo results revealed a highly significant inhibition of PDE activity (P< 0·001) after coffee intervention that was not directly dependent on the caffeine content of coffee. Although our in vitro and in vivo findings suggest that caffeine plays some role in the modulation of platelet cAMP status, other natural and roasting-associated compounds such as pyrazines and other currently unidentified species also appear to contribute significantly. In conclusion, moderate consumption of coffee can modulate platelet PDE activity and cAMP concentrations in humans, which may contribute to the putative beneficial health effects of coffee. Further detailed mechanistic investigations will be required to substantiate these beneficial effects and to elucidate the underlying mechanisms. PMID:25247601

  18. Dibutyryl cyclic AMP does not influence glomerular collagen or basement membrane production in vitro.

    PubMed

    Uw, V Y; Cohen, M P

    1980-02-01

    Glomeruli isolated from normal rat renal cortex were incubated for 3 hr with radiolabeled proline in the presence or absence of dibutyryl cyclic AMP. Following incubation, glomerular basement membranes were purified with osmotic lysis followed by selective solubilization of the cell membranes and intracellular proteins with detergents. This technique permitted quantitative recovery of radiolabeled membranes synthesized under different incubational conditions. Dibutyryl cyclic AMP did not affect the incorporation of radioactive precursor glomerular basement membrane (control = 14.72 +/- 1.08 cpm/microgram of membrane protein; cyclic AMP = 14.43 +/- 1.13). Nondialyzable [14C]protein and hydroxy[14C]proline were also measured in the media and in the various glomerular cell fractions obtained during isolation of the basement membranes. Protein ([14C]proline) and collagen (OH[14C]proline) secretion into the media in incubations with cyclic AMP did not differ from that in control incubations. OH[14C]proline content was greatest (congruent to 23% in the water-soluble fraction recovered after osmotic lysis, but significant amounts of OH[14C]proline were also associated with the detergent-solubilized cell fractions. Dibutyryl cyclic AMP had no effect on either glomerular protein or collagen synthesis in these experiments. The results suggest that total glomerular basement membrane production in mixed cell populations is not modulated via a cyclic AMP--coordinated mechanism but do not exclude the possibility that cyclic AMP modulates the amount or kind of collagen synthesis by individual glomerular cell types. PMID:6243687

  19. Autoregulation of PhoP/PhoQ and positive regulation of the cyclic AMP receptor protein-cyclic AMP complex by PhoP in Yersinia pestis.

    PubMed

    Zhang, Yiquan; Wang, Li; Han, Yanping; Yan, Yanfeng; Tan, Yafang; Zhou, Lei; Cui, Yujun; Du, Zongmin; Wang, Xiaoyi; Bi, Yujing; Yang, Huiying; Song, Yajun; Zhang, Pingping; Zhou, Dongsheng; Yang, Ruifu

    2013-03-01

    Yersinia pestis is one of the most dangerous bacterial pathogens. PhoP and cyclic AMP receptor protein (CRP) are global regulators of Y. pestis, and they control two distinct regulons that contain multiple virulence-related genes. The PhoP regulator and its cognate sensor PhoQ constitute a two-component regulatory system. The regulatory activity of CRP is triggered only by binding to its cofactor cAMP, which is synthesized from ATP by adenylyl cyclase (encoded by cyaA). However, the association between the two regulatory systems PhoP/PhoQ and CRP-cAMP is still not understood for Y. pestis. In the present work, the four consecutive genes YPO1635, phoP, phoQ, and YPO1632 were found to constitute an operon, YPO1635-phoPQ-YPO1632, transcribed as a single primary RNA, whereas the last three genes comprised another operon, phoPQ-YPO1632, transcribed with two adjacent transcriptional starts. Through direct PhoP-target promoter association, the transcription of these two operons was stimulated and repressed by PhoP, respectively; thus, both positive autoregulation and negative autoregulation of PhoP/PhoQ were detected. In addition, PhoP acted as a direct transcriptional activator of crp and cyaA. The translational/transcriptional start sites, promoter -10 and -35 elements, PhoP sites, and PhoP box-like sequences were determined for these PhoP-dependent genes, providing a map of the PhoP-target promoter interaction. The CRP and PhoP regulons have evolved to merge into a single regulatory cascade in Y. pestis because of the direct regulatory association between PhoP/PhoQ and CRP-cAMP. PMID:23264579

  20. Hydrosmotic effect of angiotensin II in the toad skin: role of cyclic AMP.

    PubMed

    Coviello, A; Brauckmann, E S; de Atenor, M S; Apud, J A; Causarano, J

    1975-01-01

    The mechanism of action of the hydrosmotic response of the isolated skin of the toad Bufo arenarum Hensel to angiotensin II was studied by means of an indirect pharmacological approach. Angiotensin II (2.10(-10) M), vasopressin (2.10(-13) M) and theophylline (10(-4) and 10(-3) M) in subliminal doses produced a significant increase on water permeability when added in different paired combinations. Angiotensin II (2.10(-7) M) and vasopressin (2.10(-8) M) in doses producing significant effects on water permeability increased the response to submaximal doses of epinephrine (10(-6) M) but not to higher doses (10(-5) M). Acid pH (6.4) and prostaglandin E1 (2.10(-7) M) reduced significantly the hydrosmotic response to angiotensin II, but in contrast with the toad bladder, the effect was not completely abolished. Present results support the view that the hydrosmotic effect of angiotensin II in toad skin is mediated by the adenylate cyclase - cyclic AMP system. PMID:189568

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

    PubMed Central

    Almblad, Henrik; Harrison, Joe J.; Rybtke, Morten; Groizeleau, Julie; Givskov, Michael; Parsek, Matthew R.

    2015-01-01

    ABSTRACT The opportunistic human pathogen Pseudomonas aeruginosa expresses numerous acute virulence factors in the initial phase of infection, and during long-term colonization it undergoes adaptations that optimize survival in the human host. Adaptive changes that often occur during chronic 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 (cAMP), 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 by inhibition of adenylate cyclase production or activity and that it is not caused by activation of cAMP phosphodiesterase activity. In addition to the studies of the RSCVs, we present evidence that the deeper layers of wild-type P. aeruginosa biofilms have high c-di-GMP levels and low cAMP levels. IMPORTANCE Our work suggests that cross talk between c-di-GMP and cAMP signaling pathways results in downregulation of acute virulence factors in P. aeruginosa biofilm infections. Knowledge about this cross-regulation adds to our understanding of virulence traits and immune evasion by P. aeruginosa in chronic infections and may provide new approaches to eradicate biofilm infections. PMID:25897033

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

    PubMed Central

    Yamashita, Haruka; Goto, Mayu; Matsui-Yuasa, Isao; Kojima-Yuasa, Akiko

    2015-01-01

    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. PMID:26096275

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

    PubMed

    Yamashita, Haruka; Goto, Mayu; Matsui-Yuasa, Isao; Kojima-Yuasa, Akiko

    2015-06-01

    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. PMID:26096275

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

    SciTech Connect

    Kroll, K.; Schrader, J.

    1986-03-01

    In order to quantify the activation of adenylate cyclase of the coronary endothelium in vivo, endothelial adenine nucleotides of isolated guinea pig hearts were selectively pre-labeled by intracoronary infusion of tritiated (H3)-adenosine, and the coronary efflux of H3-cAMP was measured. The adenosine receptor agonist, NECA (12 ..mu..M), increased total cAMP release 4 fold, and raised H3-cAMP release 22 fold. Several classes of coronary vasodilators (adenosine, L-PIA, D-PIA, the beta 2-adrenergic agonist procaterol, and PGE1) caused dose-dependent increases in endothelial-derived H3-cAMP release. These increases were accompanied by decreases in vascular resistance, at agonist doses without positive intropic effects. Hypoxic perfusion also raised H3-cAMP release, and this was antagonized by theophylline. It is concluded: (1) cyclic AMP formation by coronary endothelium can dominate total cAMP production by the heart; (2) coronary endothelial adenylate cyclase-coupled receptors for adenosine (A2), catecholamines (beta2) and prostaglandins are activated in parallel with coronary vasodilation; (3) endothelial adenylate cyclase can be activated by endogenous adenosine.

  5. Regulation of ciliary motility by membrane potential in Paramecium: a role for cyclic AMP.

    PubMed

    Bonini, N M; Gustin, M C; Nelson, D L

    1986-01-01

    The membrane potential of Paramecium controls the frequency and direction of the ciliary beat, thus determining the cell's swimming behavior. Stimuli that hyperpolarize the membrane potential increase the ciliary beat frequency and therefore increase forward swimming speed. We have observed that 1) drugs that elevate intracellular cyclic AMP increased swimming speed 2-3-fold, 2) hyperpolarizing the membrane potential by manipulation of extracellular cations (e.g., K+) induced both a transient increase in, and a higher sustained level of cyclic AMP compared to the control, and 3) the swimming speed of detergent-permeabilized cells in MgATP was stimulated 2-fold by the addition of cyclic AMP. Our results suggest that the membrane potential can regulate intracellular cAMP in Paramecium and that control of swimming speed by membrane potential may in part be mediated by cAMP. PMID:2427226

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

    NASA Technical Reports Server (NTRS)

    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.

  7. Cyclic AMP Represents a Crucial Component of Treg Cell-Mediated Immune Regulation.

    PubMed

    Klein, Matthias; Bopp, Tobias

    2016-01-01

    T regulatory (Treg) cells are one of the key players in the immune tolerance network, and a plethora of manuscripts have described their development and function in the course of the last two decades. Nevertheless, it is still a matter of debate as to which mechanisms and agents are employed by Treg cells, providing the basis of their suppressive potency. One of the important candidates is cyclic AMP (cAMP), which is long known as a potent suppressor at least of T cell activation and function. While this suppressive function by itself is widely accepted, the source and the mechanism of action of cAMP are less clear, and a multitude of seemingly contradictory data allow for, in principle, two different scenarios of cAMP-mediated suppression. In one scenario, Treg cells contain high amounts of cAMP and convey this small molecule via gap junction intercellular communication directly to the effector T cells (Teff) leading to their suppression. Alternatively, it was shown that Treg cells represent the origin of considerable amounts of adenosine, which trigger the adenylate cyclases in Teff cells via A2A and A2B receptors, thus strongly increasing intracellular cAMP. This review will present and discuss initial findings and recent developments concerning the function of cAMP for Treg cells and its impact on immune regulation. PMID:27621729

  8. Cyclic AMP Represents a Crucial Component of Treg Cell-Mediated Immune Regulation

    PubMed Central

    Klein, Matthias; Bopp, Tobias

    2016-01-01

    T regulatory (Treg) cells are one of the key players in the immune tolerance network, and a plethora of manuscripts have described their development and function in the course of the last two decades. Nevertheless, it is still a matter of debate as to which mechanisms and agents are employed by Treg cells, providing the basis of their suppressive potency. One of the important candidates is cyclic AMP (cAMP), which is long known as a potent suppressor at least of T cell activation and function. While this suppressive function by itself is widely accepted, the source and the mechanism of action of cAMP are less clear, and a multitude of seemingly contradictory data allow for, in principle, two different scenarios of cAMP-mediated suppression. In one scenario, Treg cells contain high amounts of cAMP and convey this small molecule via gap junction intercellular communication directly to the effector T cells (Teff) leading to their suppression. Alternatively, it was shown that Treg cells represent the origin of considerable amounts of adenosine, which trigger the adenylate cyclases in Teff cells via A2A and A2B receptors, thus strongly increasing intracellular cAMP. This review will present and discuss initial findings and recent developments concerning the function of cAMP for Treg cells and its impact on immune regulation.

  9. Cyclic AMP Signaling through Epac Axis Modulates Human Hemogenic Endothelium and Enhances Hematopoietic Cell Generation.

    PubMed

    Saxena, Shobhit; Rönn, Roger E; Guibentif, Carolina; Moraghebi, Roksana; Woods, Niels-Bjarne

    2016-05-10

    Hematopoietic cells emerge from hemogenic endothelium in the developing embryo. Mechanisms behind human hematopoietic stem and progenitor cell development remain unclear. Using a human pluripotent stem cell differentiation model, we report that cyclic AMP (cAMP) induction dramatically increases HSC-like cell frequencies. We show that hematopoietic cell generation requires cAMP signaling through the Exchange proteins activated by cAMP (cAMP-Epac) axis; Epac signaling inhibition decreased both hemogenic and non-hemogenic endothelium, and abrogated hematopoietic cell generation. Furthermore, in hematopoietic progenitor and stem-like cells, cAMP induction mitigated oxidative stress, created a redox-state balance, and enhanced C-X-C chemokine receptor type 4 (CXCR4) expression, benefiting the maintenance of these primitive cells. Collectively, our study provides insights and mechanistic details on the previously unrecognized role of cAMP signaling in regulating human hematopoietic development. These findings advance the mechanistic understanding of hematopoietic development toward the development of transplantable human hematopoietic cells for therapeutic needs. PMID:27117782

  10. Role of cyclic AMP in the maturation of Ciona intestinalis oocytes.

    PubMed

    Silvestre, Francesco; Gallo, Alessandra; Cuomo, Annunziata; Covino, Tiziana; Tosti, Elisabetta

    2011-11-01

    Immature oocytes are arrested at prophase I of the meiotic process and maturation onset is indicated by oocyte nuclear disassembly (germinal vesicle breakdown or GVBD). Signaling pathways that elevate intracellular cyclic AMP (cAMP) may either prevent or induce oocyte maturation depending on the species. In some marine invertebrates and, in particular, in ascidian oocytes, cAMP triggers GVBD rather than blocking it. In this paper, we tested different cAMP elevators in fully grown oocytes at the germinal vesicle stage (GV) of the ascidian Ciona intestinalis. We demonstrated that through the activation of adenylate cyclase or the inhibition and phosphodiesterases the oocyte remained at the GV stage. This effect was reversible as the GV-arrested oocytes, rinsed and incubated in sea water, are able to undergo spontaneous maturation and extrusion of follicle cells. In addition, oocytes acquire the ability to be fertilized and start early development. However, morphology of follicle cells, embryos and larvae from in vitro matured oocytes showed different morphology from those derived from in vivo mature oocytes. The role and the transduction mechanism of cAMP in the regulation of oocyte maturation were discussed. Finally, we indicated a variation of biological mechanisms present in the ascidian species; moreover, we sustain evidence proving that tunicates share some biological mechanisms with vertebrates. This information provided new hints on the importance of ascidians in the evolution of chordates. PMID:20810008

  11. Inhibition of the Raf-1 kinase by cyclic AMP agonists causes apoptosis of v-abl-transformed cells.

    PubMed Central

    Weissinger, E M; Eissner, G; Grammer, C; Fackler, S; Haefner, B; Yoon, L S; Lu, K S; Bazarov, A; Sedivy, J M; Mischak, H; Kolch, W

    1997-01-01

    Here we investigate the role of the Raf-1 kinase in transformation by the v-abl oncogene. Raf-1 can activate a transforming signalling cascade comprising the consecutive activation of Mek and extracellular-signal-regulated kinases (Erks). In v-abl-transformed cells the endogenous Raf-1 protein was phosphorylated on tyrosine and displayed high constitutive kinase activity. The activities of the Erks were constitutively elevated in both v-raf- and v-abl-transformed cells. In both cell types the activities of Raf-1 and v-raf were almost completely suppressed after activation of the cyclic AMP-dependent kinase (protein kinase A [PKA]), whereas the v-abl kinase was not affected. Raf inhibition substantially diminished the activities of Erks in v-raf-transformed cells but not in v-abl-transformed cells, indicating that v-abl can activate Erks by a Raf-1-independent pathway. PKA activation induced apoptosis in v-abl-transformed cells while reverting v-raf transformation without severe cytopathic effects. Overexpression of Raf-1 in v-abl-transformed cells partially protected the cells from apoptosis induced by PKA activation. In contrast to PKA activators, a Mek inhibitor did not induce apoptosis. The diverse biological responses correlated with the status of c-myc gene expression. v-abl-transformed cells featured high constitutive levels of expression of c-myc, which were not reduced following PKA activation. Myc activation has been previously shown to be essential for transformation by oncogenic Abl proteins. Using estrogen-regulated c-myc and temperature-sensitive Raf-1 mutants, we found that Raf-1 activation could protect cells from c-myc-induced apoptosis. In conclusion, these results suggest (i) that Raf-1 participates in v-abl transformation via an Erk-independent pathway by providing a survival signal which complements c-myc in transformation, and (ii) that cAMP agonists might become useful for the treatment of malignancies where abl oncogenes are involved, such as

  12. Sustained cyclic AMP production by parathyroid hormone receptor endocytosis

    PubMed Central

    Ferrandon, Sébastien; Feinstein, Timothy N; Castro, Marian; Wang, Bin; Bouley, Richard; Potts, John T; Gardella, Thomas J; Vilardaga, Jean-Pierre

    2011-01-01

    Cell signaling mediated by the G protein-coupled parathyroid hormone receptor type 1 (PTHR) is fundamental to bone and kidney physiology. It has been unclear how the two ligand systems—PTH, endocrine and homeostatic, and PTH-related peptide (PTHrP), paracrine—can effectively operate with only one receptor and trigger different durations of the cAMP responses. Here we analyze the ligand response by measuring the kinetics of activation and deactivation for each individual reaction step along the PTHR signaling cascade. We found that during the time frame of G protein coupling and cAMP production, PTHrP1–36 action was restricted to the cell surface, whereas PTH1–34 had moved to internalized compartments where it remained associated with the PTHR and Gαs, potentially as a persistent and active ternary complex. Such marked differences suggest a mechanism by which PTH and PTHrP induce differential responses, and these results indicate that the central tenet that cAMP production originates exclusively at the cell membrane must be revised. PMID:19701185

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  14. Cyclic AMP Receptor Protein Regulates Pheromone-Mediated Bioluminescence at Multiple Levels in Vibrio fischeri ES114

    PubMed Central

    Lyell, Noreen L.; Colton, Deanna M.; Bose, Jeffrey L.; Tumen-Velasquez, Melissa P.; Kimbrough, John H.

    2013-01-01

    Bioluminescence in Vibrio fischeri ES114 is activated by autoinducer pheromones, and this regulation serves as a model for bacterial cell-cell signaling. As in other bacteria, pheromone concentration increases with cell density; however, pheromone synthesis and perception are also modulated in response to environmental stimuli. Previous studies suggested that expression of the pheromone-dependent bioluminescence activator LuxR is regulated in response to glucose by cyclic AMP (cAMP) receptor protein (CRP) (P. V. Dunlap and E. P. Greenberg, J. Bacteriol. 164:45–50, 1985; P. V. Dunlap and E. P. Greenberg, J. Bacteriol. 170:4040–4046, 1988; P. V. Dunlap, J. Bacteriol. 171:1199–1202, 1989; and W. F. Friedrich and E. P. Greenberg, Arch. Microbiol. 134:87–91, 1983). Consistent with this model, we found that bioluminescence in V. fischeri ES114 is modulated by glucose and stimulated by cAMP. In addition, a Δcrp mutant was ∼100-fold dimmer than ES114 and did not increase luminescence in response to added cAMP, even though cells lacking crp were still metabolically capable of producing luminescence. We further discovered that CRP regulates not only luxR but also the alternative pheromone synthase gene ainS. We found that His-tagged V. fischeri CRP could bind sequences upstream of both luxR and ainS, supporting bioinformatic predictions of direct regulation at both promoters. Luminescence increased in response to cAMP if either the ainS or luxR system was under native regulation, suggesting cAMP-CRP significantly increases luminescence through both systems. Finally, using transcriptional reporters in transgenic Escherichia coli, we elucidated two additional regulatory connections. First, LuxR-independent basal transcription of the luxI promoter was enhanced by CRP. Second, the effect of CRP on the ainS promoter depended on whether the V. fischeri regulatory gene litR was also introduced. These results suggest an integral role for CRP in pheromone signaling that

  15. Analysis of the spacer DNA between the cyclic AMP receptor protein binding site and the lac promoter.

    PubMed Central

    Flatow, U; Rajendrakumar, G V; Garges, S

    1996-01-01

    The role of the spacer region DNA between the cyclic AMP receptor protein (CRP) site and the RNA polymerase in the lac promoter was examined. We wanted to determine whether the wild-type DNA sequence of this region was an absolute requirement for CRP activation of lac transcription. The sequence of a 9-bp stretch of the spacer, from -41 to -49 relative to the start of transcription, was randomized, and the effect of randomization on lac expression was investigated in vitro and in vivo. We found that the spacer contains no specific sequence determinants for CRP activation of lac transcription; fewer than 1% of the mutants displayed greater than a 50% decrease in CRP activation of lac transcription. PMID:8636052

  16. Cyclic AMP-dependent protein kinase interferes with GTP. gamma. S stimulated IP sub 3 formation in differentiated HL-60 cell membranes

    SciTech Connect

    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 {sup 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 {gamma}S-stimulated inositol trisphosphate (IP{sub 3}) formation by about 30%, but had no influence on Ca{sup 2+}-stimulated IP{sub 3} formation. And autoradiography in the phosphorylation experiments of solubilized HL-60 cell membranes by PKA showed some {sup 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.

  17. Cyclic AMP-elevating agents down-regulate the oxidative burst induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) in adherent neutrophils.

    PubMed

    Ottonello, L; Morone, M P; Dapino, P; Dallegri, F

    1995-09-01

    Human neutrophils, plated on fibronectin-precoated wells, were found to release large quantities of superoxide anion (O2-) in response to GM-CSF. O2- production was reduced by prostaglandin E2 (PGE2) and the phosphodiesterase type IV (PDE IV) inhibitor RO 20-1724. Both agents are known to increase intracellular cyclic AMP (cAMP) levels by inducing its production (PGE2) or blocking its catabolism (RO 20-1724). When added in combination, PGE2 and RO 20-1724 had a marked synergistic inhibitory effect, which was reproduced by replacing PGE2 with a direct activator of adenylate cyclase, i.e. forskolin (FK). Moreover, the neutrophil response to GM-CSF was inhibited by a membrane-permeable analogue of cAMP in a dose-dependent manner. As GM-CSF and PGE2 are known to be generated at tissue sites of inflammation, the results suggest the existence of a PGE2-dependent regulatory pathway potentially capable of controlling the neutrophil response to GM-CSF, in turn limiting the risk of local oxidative tissue injury. Moreover, owing to its susceptibility to amplification by RO 20-1724, the PGE2-dependent pathway and in particular PDE-IV may represent a pharmacological target to reduce the generation of histotoxic oxidants by GM-CSF-responding neutrophils. PMID:7664497

  18. Cyclic AMP-elevating agents down-regulate the oxidative burst induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) in adherent neutrophils.

    PubMed Central

    Ottonello, L; Morone, M P; Dapino, P; Dallegri, F

    1995-01-01

    Human neutrophils, plated on fibronectin-precoated wells, were found to release large quantities of superoxide anion (O2-) in response to GM-CSF. O2- production was reduced by prostaglandin E2 (PGE2) and the phosphodiesterase type IV (PDE IV) inhibitor RO 20-1724. Both agents are known to increase intracellular cyclic AMP (cAMP) levels by inducing its production (PGE2) or blocking its catabolism (RO 20-1724). When added in combination, PGE2 and RO 20-1724 had a marked synergistic inhibitory effect, which was reproduced by replacing PGE2 with a direct activator of adenylate cyclase, i.e. forskolin (FK). Moreover, the neutrophil response to GM-CSF was inhibited by a membrane-permeable analogue of cAMP in a dose-dependent manner. As GM-CSF and PGE2 are known to be generated at tissue sites of inflammation, the results suggest the existence of a PGE2-dependent regulatory pathway potentially capable of controlling the neutrophil response to GM-CSF, in turn limiting the risk of local oxidative tissue injury. Moreover, owing to its susceptibility to amplification by RO 20-1724, the PGE2-dependent pathway and in particular PDE-IV may represent a pharmacological target to reduce the generation of histotoxic oxidants by GM-CSF-responding neutrophils. PMID:7664497

  19. Capsaicinoids regulate airway anion transporters through Rho kinase- and cyclic AMP-dependent mechanisms.

    PubMed

    Hibino, Yoshitaka; Morise, Masahiro; Ito, Yasushi; Mizutani, Takefumi; Matsuno, Tadakatsu; Ito, Satoru; Hashimoto, Naozumi; Sato, Mitsuo; Kondo, Masashi; Imaizumi, Kazuyoshi; Hasegawa, Yoshinori

    2011-10-01

    To investigate the effects of capsaicinoids on airway anion transporters, we recorded and analyzed transepithelial currents in human airway epithelial Calu-3 cells. Application of capsaicin (100 μM) attenuated vectorial anion transport, estimated as short-circuit currents (I(SC)), before and after stimulation by forskolin (10 μM) with concomitant reduction of cytosolic cyclic AMP (cAMP) levels. The capsaicin-induced inhibition of I(SC) was also observed in the response to 8-bromo-cAMP (1 mM, a cell-permeable cAMP analog) and 3-isobutyl-1-methylxanthine (1 mM, an inhibitor of phosphodiesterases). The capsaicin-induced inhibition of I(SC) was attributed to suppression of bumetanide (an inhibitor of the basolateral Na(+)-K(+)-2 Cl(-) cotransporter 1)- and 4,4'-dinitrostilbene-2,2'-disulfonic acid (an inhibitor of basolateral HCO(3)(-)-dependent anion transporters)-sensitive components, which reflect anion uptake via basolateral cAMP-dependent anion transporters. In contrast, capsaicin potentiated apical Cl(-) conductance, which reflects conductivity through the cystic fibrosis transmembrane conductance regulator, a cAMP-regulated Cl(-) channel. All these paradoxical effects of capsaicin were mimicked by capsazepine. Forskolin application also increased phosphorylated myosin phosphatase target subunit 1, and the phosphorylation was prevented by capsaicin and capsazepine, suggesting that these capsaicinoids assume aspects of Rho kinase inhibitors. We also found that the increments in apical Cl(-) conductance were caused by conventional Rho kinase inhibitors, Y-27632 (20 μM) and HA-1077 (20 μM), with selective inhibition of basolateral Na(+)-K(+)-2 Cl(-) cotransporter 1. Collectively, capsaicinoids inhibit cAMP-mediated anion transport through down-regulation of basolateral anion uptake, paradoxically accompanied by up-regulation of apical cystic fibrosis transmembrane conductance regulator-mediated anion conductance. The latter is mediated by inhibition of Rho

  20. Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells.

    PubMed Central

    Gard, D L; Lazarides, E

    1982-01-01

    The intermediate filament proteins desmin and vimentin and the muscle tropomyosins were the major protein phosphate acceptors in 8-day-old myotubes incubated for 4 h in medium containing radiolabeled phosphate. The addition of isoproterenol or 8-bromo-cyclic AMP (BrcAMP) resulted in a two- to threefold increase in incorporation of 32PO4 into both desmin and vimentin, whereas no changes in the incorporation of 32PO4 into tropomyosin or other cellular proteins were observed. The BrcAMP- or hormonally induced increase in 32PO4 incorporation into desmin and vimentin was independent of protein synthesis and was not caused by stimulation of protein phosphate turnover. In addition, BrcAMP did not induce significant changes in the specific activity of the cellular ATP pool. These data suggest that the observed increase in 32PO4 incorporation represented an actual increase in phosphorylation of the intermediate filament proteins desmin and vimentin. Two-dimensional tryptic analysis of desmin from 8-day-old myotubes revealed five phosphopeptides of which two showed a 7- to 10-fold increase in 32PO4 incorporation in BrcAMP-treated myotubes. Four of the phosphopeptides identified in desmin labeled in vivo were also observed in desmin phosphorylated in vitro by bovine heart cAMP-dependent protein kinase. Although phosphorylation of desmin and vimentin was apparent in myogenic cells at all stages of differentiation, BrcAMP- and isoproterenol-induced increases in phosphorylation of these proteins were restricted to mature myotubes. These data strongly suggest that in vivo phosphorylation of the intermediate filament proteins desmin and vimentin is catalyzed by the cAMP-dependent protein kinases and that such phosphorylation may be regulated during muscle differentiation. Images PMID:6294504

  1. The tib adherence locus of enterotoxigenic Escherichia coli is regulated by cyclic AMP receptor protein.

    PubMed

    Espert, Shirley M; Elsinghorst, Eric A; Munson, George P

    2011-03-01

    Enterotoxigenic Escherichia coli (ETEC) is a Gram-negative enteric pathogen that causes profuse watery diarrhea through the elaboration of heat-labile and/or heat-stable toxins. Virulence is also dependent upon the expression of adhesive pili and afimbrial adhesins that allow the pathogen to adhere to the intestinal epithelium or mucosa. Both types of enterotoxins are regulated at the level of transcription by cyclic AMP (cAMP) receptor protein (CRP). To further our understanding of virulence gene regulation, an in silico approach was used to identify putative CRP binding sites in the genome of H10407 (O78:H11), an ETEC strain that was originally isolated from the stool of a Bangledeshi patient with cholera-like symptoms circa 1971. One of the predicted binding sites was located within an intergenic region upstream of tibDBCA. TibA is an autotransporter and afimbrial adhesin that is glycosylated by TibC. Expression of the TibA glycoprotein was abolished in an H10407 crp mutant and restored when crp was provided in trans. TibA-dependent aggregation was also abolished in a cyaA::kan strain and restored by addition of exogenous cAMP to the growth medium. DNase I footprinting confirmed that the predicted site upstream of tibDBCA is bound by CRP. Point mutations within the CRP binding site were found to abolish or significantly impair CRP-dependent activation of the tibDB promoter. Thus, these studies demonstrate that CRP positively regulates the expression of the glycosylated afimbrial adhesin TibA through occupancy of a binding site within tibDBp. PMID:21216994

  2. Transcriptional regulation of 2',3'-cyclic nucleotide 3'-phosphodiesterase gene expression by cyclic AMP in C6 cells.

    PubMed

    Gravel, M; Gao, E; Hervouet-Zeiber, C; Parsons, V; Braun, P E

    2000-11-01

    It was recently shown that the two transcripts encoding the isoforms of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP1 and CNP2) are differentially regulated during the process of oligodendrocyte maturation. In oligodendrocyte precursors, only CNP2 mRNA is present, whereas in differentiating oligodendrocytes, both CNP1 and CNP2 mRNAs are expressed. This pattern of CNP expression is likely due to stage-specific transcriptional regulation of the two CNP promoters during the process of oligodendrocyte differentiation. Here, we report the influence of increased intracellular cyclic AMP (cAMP) levels on the transcription of both CNP1 and CNP2 mRNAs in rat C6 glioma cells. We found that the transcription of CNP1 mRNA was significantly increased in comparison with that of CNP2 mRNA in cells treated with cAMP analogues to elevate intracellular cAMP levels. This up-regulation of CNP1 expression (a) is due to an increase of transcription, (b) requires de novo protein synthesis, and (c) requires the activity of protein kinase A. These results are physiologically significant and support the idea that a cAMP-mediated pathway is part of the molecular mechanisms regulating the expression of CNP1 in oligodendrocytes. The regulation of CNP1 promoter activity by cAMP was then investigated in stably transfected C6 cell lines containing various deletions of the CNP promoter directing the bacterial chloramphenicol acetyltransferase gene. We showed that the sequence between nucleotides -126 and -102 was essential for the cAMP-dependent induction of CNP1 expression. Gel retardation analysis showed that two protein-DNA complexes are formed between this sequence and nuclear factors from C6 cells treated or not treated with cAMP. This suggests that the induction of CNP1 mRNA transcription is not mediated by changes in binding of nuclear factors that interact directly with the -126/-102 sequence. Sequence analysis of this region revealed the presence of a putative activator protein-2 (AP

  3. Nucleotide sequences of fic and fic-1 genes involved in cell filamentation induced by cyclic AMP in Escherichia coli.

    PubMed Central

    Kawamukai, M; Matsuda, H; Fujii, W; Utsumi, R; Komano, T

    1989-01-01

    The nucleotide sequences of fic-1 involved in the cell filamentation induced by cyclic AMP in Escherichia coli and its normal counterpart fic were analyzed. The open reading frame of both fic-1 and fic coded for 200 amino acids. The Gly at position 55 in the Fic protein was changed to Arg in the Fic-1 protein. The promoter activity of fic was confirmed by fusing fic and lacZ. The gene downstream from fic was found to be pabA (p-aminobenzoate). There is an open reading frame (ORF190) coding for 190 amino acids upstream from the fic gene. Computer-assisted analysis showed that Fic has sequence similarity with part of CDC28 of Saccharomyces cerevisiae, CDC2 of Schizosaccharomyces pombe, and FtsA of E. coli. In addition, ORF190 has sequence similarity with the cyclosporin A-binding protein cyclophilin. PMID:2546924

  4. Cyclic-AMP-dependent protein kinase A regulates apoptosis by stabilizing the BH3-only protein Bim.

    PubMed

    Moujalled, Diane; Weston, Ross; Anderton, Holly; Ninnis, Robert; Goel, Pranay; Coley, Andrew; Huang, David C S; Wu, Li; Strasser, Andreas; Puthalakath, Hamsa

    2011-01-01

    The proapoptotic Bcl2 homology domain 3(BH3)-only protein Bim is controlled by stringent post-translational regulation, predominantly through alterations in phosphorylation status. To identify new kinases involved in its regulation, we carried out a yeast two-hybrid screen using a non-spliceable variant of the predominant isoform--Bim(EL)--as the bait and identified the regulatory subunit of cyclic-AMP-dependent protein kinase A--PRKAR1A--as an interacting partner. We also show that protein kinase A (PKA) is a Bim(EL) isoform-specific kinase that promotes its stabilization. Inhibition of PKA or mutation of the PKA phosphorylation site within Bim(EL) resulted in its accelerated proteasome-dependent degradation. These results might have implications for human diseases that are characterized by abnormally increased PKA activity, such as the Carney complex and dilated cardiomyopathy. PMID:21151042

  5. Glucagon, cyclic AMP and adrenaline stimulate the degradation of low-density lipoprotein by cultured rat hepatocytes.

    PubMed Central

    Brown, N F; Salter, A M; Fears, R; Brindley, D N

    1989-01-01

    Rat hepatocytes were preincubated for 16 h with hormones or drugs and then for a further 8 h with 125I-human low-density lipoprotein (LDL). Glucagon (via cyclic AMP) and adrenaline (via cyclic AMP and alpha-effects) increased the binding of 125I-LDL to the LDL receptor, and the degradation of LDL to [125I]iodotyrosine. The effects on degradation were antagonized by dexamethasone, and the action of cyclic AMP on binding and degradation was inhibited by actinomycin D. The results are discussed in relation to the control of lipoprotein metabolism in diabetes. PMID:2552996

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

    NASA Technical Reports Server (NTRS)

    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. Second messenger-dependent protein kinases and protein synthesis regulate endogenous secretin receptor responsiveness

    PubMed Central

    Ghadessy, Roxana S; Kelly, Eamonn

    2002-01-01

    The present study investigated the role of second messenger-dependent protein kinase A (PKA) and C (PKC) in the regulation of endogenous secretin receptor responsiveness in NG108-15 mouse neuroblastoma×rat glioma hybrid cells. In whole cell cyclic AMP accumulation studies, activation of PKC either by phorbol 12-myristate 13-acetate (PMA) or by purinoceptor stimulation using uridine 5′-triphosphate (UTP) decreased secretin receptor responsiveness. PKC activation also inhibited forskolin-stimulated cyclic AMP accumulation but did not affect cyclic AMP responses mediated by the prostanoid-IP receptor agonist iloprost, or the A2 adenosine receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA). In additivity experiments, saturating concentrations of secretin and iloprost were found to be additive in terms of cyclic AMP accumulation, whereas saturating concentrations of NECA and iloprost together were not. This suggests compartmentalization of Gs-coupling components in NG108-15 cells and possible heterologous regulation of secretin receptor responsiveness at the level of adenylyl cyclase activation. Cells exposed to the PKA inhibitor H-89, exhibited a time-dependent increase in secretin receptor responsiveness compared to control cells. This effect was selective since cyclic AMP responses to forskolin, iloprost and NECA were not affected by H-89 treatment. Furthermore, treatment with the protein synthesis inhibitor cycloheximide produced a time-dependent increase in secretin receptor responsiveness. Together these results indicate that endogenous secretin receptor responsiveness is regulated by PKC, PKA and protein neosynthesis in NG108-15 cells. PMID:11959806

  8. Endogenous expression of histamine H1 receptors functionally coupled to phosphoinositide hydrolysis in C6 glioma cells: regulation by cyclic AMP.

    PubMed Central

    Peakman, M C; Hill, S J

    1994-01-01

    1. The effects of histamine receptor agonists and antagonists on phospholipid hydrolysis in rat-derived C6 glioma cells have been investigated. 2. Histamine H1 receptor-stimulation caused a concentration-dependent increase in the accumulation of total [3H]-inositol phosphates in cells prelabelled with [3H]-myo-inositol. The rank order of agonist potencies was histamine (EC50 = 24 microM) > N alpha-methylhistamine (EC50 = 31 microM) > 2-thiazolylethylamine (EC50 = 91 microM). 3. The response to 0.1 mM histamine was antagonized in a concentration-dependent manner by the H1-antagonists, mepyramine (apparent Kd = 1 nM) and (+)-chlorpheniramine (apparent Kd = 4 nM). In addition, (-)-chlorpheniramine was more than two orders of magnitude less potent than its (+)-stereoisomer. 4. Elevation of intracellular cyclic AMP accumulation with forskolin (10 microM, EC50 = 0.3 microM), isoprenaline (1 microM, EC50 = 4 nM) or rolipram (0.5 mM), significantly reduced the histamine-mediated (0.1 mM) inositol phosphate response by 37%, 43% and 26% respectively. In contrast, 1,9-dideoxyforskolin did not increase cyclic AMP accumulation and had no effect on the phosphoinositide response to histamine. 5. These data indicate the presence of functionally coupled, endogenous histamine H1 receptors in C6 glioma cells. Furthermore, the results also indicate that H1 receptor-mediated phospholipid hydrolysis is inhibited by the elevation of cyclic AMP levels in these cells. PMID:7889313

  9. Regulation of hippocampus-dependent memory by cyclic AMP-dependent protein kinase

    PubMed Central

    Abel, Ted; Nguyen, Peter V.

    2010-01-01

    The hippocampus is crucial for the consolidation of new declarative long-term memories. Genetic and behavioral experimentation have revealed that several protein kinases are critical for the formation of hippocampus-dependent long-term memories. Cyclic-AMP dependent protein kinase (PKA) is a serine–threonine kinase that has been strongly implicated in the expression of specific forms of hippocampus-dependent memory. We review evidence that PKA is required for hippocampus-dependent memory in mammals, and we highlight some of the proteins that have been implicated as targets of PKA. Future directions and open questions regarding the role of PKA in memory storage are also described. PMID:18394470

  10. Mechanisms of cyclic AMP/protein kinase A- and glucocorticoid-mediated apoptosis using S49 lymphoma cells as a model system

    PubMed Central

    Keshwani, Malik M.; Kanter, Joan R.; Ma, Yuliang; Wilderman, Andrea; Darshi, Manjula; Insel, Paul A.; Taylor, Susan S.

    2015-01-01

    Cyclic AMP/protein kinase A (cAMP/PKA) and glucocorticoids promote the death of many cell types, including cells of hematopoietic origin. In wild-type (WT) S49 T-lymphoma cells, signaling by cAMP and glucocorticoids converges on the induction of the proapoptotic B-cell lymphoma-family protein Bim to produce mitochondria-dependent apoptosis. Kin–, a clonal variant of WT S49 cells, lacks PKA catalytic (PKA-Cα) activity and is resistant to cAMP-mediated apoptosis. Using sorbitol density gradient fractionation, we show here that in kin– S49 cells PKA-Cα is not only depleted but the residual PKA-Cα mislocalizes to heavier cell fractions and is not phosphorylated at two conserved residues (Ser338 or Thr197). In WT S49 cells, PKA-regulatory subunit I (RI) and Bim coimmunoprecipitate upon treatment with cAMP analogs and forskolin (which increases endogenous cAMP concentrations). By contrast, in kin– cells, expression of PKA-RIα and Bim is prominently decreased, and increases in cAMP do not increase Bim expression. Even so, kin– cells undergo apoptosis in response to treatment with the glucocorticoid dexamethasone (Dex). In WT cells, glucorticoid-mediated apoptosis involves an increase in Bim, but in kin– cells, Dex-promoted cell death appears to occur by a caspase 3-independent apoptosis-inducing factor pathway. Thus, although cAMP/PKA-Cα and PKA-R1α/Bim mediate apoptotic cell death in WT S49 cells, kin– cells resist this response because of lower levels of PKA-Cα and PKA-RIα subunits as well as Bim. The findings for Dex-promoted apoptosis imply that these lymphoma cells have adapted to selective pressure that promotes cell death by altering canonical signaling pathways. PMID:26417071

  11. Effects of granulocyte-macrophage colony-stimulating factor and cyclic AMP interaction on human neutrophil apoptosis.

    PubMed Central

    Tortorella, C; Piazzolla, G; Spaccavento, F; Antonaci, S

    1998-01-01

    The current study was undertaken to evaluate the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and cyclic AMP (cAMP) signaling interaction on human neutrophil apoptosis, either occurring spontaneously or induced by Fas antigen activation. Results show that GM-CSF, dibutyryl cAMP (a cAMP analog) and forskolin (an adenylate cyclase activator) are all able to suppress spontaneous neutrophil cell death. Of note however, when GM-CSF is used in combination with cAMP-elevating agents, an additive effect on neutrophil survival is observed with dibutyryl cAMP only, whereas supplementation of cell cultures with GM-CSF and forskolin results in a progressive reduction of antiapoptotic effects exerted by the single compounds. Moreover, although dibutyryl cAMP and forskolin do not affect Fas-triggered apoptotic events, they are still able to modulate the GM-CSF capacity to prolong neutrophil survival following anti-Fas IgM cell challenge, with effects similar to those respectively exerted on spontaneous neutrophil apoptosis. The data indicate that GM-CSF may negatively modulate the cAMP-mediated antiapoptotic pathway in human neutrophils, likely via the inhibition of adenylate cyclase activity. This would prevent an abnormal neutrophil survival as a result of cAMP signaling stimulation, which provides a novel insight into the role of GM-CSF as a physiological regulator of myeloid cell turnover. PMID:9927231

  12. Comparison of indolidan analog binding sites of drug antibody and sarcoplasmic reticulum with inhibition of cyclic AMP phosphodiesterase.

    PubMed

    Ashikaga, T; Robertson, D W; Sportsman, R J; Strada, S J; Thompson, W J

    1996-01-01

    Dihydropyridazinone(DHP) derivatives such as indolidan are positive inotropic agents that show inhibition of cyclic AMP phosphodiesterase(PDE) activity. Indolidan inhibition is selective for PDE3 among the seven PDE gene families. DHP derivatives and related analogs have been used to define critical regions of the active site of PDE3 isoforms and radiolabeled analogs have been used to define indolidan sarcoplasmic reticulum (SR) receptor sites. We report here studies comparing the structure-activity relationships (SAR) for PDE3 inhibition with indolidan binding to two types of sites: canine SR and a monoclonal antibody derived against indolidan conjugated to a hemocyanin. SR and monoclonal antibody binding both fit singlesite, high affinity models (IC50 = 1.2 and 62 nM) that were near 52 and 360 times that of SR PDE3. Indolidan and thirteen analogs showed similar competition with either SR 3H-LY186126 binding or SR PDE3 inhibition. Antibody binding maintained selectivity but showed a different rank order potency for SR binding. Indole ring C3 methylation increased and DHP ring C4' methylation decreased indolidan monoclonal antibody binding while both substitutions increased SR binding. These studies support the hypothesis that SR PDE3 is a cardiotonic receptor site in myocardial membranes and indicate that models of the structural features of binding sites derived from inhibitor data alone could produce models with limited topography relative to the natural ligand. PMID:8968964

  13. Improvement on the competitive binding assay for the measurement of cyclic AMP by using ammonium sulphate precipitation.

    PubMed Central

    Santa-Coloma, T A; Bley, M A; Charreau, E H

    1987-01-01

    The protein-binding assay developed by Brown, Albano, Ekins, Sgherzi & Tampion [(1971) Biochem. J. 121, 561-562] and Brown, Ekins & Albano [(1972) Adv. Cyclic Nucleotide Res. 2, 25-40] was modified by using precipitation with (NH4)2SO4 of the protein-cyclic AMP complex instead of adsorption of the free nucleotide on charcoal. The half-life of the protein-cyclic AMP complex obtained in the presence of charcoal was lower than that of the (NH4)2SO4-precipitated complex. In consequence, owing to the great stability of the precipitated protein-cyclic AMP complex, this method allows more accurate and reproducible determinations. PMID:2822033

  14. Characterization of soluble cyclic AMP phosphodiesterases and partial purification of a major form in human leiomyoma of the uterus.

    PubMed

    Robinson, M F; Levin, J; Savage, N

    1987-01-01

    Human leiomyoma of the uterus contained seven forms of cyclic AMP phosphodiesterase in the crude cytosol as revealed by a specific activity stain on non-denaturing polyacrylamide gel electrophoresis. The enzymes from human myometrium and normal uterus showed an identical activity pattern. Ferguson plot analysis showed four different molecular weight species of Mr 229,000 +/- 4,000, 186,000 +/- 4,000, 174,000 +/- 4,000 and 162,000 +/- 4,000. The Mr 174,000 species comprised four differently charged forms. Sucrose density gradient centrifugation of the crude cytosol revealed the presence of three molecular weight species sedimenting at 11.8S, 8.1S and 3.6S. The Michaelis constant (Km) for the band 1 form which displayed linear kinetics was 5 microM and the band 2 form which produced non-linear kinetics had Km values of 5.8 and 37 microM. PMID:2820644

  15. Calcium channel ligand binding to intact, concanavalin A and cyclic AMP-treated cells of the immune system.

    PubMed

    Pinchuk, G V; Pinchuk, L N; Tkachenko, Y V; Rudenko, A E

    1990-12-01

    To examine whether the cells of immune system express calcium channel-forming proteins, we studied the binding of calcium channel ligands, known to detect certain types of the above channels in excitable tissues, to murine splenic and human peripheral blood mononuclear cells. Specific (i.e., displaceable by excess cold ligand) binding of the 3H-labelled dihydropyridine drugs PN200-110 and nitrendipine, was not detected in these cells. Specific binding of a phenylalkylamine drug, [3H]verapamil, was detected, but cannot be attributed to the existence of certain specialized receptors, since multiple [3H]verapamil binding sites (about 10(6) per cell) appeared to be occupied. [3H]Verapamil binding to murine splenic mononuclear cells was inhibited following exposure to either the polyclonal T-cell activator, concanavalin A, or a cell-permeable analogue of the second messenger, cyclic AMP, suggesting that processes of lymphocyte activation and/or intracellular signalling may down-modulate at least some of calcium channel ligand binding sites. PMID:1964929

  16. REVIEW: Role of cyclic AMP signaling in the production and function of the incretin hormone glucagon-like peptide-1

    NASA Astrophysics Data System (ADS)

    Yu, Zhiwen; Jin, Tianru

    2008-01-01

    Pancreatic cells express the proglucagon gene (gcg) and thereby produce the peptide hormone glucagon, which stimulates hepatic glucose production and thereby increases blood glucose levels. The same gcg gene is also expressed in the intestinal endocrine L cells and certain neural cells in the brain. In the gut, gcg expression leads to the production of glucagon-like peptide-1 (GLP-1). This incretin hormone stimulates insulin secretion when blood glucose level is high. In addition, GLP-1 stimulates pancreatic cell proliferation, inhibits cell apoptosis, and has been utilized in the trans-differentiation of insulin producing cells. Today, a long-term effective GLP-1 receptor agonist has been developed as a drug in treating diabetes and potentially other metabolic disorders. Extensive investigations have shown that the expression of gcg and the production of GLP-1 can be activated by the elevation of the second messenger cyclic AMP (cAMP). Recent studies suggest that in addition to protein kinase A (PKA), exchange protein activated by cAMP (Epac), another effector of cAMP signaling, and the crosstalk between PKA and Wnt signaling pathway, are also involved in cAMP-stimulated gcg expression and GLP-1 production. Furthermore, functions of GLP-1 in pancreatic cells are mainly mediated by cAMP-PKA, cAMP-Epac and Wnt signaling pathways as well.

  17. Cyclic AMP-elevating agents prolong or inhibit eosinophil survival depending on prior exposure to GM-CSF.

    PubMed Central

    Hallsworth, M. P.; Giembycz, M. A.; Barnes, P. J.; Lee, T. H.

    1996-01-01

    1. Purified human eosinophils survived for up to 7 days when cultured in vitro in the presence of 1 ng ml-1 granulocyte-macrophage colony stimulating factor (GM-CSF) with a viability of 73%. In the absence of GM-CSF, eosinophil viability decreased after one day in culture, and only 4% of cells were viable by day 4. 2. Culture of eosinophils with cholera toxin produced a concentration-dependent decrease in GM-CSF-induced survival at 7 days (IC50 = 7 ng ml-1) which was associated with a 6 fold increase in the intracellular cyclic AMP concentration. This inhibition of cell survival could be prevented by the addition of the protein kinase A inhibitor, H89 (10(-6)M). 3. When eosinophils were cultured with dibutyryl cyclic AMP, there was a concentration-dependent inhibition of GM-CSF-induced survival at 7 days with an IC50 of 200 microM. The related cyclic nucleotide analogue, dibutyryl cyclic GMP did not inhibit GM-CSF-induced eosinophil survival over the same concentration range. 4. Culture of eosinophils with forskolin, or with the phosphodiesterase inhibitors, rolipram and SK&F94120, had no effect on GM-CSF-induced eosinophil survival at any concentration examined. 5. After 7 days' culture in the absence of GM-CSF, fractionation of eosinophil DNA on agarose gels demonstrated a 'ladder' pattern characteristic of apoptosis. GM-CSF prevented DNA fragmentation and this protection could be overcome by both cholera toxin and dibutyryl cyclic AMP. 6. GM-CSF did not affect intracellular cyclic AMP concentrations in unstimulated eosinophils or in cells stimulated by cholera toxin. Thus, GM-CSF does not apparently increase eosinophil survival by affecting cyclic AMP levels. 7. In the absence of GM-CSF both cholera toxin and dibutyryl cyclic AMP decreased the rate of eosinophil death, when compared to cells cultured with medium alone. The t1/2 values for cell death were 1.63 +/- 0.3, 2.46 +/- 0.3 and 4.62 +/- 1.0 days for cells cultured in the presence of medium, cholera toxin

  18. Cyclic AMP-dependent protein kinase (cAPK) regulatory subunits are packaged and secreted by many exocrine and endocrine cells

    SciTech Connect

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

    1986-05-01

    Regulatory (R) subunits of cAPK were identified by us as components of rat and human saliva by photoaffinity labeling with (/sup 32/P)-8-azido cyclic AMP. Photoaffinity labeling of purified rat parotid granule contents and immunogold labeling of thin sections with monoclonal antibodies showed the presence of R subunits in granules. The authors now report that cAPK R subunits are present in secretory granules and are apparently secreted by many exocrine and endocrine cell types. Labeling of thin sections of rat tissues with antibody to R subunits and protein A-gold shows gold particles over secretory granules of endocrine cells of the pituitary, pancreas and intestine. Zymogen granules of exocrine pancreatic acinar cells, the dense cores of secretory granules of seminal vesicle epithelial cells and secretory product in the seminal vesicle lumina were prominently labeled with gold. Photoaffinity labeling shows that pancreatic secretions and seminal vesicle contents have cAPK components. Phosphorylative modification of cellular proteins by cAMP controls hormonally stimulated protein secretion by many cell types. Although no catalytic activity was detected, identification of R subunits in granules and as secretory products indicates that they may have multiple roles in cellular mechanisms of action of cyclic AMP-mediated events in secretory cells.

  19. Inducible cyclic AMP early repressor produces reactivation of latent herpes simplex virus type 1 in neurons in vitro.

    PubMed

    Colgin, M A; Smith, R L; Wilcox, C L

    2001-03-01

    Herpes simplex virus type 1 (HSV-1) establishes a latent infection in neurons of the peripheral nervous system. During latent HSV-1 infection, viral gene expression is limited to latency-associated transcripts (LAT). HSV-1 remains latent until an unknown mechanism induces reactivation. The ability of the latent virus to periodically reactivate and be shed is essential to the transmission of disease. In vivo, the stimuli that induce reactivation of latent HSV-1 include stress, fever, and UV damage to the skin at the site of initial infection. In vitro, in primary neurons harboring latent HSV-1, nerve growth factor (NGF) deprivation or forskolin treatment induces reactivation. However, the mechanism involved in the induction of reactivation remains poorly understood. An in vitro neuronal model of HSV-1 latency was used to investigate potential mechanisms involved in the induction of reactivation of latent HSV-1. In situ hybridization analysis of neuronal cultures harboring latent HSV-1 showed a marked, rapid decrease in the percentage of LAT-positive neurons following induction of reactivation by NGF deprivation or forskolin treatment. Western blot analysis showed a corresponding increase in expression of the cellular transcription factor inducible cyclic AMP early repressor (ICER) during reactivation. In transient-transfection assays, ICER downregulated LAT promoter activity. Expression of ICER from a recombinant adenoviral vector induced reactivation and decreased the percentage of LAT-positive neurons in neuronal cultures harboring latent HSV-1. These results indicate that ICER represses LAT expression and induces reactivation of latent HSV-1. PMID:11222716

  20. Roles of Intracellular Cyclic AMP Signal Transduction in the Capacitation and Subsequent Hyperactivation of Mouse and Boar Spermatozoa

    PubMed Central

    HARAYAMA, Hiroshi

    2013-01-01

    It is not until accomplishment of a variety of molecular changes during the transit through the female reproductive tract that mammalian spermatozoa are capable of exhibiting highly activated motility with asymmetric whiplash beating of the flagella (hyperactivation) and undergoing acrosomal exocytosis in the head (acrosome reaction). These molecular changes of the spermatozoa are collectively termed capacitation and promoted by bicarbonate, calcium and cholesterol acceptors. Such capacitation-promoting factors can stimulate intracellular cyclic AMP (cAMP) signal transduction in the spermatozoa. Meanwhile, hyperactivation and the acrosome reaction are essential to sperm fertilization with oocytes and are apparently triggered by a sufficient increase of intracellular Ca2+ in the sperm flagellum and head, respectively. Thus, it is necessary to investigate the relationship between cAMP signal transduction and calcium signaling cascades in the spermatozoa for the purpose of understanding the molecular basis of capacitation. In this review, I cover updated insights regarding intracellular cAMP signal transduction, the acrosome reaction and flagellar motility in mammalian spermatozoa and then account for possible roles of intracellular cAMP signal transduction in the capacitation and subsequent hyperactivation of mouse and boar spermatozoa. PMID:24162806

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

    PubMed

    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. PMID:7971267

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

    PubMed Central

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

    1994-01-01

    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. Images PMID:7971267

  3. Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3′, 3′-cGAMP)

    PubMed Central

    Hallberg, Zachary F.; Wang, Xin C.; Wright, Todd A.; Nan, Beiyan; Ad, Omer; Yeo, Jongchan; Hammond, Ming C.

    2016-01-01

    Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3′, 3′-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling. PMID:26839412

  4. In vitro and in vivo characterization of the Pseudomonas aeruginosa cyclic AMP (cAMP) phosphodiesterase CpdA, required for cAMP homeostasis and virulence factor regulation.

    PubMed

    Fuchs, Erin L; Brutinel, Evan D; Klem, Erich R; Fehr, Anthony R; Yahr, Timothy L; Wolfgang, Matthew C

    2010-06-01

    Cyclic AMP (cAMP) is an important second messenger signaling molecule that controls a wide variety of eukaryotic and prokaryotic responses to extracellular cues. For cAMP-dependent signaling pathways to be effective, the intracellular cAMP concentration is tightly controlled at the level of synthesis and degradation. In the opportunistic human pathogen Pseudomonas aeruginosa, cAMP is a key regulator of virulence gene expression. To better understand the role of cAMP homeostasis in this organism, we identified and characterized the enzyme CpdA, a putative cAMP phosphodiesterase. We demonstrate that CpdA possesses 3',5'-cAMP phosphodiesterase activity in vitro and that it utilizes an iron-dependent catalytic mechanism. Deletion of cpdA results in the accumulation of intracellular cAMP and altered regulation of P. aeruginosa virulence traits. Further, we demonstrate that the cAMP-dependent transcription factor Vfr directly regulates cpdA expression in response to intracellular cAMP accumulation, thus providing a feedback mechanism for controlling cAMP levels and fine-tuning virulence factor expression. PMID:20348254

  5. Prostaglandin E2 inhibits apoptosis in human neutrophilic polymorphonuclear leukocytes: role of intracellular cyclic AMP levels.

    PubMed

    Ottonello, L; Gonella, R; Dapino, P; Sacchetti, C; Dallegri, F

    1998-08-01

    Human neutrophilic polymorphonuclear leukocytes (neutrophils) are terminally differentiated cells that die by undergoing apoptosis. At present, the intracellular pathways governing this process are only partially known. In particular, although the adenylate cyclase-dependent generation of cyclic AMP (cAMP) has been implicated in the triggering of apoptosis in lymphoid cells, the role of the intracellular cAMP pathway in neutrophil apoptosis remains controversial. In the present study, we found that two cAMP-elevating agents, prostaglandin E2 (PGE2) and the phosphodiesterase type IV inhibitor RO 20-1724, inhibit neutrophil apoptosis without inducing cell necrosis. When administered in combination, PGE2 and RO 20-1724 displayed additive effects. Moreover, neutrophil apoptosis was inhibited by a membrane-permeable analog of cAMP, dibutyryl-cAMP, in a dose-dependent manner. Finally, treatment of neutrophils with the protein kinase A inhibitor H-89 prevented PGE2- and RO 20-1724-induced inhibition of cell apoptosis. In conclusion, taking into account that PGE2 and other cAMP-elevating agents are well known downregulators of neutrophil functions, our results suggest that conditions favoring a state of functional rest, such as intracellular cAMP elevation, prolong the life span of neutrophils by delaying apoptosis. PMID:9694511

  6. GEMM-I riboswitches from Geobacter sense the bacterial second messenger cyclic AMP-GMP

    PubMed Central

    Kellenberger, Colleen A.; Wilson, Stephen C.; Hickey, Scott F.; Gonzalez, Tania L.; Su, Yichi; Hallberg, Zachary F.; Brewer, Thomas F.; Iavarone, Anthony T.; Carlson, Hans K.; Hsieh, Yu-Fang; Hammond, Ming C.

    2015-01-01

    Cyclic dinucleotides are an expanding class of signaling molecules that control many aspects of bacterial physiology. A synthase for cyclic AMP-GMP (cAG, also referenced as 3′-5′, 3′-5′ cGAMP) called DncV is associated with hyperinfectivity of Vibrio cholerae but has not been found in many bacteria, raising questions about the prevalence and function of cAG signaling. We have discovered that the environmental bacterium Geobacter sulfurreducens produces cAG and uses a subset of GEMM-I class riboswitches (GEMM-Ib, Genes for the Environment, Membranes, and Motility) as specific receptors for cAG. GEMM-Ib riboswitches regulate genes associated with extracellular electron transfer; thus cAG signaling may control aspects of bacterial electrophysiology. These findings expand the role of cAG beyond organisms that harbor DncV and beyond pathogenesis to microbial geochemistry, which is important to environmental remediation and microbial fuel cell development. Finally, we have developed an RNA-based fluorescent biosensor for live-cell imaging of cAG. This selective, genetically encodable biosensor will be useful to probe the biochemistry and cell biology of cAG signaling in diverse bacteria. PMID:25848022

  7. Role of insulin during exercise-induced glycogenesis in muscle: effect on cyclic AMP.

    PubMed

    Ivy, J L

    1977-12-01

    Skeletal muscle cyclic AMP (cAMP) content and glycogen synthesis were investigated in male rats subjected to exhaustive exercise, alloxan diabetes, and combinations of these conditions. After an exhaustive swim or control treatment of wading, randomly selected animals were administered 500 mg glucose via stomach tube. Two hours after glucose administration, gastrocnemius glycogen levels rose from 1.31 to 10.67 mg/g wet wt in fatigued nondiabetics (FND), producing a 94% supercompensation above control values. Glycogen of fatigued diabetics (FD) increased from 0.88 to 4.21 mg/g wet wt during the first 2 hr after glucose administration and did not reach control values for 24 h. In conjunction with these glycogen changes, cAMP increased from 1.23 to 2.59 and 1.47 to 2.81 pmol/mg wet wt for FND and FD, respectively (P less than 0.05). No difference in cAMP levels between diabetics and nondiabetics was found. These in vivo data suggest that insulin may not be essential for muscle glycogen synthesis, but that after glycogen depletion it plays a prominent role in supercompensation. Also, this hormone's mechanism of action in skeletal muscle does not appear to be mediated through alteration in the tissue cAMP concentration. PMID:202169

  8. Glucocorticoid Receptor, C/EBP, HNF3, and Protein Kinase A Coordinately Activate the Glucocorticoid Response Unit of the Carbamoylphosphate Synthetase I Gene

    PubMed Central

    Christoffels, Vincent M.; Grange, Thierry; Kaestner, Klaus H.; Cole, Timothy J.; Darlington, Gretchen J.; Croniger, Colleen M.; Lamers, Wouter H.

    1998-01-01

    A single far-upstream enhancer is sufficient to confer hepatocyte-specific, glucocorticoid- and cyclic AMP-inducible periportal expression to the carbamoylphosphate synthetase I (CPS) gene. To identify the mechanism of hormone-dependent activation, the composition and function of the enhancer have been analyzed. DNase I protection and gel mobility shift assays revealed the presence of a cyclic AMP response element, a glucocorticoid response element (GRE), and several sites for the liver-enriched transcription factor families HNF3 and C/EBP. The in vivo relevance of the transcription factors interacting with the enhancer in the regulation of CPS expression in the liver was assessed by the analysis of knockout mice. A strong reduction of CPS mRNA levels was observed in glucocorticoid receptor- and C/EBPα-deficient mice, whereas the CPS mRNA was normally expressed in C/EBPβ knockout mice and in HNF3α and -γ double-knockout mice. (The role of HNFβ could not be assessed, because the corresponding knockout mice die at embryonic day 10). In hepatoma cells, most of the activity of the enhancer is contained within a 103-bp fragment, which depends for its activity on the simultaneous occupation of the GRE, HNF3, and C/EBP sites, thus meeting the requirement of a glucocorticoid response unit. In fibroblast-like CHO cells, on the other hand, the GRE in the CPS enhancer does not cooperate with the C/EBP and HNF3 elements in transactivation of the CPS promoter. In both hepatoma and CHO cells, stimulation of expression by cyclic AMP depends mainly on the integrity of the glucocorticoid pathway, demonstrating cross talk between this pathway and the cyclic AMP (protein kinase A) pathway. PMID:9774647

  9. Roles of intracellular Ca2+ and cyclic AMP in mast cell histamine release induced by radiographic contrast media.

    PubMed

    Saito, Mami; Itoh, Yoshinori; Yano, Takahisa; Sendo, Toshiaki; Goromaru, Takeshi; Sakai, Naoko; Oishi, Ryozo

    2003-04-01

    Mast cell histamine release is considered to be associated with the etiology of anaphylactoid reactions to iodinated radiographic contrast media (RCM). In the present study, the effects of various ionic and non-ionic RCM on histamine release from mast cells were compared, and the possible mechanisms of the histamine release were subsequently determined. Both ionic (ioxaglate and amidotrizoate) and non-ionic (iohexol, ioversol, iomeprol, iopamidol and iotrolan) RCM increased histamine release from the dissociated rat pulmonary cells, whereby ionic materials were more potent than non-ionic agents. There was no significant correlation between the extent of histamine release and the osmolarity of each RCM solution. In addition, hyperosmotic mannitol solution (1000 mOsm/kg) caused no marked histamine release. Thus, it is unlikely that the hyperosmolarity of RCM solutions contributes to the histamine release. RCM also stimulated, but to a lesser extent, the histamine release from rat peritoneal cells. The RCM-induced histamine release from both types of cells was inhibited by dibutyl cyclic AMP or combined treatment with forskolin and 3-isobutyl-1-methylxanthine. Corresponding to these results, RCM markedly reduced the cellular cyclic AMP content. On the other hand, the removal of intracellular but not the extracellular Ca2+ attenuated the RCM-induced mast cell histamine release. From these findings, it is suggested that the decrease in cellular cyclic AMP content and an increase in intracellular Ca2+ contribute at least in part to the RCM-induced mast cell histamine release. PMID:12690428

  10. Effects of membrane polyunsaturated fatty acids on opiate peptide inhibition of basal and prostaglandin E1-stimulated cyclic AMP formation in intact N1E-115 neuroblastoma cells.

    PubMed

    Murphy, M G; Moak, C M; Rao, B G

    1987-12-01

    The effects of membrane polyunsaturated fatty acids (PUFA) on opiate peptide-mediated inhibition of basal and prostaglandin E1-stimulated cyclic AMP formation were examined in intact N1E-115 neuroblastoma cells. Addition of opiate peptides such as methionine 5-enkephalin (metEnk) to control cultures and to cultures that had been supplemented for 48 hr with 50 microM linoleic acid resulted in dose-dependent decreases in cAMP formation; these decreases were blocked by naloxone. Maximum inhibition of basal cyclase activity was 50-55% in both control and PUFA-enriched cells; however, half-maximal inhibition required ten times more metEnk in supplemented cultures than in controls. This is consistent with our observation that the affinity of binding of [tyrosyl-3',5'-3H(N)](2-D-alanine-5-D-leucine)enkephalin ([3H]DADLE) to intact PUFA-enriched cells was lower than that to control cells. Receptor density was not modified as a result of supplementation. Addition of prostaglandin E1 (PGE1) to the cells produced rapid dose-dependent increases in cAMP formation. Maximum responses were higher in PUFA-enriched than in control cells (1924 and 972 pmol cAMP formed/mg protein respectively). Also, the apparent value for EC50 for PGE1 was consistently lower in supplemented cultures. MetEnk reduced PGE1-stimulated cAMP formation by 45-55% in both control and supplemented cells, and values for IC50 were similar (approximately 30 nM) in both. In the presence of the opiate peptide, values for EC50 for PGE1 were similar in control and PUFA-enriched cultures (0.07 and 0.09 microM respectively). The data from these studies suggest that membrane PUFA increase the efficiency of coupling of receptors that stimulate cAMP formation and decrease the efficiency of those that mediate inhibition. PMID:2825714

  11. Regulation of osteosarcoma EGF receptor affinity by phorbol ester and cyclic AMP

    SciTech Connect

    Borst, S.E.; Catherwood, B.D. )

    1989-04-01

    We studied the binding and degradation of 125I-labeled epidermal growth factor (EGF) by UMR-106 osteosarcoma cells and the regulation of EGF receptor affinity for EGF by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and by treatments that raise intracellular levels of cyclic AMP. Cell surface binding of (125I)EGF to A431 cells reached a plateau after a 30 minute incubation at 37 degrees C but was undetectable in UMR-106 cells. Degradation of (125I)EGF proceeded at a 50-fold higher rate in A431 cells on a per cell basis, but receptor-bound (125I)EGF was internalized and degraded at a 3.5-fold higher rate by UMR-106 cells on a per receptor basis. At 4 degrees C, (125I)EGF labeled a single class of surface binding sites in the UMR-106 cell. Treatment with TPA at 37 degrees C reduced subsequent cell surface binding of (125I)EGF at 4 degrees C a maximum of 80% with an IC50 of 1.25 ng/ml. Maximal TPA reduction of (125I)EGF binding was observed within 5-15 minutes and was due to a reduction in the affinity of cell surface receptors of (125I)EGF without a change in receptor density. Pretreatment of the cells for 4 h with 30 microM forskolin, 1 mM isobutylmethylxanthine (IBMX) plus 30 microM forskolin, or 1 mM IBMX plus 100 ng/ml parathyroid hormone (PTH) attenuated the loss in (125I)EGF binding caused by a subsequent dose of 10 ng/ml of TPA by 17% (p less than 0.0005), 39% (p less than 0.0002), and 35% (p less than 0.002), respectively.

  12. Toxoplasma gondii Cyclic AMP-Dependent Protein Kinase Subunit 3 Is Involved in the Switch from Tachyzoite to Bradyzoite Development

    PubMed Central

    Sugi, Tatsuki; Ma, Yan Fen; Tomita, Tadakimi; Murakoshi, Fumi; Eaton, Michael S.; Yakubu, Rama; Han, Bing; Tu, Vincent; Kato, Kentaro; Kawazu, Shin-Ichiro; Gupta, Nishith; Suvorova, Elena S.; White, Michael W.; Kim, Kami

    2016-01-01

    ABSTRACT Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects warm-blooded vertebrates, including humans. Asexual reproduction in T. gondii allows it to switch between the rapidly replicating tachyzoite and quiescent bradyzoite life cycle stages. A transient cyclic AMP (cAMP) pulse promotes bradyzoite differentiation, whereas a prolonged elevation of cAMP inhibits this process. We investigated the mechanism(s) by which differential modulation of cAMP exerts a bidirectional effect on parasite differentiation. There are three protein kinase A (PKA) catalytic subunits (TgPKAc1 to -3) expressed in T. gondii. Unlike TgPKAc1 and TgPKAc2, which are conserved in the phylum Apicomplexa, TgPKAc3 appears evolutionarily divergent and specific to coccidian parasites. TgPKAc1 and TgPKAc2 are distributed in the cytomembranes, whereas TgPKAc3 resides in the cytosol. TgPKAc3 was genetically ablated in a type II cyst-forming strain of T. gondii (PruΔku80Δhxgprt) and in a type I strain (RHΔku80Δhxgprt), which typically does not form cysts. The Δpkac3 mutant exhibited slower growth than the parental and complemented strains, which correlated with a higher basal rate of tachyzoite-to-bradyzoite differentiation. 3-Isobutyl-1-methylxanthine (IBMX) treatment, which elevates cAMP levels, maintained wild-type parasites as tachyzoites under bradyzoite induction culture conditions (pH 8.2/low CO2), whereas the Δpkac3 mutant failed to respond to the treatment. This suggests that TgPKAc3 is the factor responsible for the cAMP-dependent tachyzoite maintenance. In addition, the Δpkac3 mutant had a defect in the production of brain cysts in vivo, suggesting that a substrate of TgPKAc3 is probably involved in the persistence of this parasite in the intermediate host animals. PMID:27247232

  13. Identification, characterization and regional distribution in brain of RPDE-6 (RNPDE4A5), a novel splice variant of the PDE4A cyclic AMP phosphodiesterase family.

    PubMed

    McPhee, I; Pooley, L; Lobban, M; Bolger, G; Houslay, M D

    1995-09-15

    COS-7 cells were transfected with a plasmid encoding a putative splice variant of PDE4A cyclic AMP-specific phosphodiesterase, RPDE-6 (RNPDE4A5). This led to the expression of a novel, cyclic AMP-specific, rolipram-inhibited phosphodiesterase activity. In such transfected cells a novel approximately 109 kDa species was recognized by anti-peptide sera raised against a dodecapeptide whose sequence is found at the extreme C-terminus of both RPDE-6 and another PDE4A splice variant. RD1 (RNPDE4A1A). RPDE-6 activity and immunoreactivity was found distributed between both pellet (approximately 25%) and cytosol (approximately 75%) fractions of transfected COS-7 cells. Soluble and pellet RPDE-6 activities exhibited similar low Km values for cyclic AMP (approximately 2.4 microM) and were both inhibited by low concentrations of rolipram, with IC50 values for the soluble activity being lower (approximately 0.16 microM) than for the pellet activity (approximately 1.2 microM). Pellet RPDE-6 was resistant to release by either high NaCl concentrations or the detergent Triton X-100. Probing brain homogenates with the anti-(C-terminal peptide) sera identified two immunoreactive species, namely an approximately 79 kDa species reflecting RD1 and an approximately 109 kDa species that co-migrated with the immunoreactive species seen in COS cells transfected to express RPDE-6. The approximately 109 kDa species was found distributed between both the low-speed (P1) and high-speed (P2) pellet fractions as well as the cytosol fractions derived from both brain and RPDE-6-transfected COS cells. In contrast, RD1 was found exclusively in the P2 fraction. Phosphodiesterase (PDE) activity immuno-precipitated by these antisera from brain cytosol had the characteristics of COS cell-expressed RPDE-6 with KmcyclicAMP approximately 3.7 microM and IC50rolipram approximately 0.12 microM. The distribution of PDE activity immunoprecipitated from the cytosol of various brain regions paralleled that seen for

  14. Selective inhibition of cyclic AMP-dependent protein kinase by isoquinoline derivatives.

    PubMed

    Lu, Z X; Quazi, N H; Deady, L W; Polya, G M

    1996-06-01

    A large series of isoquinoline derivatives was synthesised including derivatives of isoquinoline, isoquinolino[3,4-c]furazan, 1,2-dihydro-1-oxoisoquinoline, 6-oxopyrimido[1,2-d]isoquinoline, benzo[c][1,8]-naphthyridine, pyrazino[2,3-c]isoquinoline and benzimidazo[2,1-a]isoquinoline as well as further structurally related isoquinoline derivatives and pyrido-2,3-furazans. Representatives of all of these classes of isoquinolines are potent and selective inhibitors of the cyclic AMP-dependent protein kinase (PKA) catalytic subunit (cAK) from rat liver. The most effective cAK inhibitors are a series of 1,3-di-substituted and 1,3,4-tri-substituted isoquinolines (IC50 values 30-50 nM) (compounds A1, A2, A3, A4 and A5) and 2-ethylcarboxy-3-amino-5,6-dihydro-6-oxobenzo[c] [1,8]naphthyridine (E1) (IC50 0.08 microM). Compounds A1-A5 inhibit cAK in a fashion that is competitive with respect to ATP as substrate. The isoquinoline inhibitors A1-A5 are ineffective or very poor inhibitors of wheat embryo Ca(2+)-dependent protein kinase (CDPK) and rat brain Ca(2+)-dependent protein kinase C (PKC), chicken gizzard myosin light chain kinase (MLCK) and potato tuber cyclic nucleotide-binding phosphatase (Pase). E1 is a moderately effective inhibitor of CDPK and PKC (IC50 values 30 and 61 microM, respectively). The bisisoquinoline-1(2H)-one compound B7 inhibits cAK, CDPK, PKC and MLCK (IC50 values 8, 95, 24 and 7 microM, respectively) as does J1 [2-(p-bromophenyl)pyrrolo-[2,3-c]isoquinoline-5(4H)-one] (IC50 values 2, 50, 44 and 7 microM, respectively). The very potent isoquinoline-derived cAK inhibitors found here involve substitution of the N-containing isoquinoline ring system and these inhibitors show high specificity for cAK. PMID:8839983

  15. Identification of cyclic AMP phosphodiesterases 3, 4 and 7 in human CD4+ and CD8+ T-lymphocytes: role in regulating proliferation and the biosynthesis of interleukin-2.

    PubMed Central

    Giembycz, M. A.; Corrigan, C. J.; Seybold, J.; Newton, R.; Barnes, P. J.

    1996-01-01

    1. The cyclic AMP phosphodiesterases (PDE) expressed by CD4+ and CD8+ T-lymphocytes purified from the peripheral blood of normal adult subjects were identified and characterized, and their role in modulating proliferation and the biosynthesis of interleukin (IL)-2 and interferon (IFN)-gamma evaluated. 2. In lysates prepared from both subsets, SK&F 95654 (PDE3 inhibitor) and rolipram (PDE4 inhibitor) suppressed cyclic AMP hydrolysis indicating the presence of PDE3 and PDE4 isoenzymes in these cells. Differential centrifugation and subsequent inhibitor and kinetic studies revealed that the particulate fraction contained, predominantly, a PDE3 isoenzyme. In contrast, the soluble fraction contained a PDE4 (approximately 65% of total activity) and, in addition, a novel enzyme that had the kinetic characteristics of the recently identified PDE7. 3. Reverse transcription-polymerase chain reaction (RT-PCR) studies with primer pairs designed to recognise unique sequences in the human PDE4 and PDE7 genes amplified cDNA fragments that corresponded to the predicted sizes of HSPDE4A, HSPDE4B, HSPDE54D and HSPDE7. No message was detected for HSPDE4C after 35 cycles of amplification. 4. Functionally, rolipram inhibited phytohaemagglutinin- (PHA) and anti-CD3-induced proliferation of CD4+ and CD8+ T-lymphocytes, and the elaboration of IL-2, which was associated with a three to four fold increase in cyclic AMP mass. In all experiments, however, rolipram was approximately 60 fold more potent at suppressing IL-2 synthesis than at inhibiting mitogenesis. In contrast, SK&F 95654 failed to suppress proliferation and cytokine generation, and did not elevate the cyclic AMP content in T-cells. Although inactive alone, SK&F 95654 potentiated the ability of rolipram to suppress PHA- and anti-CD3-induced T-cell proliferation, and PHA-induced IL-2 release. 5. When a combination of phorbol myristate acetate (PMA) and ionomycin were used as a co-mitogen, rolipram did not affect proliferation but

  16. Effects of prostaglandin E2, cholera toxin and 8-bromo-cyclic AMP on lipopolysaccharide-induced gene expression of cytokines in human macrophages.

    PubMed Central

    Zhong, W W; Burke, P A; Drotar, M E; Chavali, S R; Forse, R A

    1995-01-01

    Prostaglandin E2 (PGE2) appears to regulate macrophage cytokine production through the stimulatory GTP-binding protein (Gs protein)-mediated cyclic AMP (cAMP)-dependent transmembrane signal transduction pathway. In this study, we used PGE2, cholera toxin (CT; a direct G alpha s protein stimulator) and 8-bromo-cAMP (a membrane permeable cAMP analogue) to stimulate this pathway, and investigated their influence on cytokine gene expression in lipopolysaccharide (LPS)-activated human macrophages. The mRNA expression for interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), IL-6 and IL-8 were determined employing reverse transcription polymerase chain reaction (RT-PCR) using specific primers. We demonstrated that PGE2, CT and 8-bromo-cAMP inhibited the LPS-induced gene activation of TNF-alpha and IL-1 alpha, and had no effect on the gene activation of IL-1 beta and IL-8. Further, our data indicate that PGE2 suppressed the gene activation of IL-6 following LPS stimulation, but neither CT nor 8-bromo-cAMP had an effect. These data suggest that PGE2 alters LPS-stimulated gene activation of only some of the early macrophage cytokines, and does so either by a Gs transmembrane cAMP-dependent or an independent system. Images Figure 1 PMID:7751029

  17. Expression and organization of BP74, a cyclic AMP-regulated gene expressed during Dictyostelium discoideum development.

    PubMed Central

    Hopkinson, S B; Pollenz, R S; Drummond, I; Chisholm, R L

    1989-01-01

    We have characterized a cDNA and the corresponding gene for a cyclic AMP-inducible gene expressed during Dictyostelium development. This gene, BP74, was found to be first expressed about the time of aggregate formation, approximately 6 h after starvation. Accumulation of BP74 mRNA did not occur in Dictyostelium cells that had been starved in fast-shaken suspension cultures but was induced in similar cultures to which cyclic AMP pulses had been added. The BP74 cDNA and gene were characterized by DNA sequence analysis and transcriptional mapping. When the BP74 promoter region was fused with a chloramphenicol acetyltransferase reporter gene and reintroduced into Dictyostelium cells, the transfected chloramphenicol acetyltransferase gene displayed the same developmentally regulated pattern of expression as did the endogenous BP74 gene, suggesting that all of the cis-acting elements required for regulated expression were carried by a 2-kilobase cloned genomic fragment. On the basis of sequence analysis, the gene appeared to encode a protein containing a 20-residue hydrophobic sequence at the amino-terminal end and 26 copies of a 20-amino-acid repeat. Images PMID:2555685

  18. Cyclic AMP--dependent aggregation of Swiss 3T3 cells on a cellulose substratum (Cuprophan) and decreased cell membrane Rho A.

    PubMed

    Faucheux, N; Nagel, M D

    2002-06-01

    Cell surface integrin receptors and Rho family GTPases function together to mediate adhesion-dependent events in cells. We have shown that the attachment of Swiss 3T3 cells to a cellulose substratum (Cuprophan, CU) activates adenylyl cyclase, which catalyses cyclic AMP (cAMP) production. CU adsorbs vitronectin poorly, prevents cell spreading and causes cells to aggregate. By contrast, spread cells on polystyrene (PS) contain low cAMP concentrations. We have now investigated the shift between integrin signalling-Rho A and the cAMP pathway. CU did not support the formation of focal contacts and stress fibres. The plasma membranes of cells on CU had less Rho A than those of cells on PS. Also, blocking vitronectin (VN) or fibronectin (FN)-integrin receptors with echistatin, which activates cAMP production, decreased Rho A in the plasma membrane of cells attached to PS. But adsorption of VN or FN onto CU, which limits the production of the cAMP, increased the cell membrane Rho A. Adding an inhibitor of cAMP-dependent protein kinase PKA to the medium also increased the plasma membrane Rho A in aggregated cells attached to CU. These results highlight the importance of cAMP, generated by cell attachment to substratum, as a gating element in integrin-Rho A signalling. PMID:12013176

  19. Temperature Change Induces the Expression of vuuA Encoding Vulnibactin Receptor and crp Encoding Cyclic AMP Receptor Protein in Vibrio vulnificus.

    PubMed

    Kim, Choon-Mee; Ahn, Young-Joon; Kim, Seong-Jung; Yoon, Dae-Heung; Shin, Sung-Heui

    2016-07-01

    Upon entering the human body, Vibrio vulnificus, a gram-negative marine bacterium, must withstand a temperature change (TC) from 25 to 37 °C. This bacterium acquires iron mainly via the vulnibactin receptor (VuuA)-mediated iron uptake system (IUS), which is under the positive control of cyclic AMP receptor protein (CRP), a global regulator responsible for catabolite repression. In this study, we examined the effect of TC on the expression of vuuA and crp, and the reciprocal relation between VuuA-mediated IUS and CRP under iron-limited conditions. Iron limitation increased vuuA expression but decreased crp expression. TC resulted in increased vuuA and crp expression. A crp or vuuA mutation reciprocally decreased vuuA or crp expression. TC could increase vuuA or crp expression even in a crp- or vuuA-mutated background. These results indicate that TC increases the expression of both vuuA and crp by facilitating metabolism under iron-limited conditions, and that CRP and VuuA-mediated IUS interact coordinately toward optimal metabolism in V. vulnificus. PMID:27016238

  20. A spontaneous change in the intracellular cyclic AMP level in Aspergillus niger is influenced by the sucrose concentration in the medium and by light.

    PubMed Central

    Gradisnik-Grapulin, M; Legisa, M

    1997-01-01

    A spontaneous rise in intracellular cyclic AMP (cAMP) levels was observed in the early stages of Aspergillus niger growth under conditions yielding large amounts of citric acid. The amount of cAMP formed was found to depend on the initial concentration of sucrose in the medium. Under higher-sucrose conditions, the cAMP peak appeared earlier and was higher, while in lower-sucrose media a flattened peak was observed later in fermentation. Since in media with higher concentrations of sucrose intracellular citric acid starts to accumulate earlier and more rapidly, cAMP synthesis may be triggered by intracellular acidification, which is caused by the dissociation of citric acid. No spontaneous increase in cAMP concentrations could be detected when the cells were grown in continuously illuminated cultures, suggesting that A. niger phosphodiesterase (PDE) is photoregulated. More evidence for the activation of PDE by light was obtained from morphological studies under light and dark conditions in the presence of cAMP or N6,O2'-dibutyryl cAMP, and this idea was additionally supported by experiments in which PDE inhibitors were tested. PMID:9212431

  1. Genome-Wide Identification of In Vivo Binding Sites of GlxR, a Cyclic AMP Receptor Protein-Type Regulator in Corynebacterium glutamicum▿†

    PubMed Central

    Toyoda, Koichi; Teramoto, Haruhiko; Inui, Masayuki; Yukawa, Hideaki

    2011-01-01

    Corynebacterium glutamicum GlxR is a cyclic AMP (cAMP) receptor protein-type regulator. Although over 200 GlxR-binding sites in the C. glutamicum genome are predicted in silico, studies on the physiological function of GlxR have been hindered by the severe growth defects of a glxR mutant. This study identified the GlxR regulon by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analyses. In total, 209 regions were detected as in vivo GlxR-binding sites. In vitro binding assays and promoter-reporter assays demonstrated that GlxR directly activates expression of genes for aerobic respiration, ATP synthesis, and glycolysis and that it is required for expression of genes for cell separation and mechanosensitive channels. GlxR also directly represses a citrate uptake gene in the presence of citrate. Moreover, ChIP-chip analyses showed that GlxR was still able to interact with its target sites in a mutant with a deletion of cyaB, the sole adenylate cyclase gene in the genome, even though binding affinity was markedly decreased. Thus, GlxR is physiologically functional at the relatively low cAMP levels in the cyaB mutant, allowing the cyaB mutant to grow much better than the glxR mutant. PMID:21665967

  2. Regulation of the Dictyostelium glycogen phosphorylase 2 gene by cyclic AMP.

    PubMed

    Sucic, J F; Selmin, O; Rutherford, C L

    1993-01-01

    A crucial developmental event in the cellular slime mold, Dictyostelium discoideum, is glycogen degradation. The enzyme that catalyzes this degradation, glycogen phosphorylase 2 (gp-2), is developmentally regulated and cAMP appears to be involved in this regulation. We have examined several aspects of the cAMP regulation of gp-2. We show that addition of exogenous cAMP to aggregation competent amoebae induced the appearance of gp-2 mRNA. The induction of gp-2 mRNA occurred within 1 and 1.5 h after the initial exposure to cAMP. Exposure to exogenous cAMP concentrations as low as 1.0 microM could induce gp-2 mRNA. We also examined the molecular mechanism through which cAMP induction of gp-2 occurs. Induction of gp-2 appears to result from a mechanism that does not require intracellular cAMP signaling, and may occur directly through a cAMP binding protein without the requirement of any intracellular signalling. We also examined the promoter region of the gp-2 gene for cis-acting elements that are involved in the cAMP regulation of gp-2. A series of deletions of the promoter were fused to a luciferase reporter gene and then analyzed for cAMP responsiveness. The results indicated that a region from -258 nucleotides to the transcriptional start site is sufficient for essentially full activity and appears to carry all necessary cis-acting sites for cAMP induction. Further deletion of 58 nucleotides from the 5' end, results in fivefold less activity in the presence of cAMP. Deletion of the next 104 nucleotides eliminates the cAMP response entirely. PMID:8222346

  3. Steady-State Modulation of Voltage-Gated K+ Channels in Rat Arterial Smooth Muscle by Cyclic AMP-Dependent Protein Kinase and Protein Phosphatase 2B

    PubMed Central

    Brignell, Jennifer L.; Perry, Matthew D.; Nelson, Carl P.; Willets, Jonathon M.; Challiss, R. A. John; Davies, Noel W.

    2015-01-01

    Voltage-gated potassium channels (Kv) are important regulators of membrane potential in vascular smooth muscle cells, which is integral to controlling intracellular Ca2+ concentration and regulating vascular tone. Previous work indicates that Kv channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that Kv channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on Kv and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents. Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a β-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents. We also show that this modulation of Kv by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of Kv by KT5720 can be abrogated by pre-treatment with the PP2B inhibitor cyclosporin A, or inclusion of a PP2B auto-inhibitory peptide in the pipette solution. Finally, we demonstrate that tonic PKA-mediated modulation of Kv requires intact caveolae. Pre-treatment of the cells with methyl-β-cyclodextrin to deplete cellular cholesterol, or adding caveolin-scaffolding domain peptide to the pipette solution to disrupt caveolae-dependent signalling each attenuated PKA-mediated modulation of the Kv current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone. PMID:25793374

  4. A Model Based on Receptor Desensitization for Cyclic AMP Signaling in Dictyostelium Cells

    PubMed Central

    Martiel, Jean-Louis; Goldbeter, Albert

    1987-01-01

    We analyze a model based on receptor modification for the cAMP signaling system that controls aggregation of the slime mold Dictyostelium discoideum after starvation. The model takes into account both the desensitization of the cAMP receptor by reversible phosphorylation and the activation of adenylate cyclase that follows binding of extracellular cAMP to the unmodified receptor. The dynamics of the signaling system is studied in terms of three variables, namely, intracellular and extracellular cAMP, and the fraction of receptor in active state. Using parameter values collected from experimental studies on cAMP signaling and receptor phosphorylation, we show that the model accounts qualitatively and, in a large measure, quantitatively for the various modes of dynamic behavior observed in the experiments: (a) autonomous oscillations of cAMP, (b) relay of suprathreshold cAMP pulses, i.e., excitability, characterized by both an absolute and a relative refractory period, and (c) adaptation to constant cAMP stimuli. A two-variable version of the model is used to demonstrate the link between excitability and oscillations by phase plane analysis. The response of the model to repetitive stimulation allows comprehension, in terms of receptor desensitization, of the role of periodic signaling in Dictyostelium and, more generally, the function of pulsatile patterns of hormone secretion. PMID:19431710

  5. Cyclic AMP concentrations in dendritic cells induce and regulate Th2 immunity and allergic asthma

    PubMed Central

    Lee, Jihyung; Kim, Tae Hoon; Murray, Fiona; Li, Xiangli; Choi, Sara S.; Broide, David H.; Corr, Maripat; Lee, Jongdae; Webster, Nicholas J. G.; Insel, Paul A.; Raz, Eyal

    2015-01-01

    The inductive role of dendritic cells (DC) in Th2 differentiation has not been fully defined. We addressed this gap in knowledge by focusing on signaling events mediated by the heterotrimeric GTP binding proteins Gαs, and Gαi, which respectively stimulate and inhibit the activation of adenylyl cyclases and the synthesis of cAMP. We show here that deletion of Gnas, the gene that encodes Gαs in mouse CD11c+ cells (GnasΔCD11c mice), and the accompanying decrease in cAMP provoke Th2 polarization and yields a prominent allergic phenotype, whereas increases in cAMP inhibit these responses. The effects of cAMP on DC can be demonstrated in vitro and in vivo and are mediated via PKA. Certain gene products made by GnasΔCD11c DC affect the Th2 bias. These findings imply that G protein-coupled receptors, the physiological regulators of Gαs and Gαi activation and cAMP formation, act via PKA to regulate Th bias in DC and in turn, Th2-mediated immunopathologies. PMID:25605931

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

    SciTech Connect

    McInnis, Brittney; Mitchell, Jessica; Marcus, Stevan

    2010-09-03

    Research highlights: {yields} cAMP deficiency induces phosphorylation of PKA catalytic subunit (Pka1) in S. pombe. {yields} Pka1 phosphorylation is further induced by physiological stresses. {yields} Pka1 phosphorylation is not induced in cells lacking the PKA regulatory subunit. {yields} 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 cyr1{Delta} cells, but not wild type cells, a substantial proportion of Pka1 protein is hyperphosphorylated. Pka1 hyperphosphorylation is strongly induced in cyr1{Delta} 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{sup +} or cyr1{Delta} 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.

  7. Identification and characterization of the type-IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum.

    PubMed

    Shakur, Y; Wilson, M; Pooley, L; Lobban, M; Griffiths, S L; Campbell, A M; Beattie, J; Daly, C; Houslay, M D

    1995-03-15

    An antiserum was generated against a dodecapeptide whose sequence is found at the C-terminus of a cyclic AMP (cAMP)-specific, type-IVA phosphodiesterase encoded by the rat 'dunc-like' cyclic AMP phosphodiesterase (RD1) cDNA. This antiserum identified a single approximately 73 kDa protein species upon immunoblotting of cerebellum homogenates. This species co-migrated upon SDS/PAGE with a single immunoreactive species observed in COS cells transfected with the cDNA for RD1. Native RD1 in cerebellum was found to be predominantly (approximately 93%) membrane-associated and could be found in isolated synaptosome populations, in particular those enriched in post-synaptic densities. Fractionation of lysed synaptosomes on sucrose density gradients identified RD1 as co-migrating with the plasma membrane marker 5'-nucleotidase. Laser scanning confocal and digital deconvolution immunofluorescence studies done on intact COS cells transfected with RD1 cDNA showed RD1 to be predominantly localized to plasma membranes but also associated with the Golgi apparatus and intracellular vesicles. RD1-specific antisera immunoprecipitated phosphodiesterase activity from solubilized cerebellum membranes. This activity had the characteristics expected of the type-IV cAMP phosphodiesterase RD1 in that it was cAMP specific, exhibited a low Km cAMP of 2.3 microM, high sensitivity to inhibition by 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram) (Ki approximately 0.7 microM) and was unaffected by Ca2+/calmodulin and low concentrations of cyclic GMP. The phosphodiesterase activities of RD1 solubilized from both cerebellum and transfected COS cell membranes showed identical first-order thermal denaturation kinetics at 50 degrees C. Native RD1 from cerebellum was shown to be an integral protein in that it was solubilized using the non-ionic detergent Triton X-100 but not by either re-homogenization or high NaCl concentrations. The observation that hydroxylamine was unable to cause

  8. CCR-08-0827 Version 2 Targeted inhibition of cyclic AMP phosphodiesterase-4 promotes brain tumor regression

    PubMed Central

    Goldhoff, Patricia; Warrington, Nicole; Limbrick, David D.; Hope, Andrew; Woerner, B. Mark; Jackson, Erin; Perry, Arie; Piwnica-Worms, David; Rubin, Joshua B.

    2008-01-01

    Statement of Clinical Relevance Therapies that can overcome the resistance of malignant brain tumors would be a major clinical advance. Here, we investigate the role of cAMP Phosphodiesterase-4 in stimulating brain tumor growth and the therapeutic utility of cAMP Phosphodiesterase-4 inhibition in the treatment of malignant brain tumors. Cyclic AMP Phosphodiesterase-4 was widely expressed in human brain tumors of glial and neuronal lineage, and forced expression of PDE4A1 accelerated intracranial glioblastoma and medulloblastoma xenograft growth. Moreover, targeted inhibition of PDE4, in combination with standard radiation and chemotherapy, induced a unique regression of established intracranial glioblastoma xenografts. These findings identify PDE4 as a novel molecular target for brain tumor therapy and indicate that PDE4 inhibition should be evaluated in clinical trials for malignant brain tumors. Purpose As favorable outcomes from malignant brain tumors remain limited by poor survival and treatment-related toxicity, novel approaches to cure are essential. Previously, we identified the cyclic AMP phosphodiesterase-4 (PDE4) inhibitor Rolipram as a potent anti-tumor agent. Here, we investigate the role of PDE4 in brain tumors and examine the utility of PDE4 as a therapeutic target. Experimental Design Immunohistochemistry was used to evaluate the expression pattern of a subfamily of PDE4, PDE4A, in multiple brain tumor types. To evaluate the effect of PDE4A on growth, a brain-specific isoform, PDE4A1 was overexpressed in xenografts of Daoy medulloblastoma and U87 glioblastoma cells. To determine therapeutic potential of PDE4 inhibition, Rolipram, temozolomide, and radiation were tested alone and in combination on mice bearing intracranial U87 xenografts. Results We found that PDE4A is expressed in medulloblastoma, glioblastoma, oligodendroglioma, ependymoma and meningioma. Moreover, when PDE4A1 was overexpressed in Daoy medulloblastoma and U87 glioblastoma cells, in

  9. The prostaglandin E2/EP4 receptor/cyclic AMP/T-type Ca(2+) channel pathway mediates neuritogenesis in sensory neuron-like ND7/23 cells.

    PubMed

    Mitani, Kenji; Sekiguchi, Fumiko; Maeda, Takashi; Tanaka, Yukari; Yoshida, Shigeru; Kawabata, Atsufumi

    2016-03-01

    We investigated mechanisms for the neuritogenesis caused by prostaglandin E2 (PGE2) or intracellular cyclic AMP (cAMP) in sensory neuron-like ND7/23 cells. PGE2 caused neuritogenesis, an effect abolished by an EP4 receptor antagonist or inhibitors of adenylyl cyclase (AC) or protein kinase A (PKA) and mimicked by the AC activator forskolin, dibutyryl cAMP (db-cAMP), and selective activators of PKA or Epac. ND7/23 cells expressed both Cav3.1 and Cav3.2 T-type Ca(2+) channels (T-channels). The neuritogenesis induced by db-cAMP or PGE2 was abolished by T-channel blockers. T-channels were functionally upregulated by db-cAMP. The PGE2/EP4/cAMP/T-channel pathway thus appears to mediate neuritogenesis in sensory neurons. PMID:27032908

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

    PubMed Central

    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. PMID:26361388

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

    PubMed

    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. PMID:26361388

  12. [Effects of dibutyryl cyclic AMP on the gene expression during the differentiation of retinoblastoma cells (Y 79) in culture].

    PubMed

    Murakami, T; Takahashi, H; Akiya, S; Higashi, K

    1993-06-01

    Cultured human retinoblastoma cells (Y79) were induced to differentiate by dibutyryl cyclic AMP(Bt2cAMP). We examined the effects on the mRNA levels of several cellular genes when the induction of differentiation was monitored by observation of the cellular processes. Bt2cAMP(1mM) treatment produced significant extension of cellular process after 3 days. We examined the mRNA levels of N-myc gene(oncogene), Rb(anti-oncogene, retinoblastoma gene) and nucleolin (nucleolar protein) being linked with ribosome biosynthesis. The mRNA levels of all these genes decreased for 3 days after Bt2cAMP treatment. These results suggested the possibility that Y 79 cells were induced to differentiate by down-modulation of both N-myc gene expression and ribosome biosynthesis in the nucleolus following treatment of Bt2cAMP. Furthermore, the gene expression of the retinoblastoma gene is likely to be downregulated by this condition even if the product of mRNA is not functional. PMID:8392280

  13. Cyclic AMP (cAMP) Receptor Protein-cAMP Complex Regulates Heparosan Production in Escherichia coli Strain Nissle 1917.

    PubMed

    Yan, Huihui; Bao, Feifei; Zhao, Liping; Yu, Yanying; Tang, Jiaqin; Zhou, Xianxuan

    2015-11-01

    Heparosan serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The availability of heparosan is a significant concern for the cost-effective synthesis of bioengineered heparin. The carbon source is known as the pivotal factor affecting heparosan production. However, the mechanism by which carbon sources control the biosynthesis of heparosan is unclear. In this study, we found that the biosynthesis of heparosan was influenced by different carbon sources. Glucose inhibits the biosynthesis of heparosan, while the addition of either fructose or mannose increases the yield of heparosan. Further study demonstrated that the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex binds to the upstream region of the region 3 promoter and stimulates the transcription of the gene cluster for heparosan biosynthesis. Site-directed mutagenesis of the CRP binding site abolished its capability of binding CRP and eliminated the stimulative effect on transcription. (1)H nuclear magnetic resonance (NMR) analysis was further performed to determine the Escherichia coli strain Nissle 1917 (EcN) heparosan structure and quantify extracellular heparosan production. Our results add to the understanding of the regulation of heparosan biosynthesis and may contribute to the study of other exopolysaccharide-producing strains. PMID:26319872

  14. Cyclic AMP (cAMP) Receptor Protein-cAMP Complex Regulates Heparosan Production in Escherichia coli Strain Nissle 1917

    PubMed Central

    Yan, Huihui; Bao, Feifei; Zhao, Liping; Yu, Yanying; Tang, Jiaqin

    2015-01-01

    Heparosan serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The availability of heparosan is a significant concern for the cost-effective synthesis of bioengineered heparin. The carbon source is known as the pivotal factor affecting heparosan production. However, the mechanism by which carbon sources control the biosynthesis of heparosan is unclear. In this study, we found that the biosynthesis of heparosan was influenced by different carbon sources. Glucose inhibits the biosynthesis of heparosan, while the addition of either fructose or mannose increases the yield of heparosan. Further study demonstrated that the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex binds to the upstream region of the region 3 promoter and stimulates the transcription of the gene cluster for heparosan biosynthesis. Site-directed mutagenesis of the CRP binding site abolished its capability of binding CRP and eliminated the stimulative effect on transcription. 1H nuclear magnetic resonance (NMR) analysis was further performed to determine the Escherichia coli strain Nissle 1917 (EcN) heparosan structure and quantify extracellular heparosan production. Our results add to the understanding of the regulation of heparosan biosynthesis and may contribute to the study of other exopolysaccharide-producing strains. PMID:26319872

  15. Stimulation of T-cells with OKT3 antibodies increases forskolin binding and cyclic AMP accumulation.

    PubMed

    Kvanta, A; Gerwins, P; Jondal, M; Fredholm, B B

    1990-01-01

    It has recently been shown that elevation of cAMP by adenosine receptor stimulation may be potentiated by stimulation of the T-cell receptor/CD3 complex on human T-cells with the monoclonal antibody OKT3, and that this is mimicked by activation of protein kinase C [Kvanta, A. et al. (1989) Naunyn-Schmeideberg's Arch. Pharmac. 340, 715-717]. In this study the diterpene forskolin, which binds to and activates the adenylate cyclase, has been used to examine further how the CD3 complex may influence the adenylate cyclase pathway. Stimulation with OKT3 alone was found to cause a small dose-dependent increase in basal cAMP accumulation. When combining OKT3 with a concentration of forskolin (10 microM), which by itself had little effect on the cyclase activity, the cAMP accumulation was markedly potentiated. This potentiation was paralleled by an increase in [3H]forskolin binding to saponine permeabilized Jurkat cells from 24 to 41 fmol/10(6) cells. The OKT3 effect on cAMP was blocked by chelating extracellular Ca2+ with EGTA or intracellular Ca2+ with BAPTA and also by W-7, an inhibitor of calmodulin, but was unaffected by H-7, an inhibitor of protein kinase C. Even though OKT3 caused an increase in inositolphosphate turnover, and activated protein kinase C, neither phorbol 12,13 dibutyrate (PDBu) nor the Ca2(+)-ionophore A23187 could mimic the OKT3 effect, whereas a combination of PDBu and A23187 at high concentrations could potentiate forskolin stimulated cyclase activity. Together, these results indicated that stimulation of the CD3 complex could influence the adenylate cyclase by two different mechanisms, one involving activation of protein kinase C and another which does not. PMID:2177619

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

    PubMed Central

    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

  17. Cyclic AMP Stimulates Neurite Outgrowth of Lamprey Reticulospinal Neurons without Substantially Altering Their Biophysical Properties

    PubMed Central

    Pale, Timothée; Frisch, Emily B.; McClellan, Andrew D.

    2013-01-01

    Reticulospinal (RS) neurons are critical for initiation of locomotor behavior, and following spinal cord injury (SCI) in the lamprey, the axons of these neurons regenerate and restore locomotor behavior within a few weeks. For lamprey RS neurons in culture, experimental induction of calcium influx, either in the growth cone or cell body, is inhibitory for neurite outgrowth. Following SCI, these neurons partially downregulate calcium channel expression, which would be expected to reduce calcium influx and possibly provide supportive conditions for axonal regeneration. In the present study, it was tested whether activation of second messenger signaling pathways stimulates neurite outgrowth of lamprey RS neurons without altering their electrical properties (e.g. spike broadening) so as to possibly increase calcium influx and compromise axonal growth. First, activation of cAMP pathways with forskolin or dbcAMP stimulated neurite outgrowth of RS neurons in culture in a PKA-dependent manner, while activation of cGMP signaling pathways with dbcGMP inhibited outgrowth. Second, neurophysiological recordings from uninjured RS neurons in isolated lamprey brain-spinal cord preparations indicated that dbcAMP or dbcGMP did not significantly affect any of the measured electrical properties. In contrast, for uninjured RS neurons, forskolin increased action potential duration, which might have increased calcium influx, but did not significantly affect most other electrical properties. Importantly, for injured RS neurons during the period of axonal regeneration, forskolin did not significantly alter their electrical properties. Taken together, these results suggest that activation of cAMP signaling by dbcAMP stimulates neurite outgrowth, but does not alter the electrical properties of lamprey RS neurons in such a way that would be expected to induce calcium influx. In conclusion, our results suggest that activation of cAMP pathways alone, without compensation for possible

  18. Cyclic AMP-induced K+ secretion occurs independently of Cl- secretion in rat distal colon.

    PubMed

    Sandle, Geoffrey I; Rajendran, Vazhaikkurichi M

    2012-08-01

    cAMP induces both active Cl(-) and active K(+) secretion in mammalian colon. It is generally assumed that a mechanism for K(+) exit is essential to maintain cells in the hyperpolarized state, thus favoring a sustained Cl(-) secretion. Both Kcnn4c and Kcnma1 channels are located in colon, and this study addressed the questions of whether Kcnn4c and/or Kcnma1 channels mediate cAMP-induced K(+) secretion and whether cAMP-induced K(+) secretion provides the driving force for Cl(-) secretion. Forskolin (FSK)-enhanced short-circuit current (indicator of net electrogenic ion transport) and K(+) fluxes were measured simultaneously in colonic mucosa under voltage-clamp conditions. Mucosal Na(+) orthovanadate (P-type ATPase inhibitor) inhibited active K(+) absorption normally present in rat distal colon. In the presence of mucosal Na(+) orthovanadate, serosal FSK induced both K(+) and Cl(-) secretion. FSK-induced K(+) secretion was 1) not inhibited by either mucosal or serosal 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34; a Kcnn4 channel blocker), 2) inhibited (92%) by mucosal iberiotoxin (Kcnma1 channel blocker), and 3) not affected by mucosal cystic fibrosis transmembrane conductance regulator inhibitor (CFTR(inh)-172). By contrast, FSK-induced Cl(-) secretion was 1) completely inhibited by serosal TRAM-34, 2) not inhibited by either mucosal or serosal iberiotoxin, and 3) completely inhibited by mucosal CFTR(inh)-172. These results indicate that cAMP-induced colonic K(+) secretion is mediated via Kcnma1 channels located in the apical membrane and most likely contributes to stool K(+) losses in secretory diarrhea. On the other hand, cAMP-induced colonic Cl(-) secretion requires the activity of Kcnn4b channels located in the basolateral membrane and is not dependent on the concurrent activation of apical Kcnma1 channels. PMID:22648950

  19. Relationship between inhibition of cyclic AMP production in Chinese hamster ovary cells expressing the rat D2(444) receptor and antagonist/agonist binding ratios.

    PubMed Central

    Harley, E. A.; Middlemiss, D. N.; Ragan, C. I.

    1995-01-01

    1. Radioligand binding assays using [3H]-(-)-sulpiride, in the presence of 1 mM ethylenediaminetetraacetic acid (EDTA) and 100 microM guanylylimidodiphosphate (GppNHp) and [3H]-N0437 were developed to label the low and high agonist affinity states of the rD2(444) receptor (long form of the rat D2 receptor) respectively. The ratios of the affinities of compounds in these two assays (Kapp [3H]-(-)-supiride/Kapp [3H]-N-0437) were then calculated. 2. The prediction that the binding ratio reflected the functional efficacy of a compound was supported by measurement of the ability of a number of compounds acting at dopamine receptors to inhibit rD2(444)-mediated inhibition of cyclic AMP production. When the rank order of the ratios of a number of these compounds was compared to their ability to inhibit the production of cyclic AMP, a significant correlation was seen (Spearman rank correlation coefficient = 0.943, P = 0.01). 3. In conclusion, the sulpiride/N-0437 binding ratio reliably predicted the efficacy of compounds acting at dopamine receptors to inhibit cyclic AMP production mediated by the rD2(444) receptor. PMID:7582561

  20. Binding of regulatory subunits of cyclic AMP-dependent protein kinase to cyclic CMP agarose.

    PubMed

    Hammerschmidt, Andreas; Chatterji, Bijon; Zeiser, Johannes; Schröder, Anke; Genieser, Hans-Gottfried; Pich, Andreas; Kaever, Volkhard; Schwede, Frank; Wolter, Sabine; Seifert, Roland

    2012-01-01

    The bacterial adenylyl cyclase toxins CyaA from Bordetella pertussis and edema factor from Bacillus anthracis as well as soluble guanylyl cyclase α(1)β(1) synthesize the cyclic pyrimidine nucleotide cCMP. These data raise the question to which effector proteins cCMP binds. Recently, we reported that cCMP activates the regulatory subunits RIα and RIIα of cAMP-dependent protein kinase. In this study, we used two cCMP agarose matrices as novel tools in combination with immunoblotting and mass spectrometry to identify cCMP-binding proteins. In agreement with our functional data, RIα and RIIα were identified as cCMP-binding proteins. These data corroborate the notion that cAMP-dependent protein kinase may serve as a cCMP target. PMID:22808067

  1. Cyclic AMP-dependent phosphorylation of neuronal nitric oxide synthase mediates penile erection

    PubMed Central

    Hurt, K. Joseph; Sezen, Sena F.; Lagoda, Gwen F.; Musicki, Biljana; Rameau, Gerald A.; Snyder, Solomon H.; Burnett, Arthur L.

    2012-01-01

    Nitric oxide (NO) generated by neuronal NO synthase (nNOS) initiates penile erection, but has not been thought to participate in the sustained erection required for normal sexual performance. We now show that cAMP-dependent phosphorylation of nNOS mediates erectile physiology, including sustained erection. nNOS is phosphorylated by cAMP-dependent protein kinase (PKA) at serine(S)1412. Electrical stimulation of the penile innervation increases S1412 phosphorylation that is blocked by PKA inhibitors but not by PI3-kinase/Akt inhibitors. Stimulation of cAMP formation by forskolin also activates nNOS phosphorylation. Sustained penile erection elicited by either intracavernous forskolin injection, or augmented by forskolin during cavernous nerve electrical stimulation, is prevented by the NOS inhibitor l-NAME or in nNOS-deleted mice. Thus, nNOS mediates both initiation and maintenance of penile erection, implying unique approaches for treating erectile dysfunction. PMID:23012472

  2. Occurrence of cyclic AMP and related enzymes during germination of Pinus pinea seeds.

    PubMed

    Martelli, P; Lusini, P; Bovalini, L; Bartali, R; Franchi, G G; Cinci, G

    1987-01-01

    The occurrence of cAMP, adenylate cyclase and cAMP phosphodiesterase has been tested in Pinus pinea seed during germination. The study has been carried out on dormant and imbibed seeds, seedlings, endospermic residues, roots and cotyledons. cAMP has been detected by the protein binding method and its occurrence has been verified by HPLC detections. cAMP phosphodiesterase shows a very high activity at acidic pH, while being completely inactive at pH 7.4. At this pH value, well detectable levels of adenylate cyclase have been observed. Therefore, the classical pathway of synthesis and breakdown of cAMP, already accepted for animal and bacterial cells, seems to be operating in Pinus pinea plant too. PMID:3038780

  3. Xanthine effects on renal proximal tubular function and cyclic AMP metabolism.

    PubMed

    Coulson, R; Scheinman, S J

    1989-02-01

    We evaluated the renal effects of xanthines using two in vitro models: the isolated perfused rat kidney (IPRK) and cultured opossum kidney (OK) cells, a continuous cell line that resembles proximal tubule and responds to parathyroid hormone (PTH). 1,3-Diethyl-8-phenylxanthine (DPX) a potent adenosine receptor antagonist, increased urine volume, glomerular filtration rate, vascular resistance and the fractional excretions of Na, K, Ca and Pi in the IPRK. DPX lowered the Na-dependent uptake of Pi by OK cells. By comparison enprofylline, 3-propylxanthine (ENP), a weak adenosine receptor antagonist, produced a slight elevation in glomerular filtration rate but no changes in electrolyte excretion by IPRK or Pi uptake by OK cells. Both DPX and ENP produced negligible elevations in basal IPRK cAMP. A 1-nM bolus of PTH elevated urinary and perfusate cAMP 50- and 10-fold, respectively. PTH-elevated urinary and perfusate cAMP were augmented further 4- to 7-fold with DPX and 3- to 4-fold with ENP (All IPRK experiments used 50 microM xanthine). OK cells produced a 2-fold cAMP response to 10 nM PTH alone. OK cells treated with 50 microM DPX exhibited no increase in basal but a 13-fold increase in PTH-stimulated cell cAMP. The rank order of potency at 50 microM to augment OK cell cAMP with 10 nM PTH was DPX greater than 1,3-dipropyl-8-cyclopentylxanthine (DPC) greater than 1-methyl-3-isobutylxanthine greater than theobromine greater than theophylline greater than caffeine greater than ENP = no effect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2537403

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

    SciTech Connect

    Sonnenburg, W.K.

    1987-01-01

    In the rabbit cortical collecting tubule (RCCT), prostaglandin E/sub 1/ (PGE/sub 1/) and prostaglandin E/sub 2/ (PGE/sub 2/) at 1 nM inhibit arginine-vasopressin (AVP)-induced water reabsorption, while 100 nM PGE/sub 1/ and PGE/sub 2/ alone stimulate water reabsorption. Reported here are studies designed to investigate the molecular basis for the biphasic physiological action of PGE/sub 1/ and PGE/sub 2/ in the collecting duct. In freshly isolated RCCT cells, PGE/sub 1/, PGE/sub 2/, and 16,16-dimethyl-PGE/sub 2/ (DM-PGE/sub 2/) stimulated cAMP synthesis at concentrations ranging from 0.1 to 10 M. Other prostaglandins including the synthetic PGE/sub 2/ analogue, sulprostone, failed to stimulate cAMP synthesis. Moreover, sulprostone did not antagonize PGE/sub 2/-stimulated cAMP formation. In contrast, PGE/sub 2/ and sulprostone at concentrations ranging from 1 to 100 nM, inhibited AVP-induced cAMP accumulation in freshly isolated RCCT cells. PGE/sub 2/, PGE/sub 1/, DM-PGE/sub 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/sub 2/ binding to freshly isolated RCCT cell membranes was characterized. Two different classes of PGE/sub 2/ binding were detected. //sup 3/H/PGE/sub 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, //sup 3/H/ PGE/sub 2/ binding to the low affinity class of sites was decreased by GTP S; this inhibitory effect was not blocked by pertussis toxin pretreatment.

  5. Cyclic AMP-dependent Protein Lysine Acylation in Mycobacteria Regulates Fatty Acid and Propionate Metabolism*

    PubMed Central

    Nambi, Subhalaxmi; Gupta, Kallol; Bhattacharyya, Moitrayee; Ramakrishnan, Parvathy; Ravikumar, Vaishnavi; Siddiqui, Nida; Thomas, Ann Terene; Visweswariah, Sandhya S.

    2013-01-01

    Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guérin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host. PMID:23553634

  6. The cAMP response element binding protein, CREB, is a potent inhibitor of diverse transcriptional activators.

    PubMed Central

    Lemaigre, F P; Ace, C I; Green, M R

    1993-01-01

    Cyclic AMP response element binding protein (CREB) activates transcription of cAMP response element (CRE)-containing promoters following an elevation of intracellular cAMP. Here we show that CREB and the highly related protein ATF-1 are also potent transcription inhibitors. Strikingly, CREB inhibits transcription of multiple activators, whose DNA-binding domains and activation regions are unrelated to one another. Inhibition requires that the CREB dimerization and DNA-binding domains are intact. However, inhibition is not dependent upon the presence of a CRE in the promoter, and does not involve heterodimer formation between CREB and the activator. The ability of an activator protein to inhibit transcription in such a promiscuous fashion has not been previously reported. Images PMID:8332500

  7. Dose and Chemical Modification Considerations for Continuous Cyclic AMP Analog Delivery to the Injured CNS

    PubMed Central

    Fouad, Karim; Ghosh, Mousumi; Vavrek, Romana; Tse, Arthur D.

    2009-01-01

    Abstract In this investigation, two cell-permeable synthetic analogs of cAMP, dibutyryl-cAMP (db-cAMP) and 8-bromo-cAMP, which are widely used to elevate intracellular cAMP levels under experimental conditions, were investigated for their ability to dose-dependently improve histological and functional outcomes following continuous delivery in two models of incomplete spinal cord injury (SCI). The cAMP analogs were delivered via osmotic minipumps at 1–250 mM through an indwelling cortical cannula or by intrathecal infusion for up to 4 weeks after either a T8 unilateral over-hemisection or a C2-3 dorsolateral quadrant lesion, respectively. In both SCI models, continuous db-cAMP delivery was associated with histopathological changes that included sporadic micro-hemorrhage formation and cavitation, enhanced macrophage infiltration and tissue damage at regions beyond the immediate application site; no deleterious or beneficial effect of agent delivery was observed at the spinal injury site. Furthermore, these changes were accompanied by pronounced behavioral deficits that included an absence of progressive locomotor recovery, increased extensor tone, paralysis, and sensory abnormalities. These deleterious effects were not observed in saline-treated animals, in animals in which the db-cAMP dose did not exceed 1 mM, or in those animals that received a high dose (250 mM) of the alternative cAMP analog, 8-bromo-cAMP. These results demonstrate that, for continuous intraparenchymal or intrathecal administration of cAMP analogs for the study of biological or therapeutic effects within the central nervous system (CNS), consideration of the effective concentration applied as well as the potential toxicity of chemical moieties on the parent molecule and/or their activity needs to be taken into account. PMID:19397425

  8. Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α2C-adrenoceptors through the actin-binding protein filamin-2

    PubMed Central

    Motawea, Hanaa K. B.; Jeyaraj, Selvi C.; Eid, Ali H.; Mitra, Srabani; Unger, Nicholas T.; Ahmed, Amany A. E.; Flavahan, Nicholas A.

    2013-01-01

    The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α2C-ARs and also increased translocation of perinuclear α2C-ARs to intracellular F-actin and to the plasma membrane. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We utilized a yeast two-hybrid screen with human microvascular smooth muscle cells (microVSM) cDNA library and the α2C-AR COOH terminus to identify a novel interaction with the actin cross-linker filamin-2. Yeast α-galactosidase assays, site-directed mutagenesis, and coimmunoprecipitation experiments in heterologous human embryonic kidney (HEK) 293 cells and in human microVSM demonstrated that α2C-ARs, but not α2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, α2C-ARs colocalized with filamin on intracellular filaments and at the plasma membrane. Small interfering RNA-mediated knockdown of filamin-2 inhibited Rap1-induced redistribution of α2C-ARs to the cell surface and inhibited receptor function. The studies suggest that cAMP-Rap1-Rho-ROCK signaling facilitates receptor translocation and function via phosphorylation of filamin-2 Ser2113. Together, these studies extend our previous findings to show that functional rescue of α2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function. PMID:23864608

  9. Pharmacological characterization of the dopamine receptor coupled to cyclic AMP formation expressed by rat mesenteric artery vascular smooth muscle cells in culture.

    PubMed Central

    Hall, A. S.; Bryson, S. E.; Vaughan, P. F.; Ball, S. G.; Balmforth, A. J.

    1993-01-01

    1. Mesenteric artery vascular smooth muscle cells derived from male Wistar rats and grown in culture were prelabelled with [3H]-adenine and exposed to a range of dopamine receptor agonists and antagonists. Resultant [3H]-cyclic AMP formation was determined and concentration-effect curves constructed, in the presence of propranolol (10-6) M) and the phosphodiesterase inhibitor IBMX (5 x 10(-4) M). 2. Ka apparent values for D1/DA1 dopamine receptor agonists SKF 38393, fenoldopam, 6,7-ADTN, and dopamine were 0.06, 0.59, 4.06 and 5.77 x 10(-6) M respectively. Although fenoldopam and SKF 38393 were more potent than dopamine, they were partial agonists with efficacies, relative to dopamine of approximately 48% and 24% respectively. 6,7-ADTN, in contrast, behaved as a full agonist. 3. Dopamine-stimulated cyclic AMP formation was inhibited in a concentration-dependent manner by the D1/DA1 dopamine receptor selective antagonists, SCH 23390 and cis-flupenthixol (Ki values 0.53 and 36.1 x 10(-1) M respectively). In contrast, the D2/DA2 dopamine receptor selective antagonists, domperidone and (-)-sulpiride, were less potent (Ki values 2.06 and 5.82 x 10(-6) M respectively). Furthermore, the stereoisomers of SCH 23390 and cis-flupenthixol, SCH 23388 and trans-flupenthixol, were at least two orders of magnitude less potent (Ki values 0.14 and 13.2 x 10(-6) M respectively) indicating the stereoselective nature of this receptor. 4. Our results indicate that rat mesenteric artery vascular smooth muscle cells in culture express a dopamine receptor coupled to cyclic AMP formation, which has the pharmacological profile, characteristic of the D1 dopamine receptor subfamily. PMID:7902178

  10. Opposing functional effects of cyclic GMP and cyclic AMP may act through protein phosphorylation in rabbit cardiac myocytes.

    PubMed

    Yan, L; Lee, H; Huang, M W; Scholz, P M; Weiss, H R

    2000-04-01

    1. We tested the hypothesis that the negative functional effects of cyclic GMP (cGMP) oppose the positive effects of cyclic AMP (cAMP) in cardiac myocytes through interaction at the level of their respective protein kinases. 2. Cell shortening was studied using a video-edge detector. The O2 consumption of a suspension of rabbit ventricular myocytes was measured using O2 electrodes. Protein phosphorylation was measured autoradiographically following SDS-PAGE. Data were collected with: (1) 8-bromo-cGMP (8-Br-cGMP) 10(-7) or 10(-5) M; (2) 8-bromo-cAMP (8-Br-cAMP) 10(-7) or 10(-5) M; (3) 8-Br-cAMP 10(-5) M followed by 8-Br-cGMP 10(-7) or 10(-5) M; (4) 8-Br-cGMP 10(-5) M followed by 8-Br-cAMP 10(-7) or 10(-5) M; (5) 8-Br-cGMP 10(-7) or 10(-5) M followed by KT 5720 (cAMP-dependent protein kinase inhibitor) or KT 5823 (cGMP-dependent protein kinase inhibitor) 10(-6) M; and (6) 8-Br-cAMP 10(-7) or 10(-5) M followed by KT 5720 or KT 5823 10(-6) M. 3. 8-Br-cGMP 10(-5) M decreased percent shortening (Pcs) from 6.3+/-0.6 to 3.6+/-0.4% and rate of shortening (Rs) from 66.7+/-4.4 to 41.8+/-4.2 microm s(-1). 8-Br-cAMP 10(-5) M increased Pcs (from 3.7+/-0.2 to 4.8+/-0.2) and Rs (from 50.0+/-3.0 to 60.0+/-3.1). With 8-Br-cAMP 10(-5) M, 8-Br-cGMP 10(-5) M decreased Pcs and Rs less. The positive functional effects of 8-Br-cAMP 10(-7) or 10(-5) M were also diminished with 8-Br-cGMP 10(-5) M. Following 8-Br-cGMP 10(-7) or 10(-5) M, KT 5720 10(-6) M further decreased Pcs to 2.5+/-0.3 and Rs to 30.0+/-4.1. KT 5823 10(-6) M returned Pcs to 4.7+/-0.4 and Rs to 61.3+/-5.3. Following 8-Br-cAMP 10(-7) or 10(-5) M, KT 5720 decreased the elevated Pcs and Rs significantly and KT 5823 10(-6) M further increased these parameters. 4. cGMP and cAMP phosphorylated the same five protein bands. With KT 5720 or KT 5823, all of the bands were lighter at the same concentration of 8-Br-cAMP and 8-Br-cGMP. 5. We conclude that, in rabbit ventricular myocytes, the opposing functional effects of cGMP and c

  11. Analysis of the Escherichia coli gene encoding L-asparaginase II, ansB, and its regulation by cyclic AMP receptor and FNR proteins.

    PubMed

    Jennings, M P; Beacham, I R

    1990-03-01

    Escherichia coli contains two L-asparaginase isozymes: L-asparaginase I, a low-affinity enzyme located in the cytoplasm, and L-asparaginase II, a high-affinity secreted enzyme. A molecular genetic analysis of the gene (ansA) encoding the former enzyme has previously been reported. We now present a molecular study of the gene, ansB, encoding L-asparaginase II. This gene was isolated by using oligonucleotide probes, whose sequences were based on the previously determined amino acid sequence. The nucleotide sequence of ansB, including 5'- and 3'-untranslated regions, was determined. The amino acid sequence of L-asparaginase II, deduced from this nucleotide sequence, contains differences at 11 positions when compared with the previously determined amino acid sequence. The deduced amino acid sequence also reveals a typical secretory signal peptide of 22 residues. A single region of sequence similarity is observed when ansA and ansB are compared. The transcriptional start site in ansB was determined, allowing the identification of the promoter region. The regulation of ansB was studied by using ansB'-'lacZ fusions, together with a deletion analysis of the 5' region upstream of the promoter. Regulation by cyclic AMP receptor protein and anaerobiosis (FNR protein) was confirmed, and the presence of nucleotide sequence motifs, with homology to cyclic AMP receptor protein and FNR protein-binding sites, investigated. PMID:2407723

  12. Cyclic AMP receptor protein (CRP) regulates the expression of cspA, cspB, cspG and cspI, members of cspA family, in Escherichia coli.

    PubMed

    Uppal, Sheetal; Jawali, Narendra

    2015-04-01

    Escherichia coli K-12 contains nine paralogs of CspA, CspA-CspI, collectively known as CspA family of cold-shock proteins (CSPs). In spite of the high degree of similarity among themselves, only five (cspA, B, E, G and I) are induced during cold-stress. In the present study, we show that cspB, cspG and cspI, the members of cspA family, known to be induced in response to cold shock, are regulated by cyclic AMP receptor protein (CRP) , a global regulator involved in sugar metabolism, during growth at 37 °C as well as at 15 °C, as seen by green fluorescent protein (gfp) promoter fusions assays. Interestingly, cspA is selectively regulated by CRP during growth at 15 °C but not at 37 °C. The regulation of cspA, cspB, cspG and cspI by CRP was found to be through an indirect mechanism as determined by electrophoretic mobility shift assay (EMSA). These results substantiate our earlier study demonstrating a role for CRP during growth at low temperature. PMID:25637299

  13. Cyclic AMP stimulation of transferrin secretion by breast cancer cell grown on extracellular matrix or in two-compartment culture chambers

    SciTech Connect

    Vandewalle, B.; Hornez, L.; Revillion, F.; Lefebvre, J. )

    1991-06-28

    Extrahepatic synthesis and secretion of transferrin (Tf), the major iron-carrying protein, have been described in normal and tumoral tissues suggesting a potential role for paracrine or autocrine function. In breast tumor cell MCF-7, we have previously shown a Tf secretion stimulated by estradiol which might confer selective growth advantages of these rapidly proliferating cells. The present work refers to possible additional Tf functions related to differentiation of breast tumor cells. We induced MCF-7 cell differentiation by the cyclic AMP derivative, dibutyryl cAMP (dB cAMP) and studied Tf secretion in different culture conditions after labeling with (35S) methionine. Our results demonstrate that dB cAMP stimulates Tf secretion only in culture environment that permits access to the basolateral surface and caters to the polarity requirements of the cell. These results suggest that Tf may also act as a modulator of cellular differentiation in breast cancer cells.

  14. Phosphorylation by protein kinase C and cyclic AMP-dependent protein kinase of synthetic peptides derived from the linker region of human P-glycoprotein.

    PubMed Central

    Chambers, T C; Pohl, J; Glass, D B; Kuo, J F

    1994-01-01

    Specific sites in the linker region of human P-glycoprotein phosphorylated by protein kinase C (PKC) were identified by means of a synthetic peptide substrate, PG-2, corresponding to residues 656-689 from this region of the molecule. As PG-2 has several sequences of the type recognized by the cyclic AMP-dependent protein kinase (PKA), PG-2 was also tested as a substrate for PKA. PG-2 was phosphorylated by purified PKC in a Ca2+/phospholipid-dependent manner, with a Km of 1.3 microM, and to a maximum stoichiometry of 2.9 +/- 0.1 mol of phosphate/mol of peptide. Sequence analysis of tryptic fragments of PG-2 phosphorylated by PKC identified Ser-661, Ser-667 and Ser-671 as the three sites of phosphorylation. PG-2 was also found to be phosphorylated by purified PKA in a cyclic AMP-dependent manner, with a Km of 21 microM, and to a maximum stoichiometry of 2.6 +/- 0.2 mol of phosphate/mol of peptide. Ser-667, Ser-671 and Ser-683 were phosphorylated by PKA. Truncated peptides of PG-2 were utilized to confirm that Ser-661 was PKC-specific and Ser-683 was PKA-specific. Further studies showed that PG-2 acted as a competitive substrate for the P-glycoprotein kinase present in membranes from multidrug-resistant human KB cells. The membrane kinase phosphorylated PG-2 mainly on Ser-661, Ser-667 and Ser-671. These results show that human P-glycoprotein can be phosphorylated by at least two protein kinases, stimulated by different second-messenger systems, which exhibit both overlapping and unique specificities for phosphorylation of multiple sites in the linker region of the molecule. Images Figure 3 Figure 5 PMID:7909431

  15. Identification of two splice variant forms of type-IVB cyclic AMP phosphodiesterase, DPD (rPDE-IVB1) and PDE-4 (rPDE-IVB2) in brain: selective localization in membrane and cytosolic compartments and differential expression in various brain regions.

    PubMed

    Lobban, M; Shakur, Y; Beattie, J; Houslay, M D

    1994-12-01

    In order to detect the two splice variant forms of type-IVB cyclic AMP phosphodiesterase (PDE) activity, DPD (type-IVB1) and PDE-4 (type-IVB2), anti-peptide antisera were generated. One set ('DPD/PDE-4-common'), generated against a peptide sequence found at the common C-terminus of these two PDEs, detected both PDEs. A second set was PDE-4 specific, being directed against a peptide sequence found within the unique N-terminal region of PDE-4. In brain, DPD was found exclusively in the cytosol and PDE-4 exclusively associated with membranes. Both brain DPD and PDE-4 activities, isolated by immunoprecipitation, were cyclic AMP-specific (KmcyclicAMP: approximately 5 microM for DPD; approximately 4 microM for PDE-4) and were inhibited by low rolipram concentrations (K1rolipram approximately 1 microM for both). Transient expression of DPD in COS-1 cells allowed identification of an approx. 64 kDa species which co-migrated on SDS/PAGE with the immunoreactive species identified in both brain cytosol and membrane fractions using the DPD/PDE-4-common antisera. The subunit size observed for PDE-4 (approx. 64 kDa) in brain membranes was similar to that predicted from the cDNA sequence, but that observed for DPD was approx. 4 kDa greater. Type-IV, rolipram-inhibited PDE activity was found in all brain regions except the pituitary, where it formed between 30 and 70% of the PDE activity in membrane and cytosolic fractions when assayed with 1 microM cyclic AMP, PDE-4 formed 40-50% of the membrane type-IV activity in all brain regions save the midbrain (approx. 20%). DPD distribution was highly restricted to certain regions, providing approx. 35% of the type-IV cytosolic activity in hippocampus and 13-21% in cortex, hypothalamus and striatum with no presence in brain stem, cerebellum, midbrain and pituitary. The combined type-IVB PDE activities of DPD and PDE-4 contributed approx. 10% of the total PDE activity in most brain regions except for the pituitary (zero) and the mid

  16. Involvement of a putative cyclic amp receptor protein (CRP)-like binding sequence and a CRP-like protein in glucose-mediated catabolite repression of thn genes in Rhodococcus sp. strain TFB.

    PubMed

    Tomás-Gallardo, Laura; Santero, Eduardo; Floriano, Belén

    2012-08-01

    Glucose catabolite repression of tetralin catabolic genes in Rhodococcus sp. strain TFB was shown to be exerted by a protein homologous to transcriptional regulators of the cyclic AMP receptor (CRP)-FNR family. The protein was detected bound to putative CRP-like boxes localized at the promoters of the thnA1 and thnS genes. PMID:22636000

  17. Effect of beta-ADrenergic Agonist on Cyclic AMP Synthesis in Chicken Skeletal Muscle Cells in Culture

    NASA Technical Reports Server (NTRS)

    Young, R. B.; Bridge, K. Y.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    Several beta-adrenergic receptor (bAR) agonists are known to cause hypertrophy of skeletal muscle tissue. Because it seems logical that these agonists exert their action on muscle through stimulation of cAMP synthesis, five bAR agonists encompassing a range in activity from strong to weak were evaluated for their ability to stimulate cAMP accumulation in embryonic chicken skeletal muscle cells in culture. Two strong agonists (epinephrine and isoproterenol), one moderate agonist (albuterol), and two weak agonists known to cause hypertrophy in animals (clenbuterol and cimaterol) were studied. Dose response curves were determined over six orders of magnitude in concentration for each agonist, and values were determined for their maximum stimulation of cAMP synthesis rate (Bmax) and the agonist concentration at which 50% stimulation of cAMP synthesis (EC50) occurred. Bmax values decreased in the following order: isoproterenol, epinephrine, albuterol, cimaterol, clenbuterol. Cimaterol and clenbuterol at their Bmax levels were approximately 15-fold weaker than isoproterenol in stimulating the rate of cAMP synthesis. In addition, the EC50 values for isoproterenol, cimaterol, clenbuterol, epinephrine, and albuterol were 360 nM, 630 nM, 900 nM, 2,470 nM, and 3,650 nM, respectively. Finally, dose response curves show that the concentrations of cimaterol and clenbuterol in culture media at concentrations known to cause significant muscle hypertrophy in animals had no detectable effect on stimulation of CAMP accumulation in chicken skeletal muscle cells.

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

    PubMed Central

    Issinger, O G; Beier, H; Speichermann, N; Flokerzi, V; Hofmann, F

    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 identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6. In 90 min between 0.7 and 1.0 mol of phosphate/mol of protein S6 was incorporated by the catalytic subunit of cyclic AMP-dependent protein kinase. Of the other proteins, S3 and S7 from the small subunit and proteins L6, L18, L19 and L35 from the large subunit were predominantly phosphorylated 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 by either kinase; (3) proteins S7 and L29 were almost exclusively phosphorylated by the cyclic AMP-dependent protein kinase; (4) protein S6 and most of the other proteins were phosphorylated about two or three times faster by the cyclic AMP-dependent than by the cyclic GMP-dependent enzyme. Images Fig. 1. Fig. 2. PMID:6246882

  19. Effects of dibutyryl cyclic AMP on the syntheses of dolichol-linked saccharides and glycoproteins in cultured hepatoma cells. Correlation with the effect on the adhesiveness of the cells.

    PubMed Central

    Okamoto, Y; Sakai, H; Sato, J; Akamatsu, N

    1983-01-01

    When the hepatoma cells (AH 70Btc, Clone 10-5) were cultured in the presence of 1 mM-dibutyryl cyclic AMP for 2 days, the incorporation of [14C]glucosamine into protein was increased over 2-fold. At the same time, dibutyryl cyclic AMP increased the incorporation of [14C]glucosamine into dolichol-linked N-acetylglucosamine and NN'-diacetylchitobiose about 1.5-fold and into dolichol-linked oligosaccharides about 3-fold. Analysis of cellular glycoproteins by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after reduction showed that dibutyryl cyclic AMP specifically enhanced the glycosylation of a fibronectin-like glycoprotein with an apparent mol.wt. of 220 000 and two other high-molecular-weight glycoproteins (apparent mol.wts. 270 000 and 185 000). Increased glycosylation of the glycoproteins with mol.wts. of 220 000 and 185 000 was shown to be linked to increased synthesis of the polypeptide portion. In addition to the above effects, dibutyryl cyclic AMP enhanced the adhesiveness of AH 70Btc cells to glass surfaces. Both the effects on the glycosylation pathway and on adhesiveness of cells were reversed by further treatment of the cells with 1 microgram of tunicamycin/ml. The results indicated that dibutyryl cyclic AMP increased the synthesis of dolichol-linked oligosaccharides and N-glycosylation of proteins in AH 70Btc cells. The enhancement of adhesiveness may be mediated by the increased synthesis of dolichol-linked oligosaccharides and also may be related to the increased synthesis of fibronectin. PMID:6309156

  20. Wound-healing effect of ginsenoside Rd from leaves of Panax ginseng via cyclic AMP-dependent protein kinase pathway.

    PubMed

    Kim, Wang-Kyun; Song, Seung-Yong; Oh, Won Keun; Kaewsuwan, Sireewan; Tran, Tien Lam; Kim, Won-Serk; Sung, Jong-Hyuk

    2013-02-28

    Panax ginseng is considered as one of the most valuable medicinal herbs in traditional medicine, and ginsenoside Rd is one of the main active ingredients in P. ginseng leaf. Although there is significant number of evidences implicated on the beneficial effects of the ginsenosides with diverse associated mechanisms, reports on the skin regeneration by the ginsenoside Rd are not sufficient. Therefore, we examined the mitogenic and protective effects of the ginsenoside Rd in the keratinocyte progenitor cells (KPCs) and human dermal fibroblasts (HDFs). Furthermore, the signaling pathways involved in the activation of KPCs and HDFs were investigated, and wound-healing effect is evaluated in vivo through animal wound models. We found that the ginsenoside Rd significantly increased the proliferation and migration level of KPCs and HDFs in a dose-dependent manner. Additionally, the cell survival was significantly increased in H2O2 treated KPCs. Moreover, the ginsenoside Rd effectively induced collagen type 1 and down-regulated matrix metalloprotinase-1 (MMP-1) in a dose-dependent manner. All of these beneficial effects are associated with an induction of intracellular cAMP levels and phosphorylated cAMP response element-binding protein expression in nucleus, which both attenuated by adenine 9-β-d-arabinofuranoside, an adenylate cyclase inhibitor. Application of the ginsenoside Rd to an excision wound in mice showed an effective healing process. As skin regeneration is mainly associated with the activation of HDFs and KPCs, P. ginseng leaf, an alternative source of the ginsenoside Rd, can be used as a natural source for skin regeneration. PMID:23399764

  1. Increase in Cellular Cyclic AMP Concentrations Reverses the Profibrogenic Phenotype of Cardiac Myofibroblasts: A Novel Therapeutic Approach for Cardiac Fibrosis

    PubMed Central

    Lu, David; Aroonsakool, Nakon; Yokoyama, Utako; Patel, Hemal H.

    2013-01-01

    Tissue fibrosis is characterized by excessive production, deposition, and contraction of the extracellular matrix (ECM). The second messenger cAMP has antifibrotic effects in fibroblasts from several tissues, including cardiac fibroblasts (CFs). Increased cellular cAMP levels can prevent the transformation of CFs into profibrogenic myofibroblasts, a critical step that precedes increased ECM deposition and tissue fibrosis. Here we tested two hypotheses: 1) myofibroblasts have a decreased ability to accumulate cAMP in response to G protein–coupled receptor (GPCR) agonists, and 2) increasing cAMP will not only prevent, but also reverse, the myofibroblast phenotype. We found that myofibroblasts produce less cAMP in response to GPCR agonists or forskolin and have decreased expression of several adenylyl cyclase (AC) isoforms and increased expression of multiple cyclic nucleotide phosphodiesterases (PDEs). Furthermore, we found that forskolin-promoted increases in cAMP or N6-phenyladenosine-cAMP, a protein kinase A–selective analog, reverse the myofibroblast phenotype, as assessed by the expression of collagen Iα1, α–smooth muscle actin, plasminogen activator inhibitor–1, and cellular contractile abilities, all hallmarks of a fibrogenic state. These results indicate that: 1) altered expression of AC and PDE isoforms yield a decrease in cAMP concentrations of cardiac myofibroblasts (relative to CFs) that likely contributes to their profibrotic state, and 2) approaches to increase cAMP concentrations not only prevent fibroblast-to-myofibroblast transformation but also can reverse the profibrotic myofibroblastic phenotype. We conclude that therapeutic strategies designed to enhance cellular cAMP concentrations in CFs may provide a means to reverse excessive scar formation following injury and to treat cardiac fibrosis. PMID:24085841

  2. Modulation of VEGF-induced endothelial cell cycle protein expression through cyclic AMP hydrolysis by PDE2 and PDE4.

    PubMed

    Favot, Laure; Keravis, Thérèse; Lugnier, Claire

    2004-09-01

    Endothelial cell proliferation in response to VEGF plays an important role in physiological and pathological angiogenesis. The role of PDE2 and PDE4 in VEGF-induced proliferation in HUVEC was investigated: 1) VEGF increased cAMP-hydrolytic activity by up-regulating the expression of PDE2 and PDE4 isozymes; 2) VEGF increased progression in cell cycle with an increase in p42/p44 MAP kinase, cyclin A and cyclin D1 expressions and with a decrease in p21 waf1/cip1 and p27 kip1 expressions; 3) EHNA (20 micro M), a selective PDE2 inhibitor, RP73401 (10 micro M), a selective PDE4 inhibitor blocked the VEGF-induced increase in p42/p44 MAP kinase expression; 4) RP73401, but not EHNA, blocked the VEGF-induced increase in cyclin A and decrease in p27 kip1 expressions; 5) EHNA, contrary to RP73401, enhanced the VEGF-induced increase of cyclin A and decrease of p27 kip1. 6) EHNA and RP73401 together blocked the VEGF-induced increase in cyclin D1 and decrease in p21 waf1/cip1 expressions; 7) Inhibition of VEGF-upregulated PDE2 and PDE4 reversed the VEGF-induced alterations in cell cycle protein expression, bringing back endothelial cells to a non-proliferating status. Consequently, PDE2 and PDE4 inhibitions were able to inhibit VEGF-induced endothelial cell proliferation by restoring cell cycle key protein expression, and might thus be useful in excessive angiogenesis. Furthermore, the differences between PDE2 and PDE4 effects may suggest compartmentalized effects. PMID:15351862

  3. Cyclic nucleotide responses and radiation-induced mitotic delay in Physarum polycephalum

    SciTech Connect

    Daniel, J.W.; Oleinick, N.L.

    1984-02-01

    The response of the plasmodial levels of cyclic AMP and cyclic GMP in Physarum polycephalum to several putative phosphodiesterase inhibitors and to ionizing radiation has been measured. Isobutylmethylxanthine (2 mM) induces a rapid transient threefold elevation of cyclic AMP alone, with maximum response in about 10 min and return to the base line in about 30 min. Theophylline (2 mM) induces a rapid, sustained twofold elevation of cyclic GMP only. Caffeine (2mM) and Ro-20-1724 (18 ..mu..M) both elicit a rapid transient rise in cyclic AMP, resembling the isobutylmethylxanthine response, and a slow transient elevation of the cyclic GMP level. Of particular interest is the rapid threefold transient elevation of the cyclic AMP, but not of the cyclic GMP, level by ..gamma.. radiation.

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

    PubMed

    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. PMID:1322129

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

    PubMed Central

    Brierley, I; Hoggett, J G

    1992-01-01

    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. Images Fig. 1. Fig. 3. Fig. 4. PMID:1322129

  6. Ligands to the platelet fibrinogen receptor glycoprotein IIb-IIIa do not affect agonist-induced second messengers Ca2+ or cyclic AMP.

    PubMed Central

    Williams, J A; Ashby, B; Daniel, J L

    1990-01-01

    Previous studies have suggested that the platelet glycoprotein complex GPIIb-IIIa, which is the putative fibrinogen receptor, regulates Ca2+ influx into platelets, possibly operating as a Ca2+ channel. We have used RGD-peptides (peptides containing the sequence Arg-Gly-Asp; disintegrins), isolated from snake venoms, that have a high affinity and specificity for the fibrinogen-binding site of GPIIb-IIIa to address the question of whether blocking this site inhibits Ca2+ movement from the extracellular medium to the cytosol. Using fura-2-loaded human platelets, we found that neither disintegrins nor a monoclonal antibody (M148) to the GPIIb-IIIa complex altered the level of cytosolic Ca2+ obtained when the cells were stimulated with various agonists in the presence of either nominal or 1 mM extracellular Ca2+. In the presence of Mn2+, an ion that quenches fura-2 fluorescence, fura-2-loaded platelets were stimulated with thrombin or ADP. Neither disintegrins nor the monoclonal antibody altered the kinetics or the amount of quenching of fura-2 fluorescence by Mn2+. These data indicate that the binding of ligands to the fibrinogen receptor is not associated with an inhibition of Ca2+ movement through a receptor-operated channel. Furthermore, the disintegrins have no effect on platelet cyclic AMP metabolism in either the presence or the absence of phosphodiesterase inhibitors. PMID:2168700

  7. Identification of sigma S-regulated genes in Salmonella typhimurium: complementary regulatory interactions between sigma S and cyclic AMP receptor protein.

    PubMed Central

    Fang, F C; Chen, C Y; Guiney, D G; Xu, Y

    1996-01-01

    sigma S (RpoS)-regulated lacZ transcriptional fusions in Salmonella typhimurium were identified from a MudJ transposon library by placing the rpoS gene under the control of the araBAD promoter and detecting lacZ expression in the presence or absence of arabinose supplementation. Western blot (immunoblot) analysis of bacteria carrying PBAD::rpoS demonstrated arabinose-dependent rpoS expression during all phases of growth. sigma S-dependent gene expression of individual gene fusions was confirmed by P22-mediated transduction of the MudJ insertions into wild-type or rpoS backgrounds. Analysis of six insertions revealed the known sigma S-regulated gene otsA, as well as five novel loci. Each of these genes is maximally expressed in stationary phase, and all but one show evidence of cyclic AMP receptor protein-dependent repression during logarithmic growth which is relieved in stationary phase. For these genes, as well as for the sigma S-regulated spvB plasmid virulence gene, a combination of rpoS overexpression and crp inactivation can result in high-level expression during logarithmic growth. The approach used to identify sigma S-regulated genes in this study provides a general method for the identification of genes controlled by trans-acting regulatory factors. PMID:8752327

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

    NASA Technical Reports Server (NTRS)

    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.

  9. Transcriptome analysis of cyclic AMP-dependent protein kinase A–regulated genes reveals the production of the novel natural compound fumipyrrole by Aspergillus fumigatus

    PubMed Central

    Macheleidt, Juliane; Scherlach, Kirstin; Neuwirth, Toni; Schmidt-Heck, Wolfgang; Straßburger, Maria; Spraker, Joseph; Baccile, Joshua A.; Schroeder, Frank C.; Keller, Nancy P.; Hertweck, Christian; Heinekamp, Thorsten; Brakhage, Axel A.

    2015-01-01

    Summary Aspergillus fumigatus is an opportunistic human pathogenic fungus causing life-threatening infections in immunocompromised patients. Adaptation to different habitats and also virulence of the fungus depends on signal perception and transduction by modules such as the cyclic AMP-dependent protein kinase A (PKA) pathway. Here, by transcriptome analysis, 632 differentially regulated genes of this important signaling cascade were identified, including 23 putative transcriptional regulators. The highest upregulated transcription factor gene was located in a previously unknown secondary metabolite gene cluster, which we named fmp, encoding an incomplete nonribosomal peptide synthetase, FmpE. Overexpression of the regulatory gene fmpR using the TetOn system led to the specific expression of the other six genes of the fmp cluster. Metabolic profiling of wild type and fmpR overexpressing strain by HPLC-DAD and HPLCHRESI-MS and structure elucidation by NMR led to identification of 5-benzyl-1H-pyrrole-2-carboxylic acid, which we named fumipyrrole. Fumipyrrole was not described as natural product yet. Chemical synthesis of fumipyrrole confirmed its structure. Interestingly, deletion of fmpR or fmpE led to reduced growth and sporulation of the mutant strains. Although fmp cluster genes were transcribed in infected mouse lungs, deletion of fmpR resulted in wild-type virulence in a murine infection model. PMID:25582336

  10. Regulation of cyclic AMP synthesis in Escherichia coli K-12: effects of the rpoD800 sigma mutation, glucose, and chloramphenicol.

    PubMed Central

    Grossman, A D; Ullmann, A; Burgess, R R; Gross, C A

    1984-01-01

    An immediate 12-fold inhibition in the rate of beta-galactosidase synthesis occurs in Escherichia coli cells containing the mutant sigma allele rpoD800 after a shift to 42 degrees C. In the present study we characterize the nature of the inhibition. The severe inhibition of beta-galactosidase synthesis was partly relieved by cyclic AMP (cAMP). We inferred that the inhibition might be mediated by a decreased intracellular concentration of cAMP. Consistent with this inference, the rate of cAMP accumulation in mutant cells after a temperature upshift was depressed relative to that in wild-type cells. Glucose and chloramphenicol, two agents known to inhibit differentially beta-galactosidase mRNA synthesis, caused a similar inhibition in the rate of cAMP accumulation. Thus, three diverse stimuli, glucose, chloramphenicol, and a temperature-sensitive sigma mutation, appear to affect beta-galactosidase synthesis by regulating the synthesis of cAMP. PMID:6325382

  11. Differential regulation of the β-adrenoceptor density and cyclic AMP level with age and sex in turkey cardiac chambers.

    PubMed

    Hoffmann, Sandra; Böhme, Julia; Kube, Christian; Haufe, Jörg; Krautwald-Junghanns, Maria-Elisabeth; Abraham, Getu

    2016-04-15

    Decreased responses of the heart to β-adrenoceptor stimulation with aging have been shown to occur merely in selected heart chambers in relation to increased catecholamine levels. However, there are no systematic studies that investigate all cardiac chambers with regard to receptor density and cAMP (adenosine 3', 5'-cyclic monophosphate) responses. We used meat-type turkey poults (British United Turkey (B.U.T.) Big 6) with increasing age because their heart seems to decrease in weight in relation to body weight and they are often used as an animal model for heart failure. The receptor density and distribution were quantified by radioligand binding analysis using (-)-[(125)I]-iodocyanopindolol and β-adrenoceptor subtype-specific antagonists (ICI 118.551 and CGP 20712 A) in membranes of four cardiac chambers (right and left atria and ventricles) of 6-week-, 12-week-, 16/21-week-, and 57-week-old B.U.T. BIG 6 turkeys. Receptor function was determined by measuring basal and stimulated cAMP production. In both sexes, the β-adrenoceptor density decreased significantly in all chambers with age without altered β-adrenoceptor subtype distribution. The receptor affinity (KD) to the radioligand was similar in hearts of all age groups. β-adrenoceptor-(isoproterenol and guanosine 5'-triphosphate), G-protein-(NaF) and catalytic unit of adenylate cyclase (forskolin, Mn(2+)) mediated cAMP responses were not chamber-dependent. Indeed, the cAMP level was significantly lower in 57-week-old hearts than in 6-week-, 12-week-, 16/21-week-old hearts. These data suggest that with increasing age and body weight, the β-adrenoceptor signal transduction pathway was highly blunted in all cardiac chambers, occurring by decreased receptor density and cAMP responses. PMID:26957056

  12. Boron and silicon: Effects on growth, plasma lipids, urinary cyclic AMP and bone and brain mineral composition of male rats

    SciTech Connect

    Seaborn, C.D.; Nielsen, F.H. . Grand Forks Human Nutrition Research Center)

    1994-06-01

    Because boron resembles silicon in its chemical properties, an experiment was performed to determine if excessive dietary boron would affect the response to silicon deprivation and, conversely, if silicon would influence the effects of an excessive intake of boron. Male weanling Sprague-Dawley rats were assigned to groups of 6 or 12 in a two-by-two factorially arranged experiment. Supplemented to a ground corn/casein diet containing 1.2 [mu]g silicon and 3 [mu]g boron per gram were silicon as sodium metasilicate at 0 or 50 [mu]g/g and boron as orthoboric acid at 0 or 500 [mu]g/g diet. At nine weeks, animals fed high dietary boron had significantly decreased final body weights, liver-weight-to-body-weight ratios, urinary cAMP concentrations, plasma triglyceride, cholesterol, glycine, valine, leucine, and lysine concentrations and skull copper, sodium, and manganese concentrations. High dietary boron also significantly increased brain-weight-to-body-weight ratios, magnesium concentrations of femur, brain, and plasma, zinc concentration of femur, and iron concentration of skull. The bone mineral findings suggest that excess dietary boron exerts subtle effects on bone composition. Dietary silicon affected blood urea nitrogen, hematocrit, hemoglobin, and the concentrations of plasma threonine and aspartic acid in animals fed excess boron. Depression of the testes-weight-to-body-weight ratio of animals fed 500 [mu]g boron per gram diet was most marked in animals not fed silicon. Although excessive dietary boron did not markedly enhanced the response of rats to silicon deprivation, dietary silicon affected their response to high dietary boron. Thus, dietary silicon apparently can influence boron toxicity.

  13. Intracellular signaling in the regulation of renal Na-K-ATPase. I. Role of cyclic AMP and phospholipase A2.

    PubMed Central

    Satoh, T; Cohen, H T; Katz, A I

    1992-01-01

    We have reported that dopamine (DA) inhibits Na-K-ATPase activity in the cortical collecting duct (CCD) by stimulating the DA1 receptor, and the present study was designed to evaluate the mechanism of this effect. Short-term exposure (15-30 min) of microdissected rat CCD to DA, a DA1 agonist (fenoldopam), vasopressin (AVP), forskolin, or dibutyryl cAMP (dBcAMP), which increase cAMP content by different mechanisms, strongly (approximately 60%) inhibited Na-K-ATPase activity. 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, completely blocked Na-K-ATPase inhibition by DA or fenoldopam, and IP20, an inhibitor peptide of cAMP-dependent protein kinase A (PKA), abolished the Na:K pump effect of all the cAMP agonists listed above. To verify whether the mechanism of pump inhibition by agents that increase cell cAMP involves phospholipase A2 (PLA2), we used mepacrine, a PLA2 inhibitor, which also abolished Na-K-ATPase inhibition by DA or fenoldopam, as well as by AVP, forskolin, or dBcAMP. Arachidonic acid (10(-7) - 10(-4) M) inhibited Na-K-ATPase activity in dose-dependent fashion. Corticosterone, which induces lipomodulin, a PLA2 inhibitor protein inactivated by PKA, equally abolished the pump effects of DA, fenoldopam, forskolin, and dBcAMP, suggesting that lipomodulin might act between PKA and PLA2 in cAMP-dependent pump regulation. We conclude that dopamine inhibits Na-K-ATPase activity in the CCD through a DA1 receptor-mediated cAMP-PKA pathway that involves the stimulation of PLA2 and arachidonic acid release, possibly mediated by inactivation of lipomodulin. This pathway is shared by other agonists that increase cell cAMP and thus stimulate PKA activity. PMID:1349027

  14. [Effect of zinc deficiency on 3',5'-cyclic-AMP content and parameters of energy metabolism in the rat].

    PubMed

    Roth, H P; Kirchgessner, M

    1983-06-01

    Loss of appetite, strongly reduced feed intake, and stop in weight gain are characteristic signs of alimentary zinc deficiency. The present paper investigates some parameters of the energy metabolism of Zn-deficient rats in order to obtain information on possible disturbances. The blood of Zn-deficient rats showed an increased activity of adenosine triphosphatase (ATPase) in comparison to ad-libitum- and pair-fed control animals. Therefore the concentration of adenosine triphosphate (ATP) was reduced and the concentration of adenosine diphosphate (ADP) increased in deficient animals. As a consequence, the ratio ATP/ADP was strongly reduced in Zn-deficient rats compared with both control groups. The concentration of adenosine monophosphate (AMP) was reduced in the blood of Zn-deficient rats. The levels of c-AMP in serum and urine were markedly increased in Zn-deficient rats in comparison with both control groups. Key enzymes of energetic utilization of carbohydrates such as fructose-1.6-biphosphatase and glucose-6-phosphate dehydrogenase were reduced in their activities in livers and kidneys of Zn-deficient animals. The results show that alimentary Zn deficiency impairs some parameters of the energy metabolism. The problems of reduced feed intake in Zn deficiency still remain unsolved. PMID:6308919

  15. Distinct PKC isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbol ester in RAW264.7 macrophages

    PubMed Central

    Lin, Wan-W; Chen, Bin C

    1998-01-01

    The modulatory effects of protein kinase C (PKC) on the activation of cytosolic phospholipase A2 (cPLA2) and adenylyl cyclase (AC) have recently been described. Since the signalling cascades associated with these events play critical roles in various functions of macrophages, we set out to investigate the crosstalk between PKC and the cPLA2 and AC pathways in mouse RAW 264.7 macrophages and to determine the involvement of individual PKC isoforms. The cPLA2 and AC pathways were studied by measuring the potentiation by the phorbol ester PMA of ionomycin-induced arachidonic acid (AA) release and prostagladin E1 (PGE1)-stimulated cyclic AMP production, respectively.PMA at 1 μM caused a significant increase in AA release both in the presence (371%) and absence (67%) of ionomycin induction, while exposure of RAW 264.7 cells to PMA increased PGE1 stimulation of cyclic AMP levels by 208%.Treatment of cells with staurosporine and Ro 31-8220 inhibited the PMA-induced potentiation of both AA release and cyclic AMP accumulation, while Go 6976 (an inhibitor of classical PKC isoforms) and LY 379196 (a specific inhibitor of PKCβ) inhibited the AA response but failed to affect the enhancement of the cyclic AMP response by PMA.Long term pretreatment of cells with PMA abolished the subsequent effect of PMA in potentiating AA release, but only inhibited the cyclic AMP response by 42%.Neither PD 98059, an inhibitor of MEK, nor genistein, an inhibitor of tyrosine kinases, had any effect on the ability of PMA to potentiate AA or cyclic AMP production.The potentiation of AA release, but not of cyclic AMP formation, by PMA was sensitive to inhibition by wortmannin. This effect was unrelated to the inhibition of PKC activation as deduced from the translocation of PKC activity to the cell membrane.Western blot analysis revealed the presence of eight PKC isoforms (α, βI, βII, δ, ε, μ λ and ξ) in RAW 264.7 cells and PMA was shown to induce the translocation of the α, βI, βII,

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

    PubMed Central

    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

  17. Temporal Analysis of the Magnaporthe Oryzae Proteome During Conidial Germination and Cyclic AMP (cAMP)-mediated Appressorium Formation*

    PubMed Central

    Franck, William L.; Gokce, Emine; Oh, Yeonyee; Muddiman, David C.; Dean, Ralph A.

    2013-01-01

    Rice blast disease caused by Magnaporthe oryzae is one of the most serious threats to global rice production. During the earliest stages of rice infection, M. oryzae conidia germinate on the leaf surface and form a specialized infection structure termed the appressorium. The development of the appressorium represents the first critical stage of infectious development. A total of 3200 unique proteins were identified by nanoLC-MS/MS in a temporal study of conidial germination and cAMP-induced appressorium formation in M. oryzae. Using spectral counting based label free quantification, observed changes in relative protein abundance during the developmental process revealed changes in the cell wall biosynthetic machinery, transport functions, and production of extracellular proteins in developing appressoria. One hundred and sixty-six up-regulated and 208 down-regulated proteins were identified in response to cAMP treatment. Proteomic analysis of a cAMP-dependent protein kinase A mutant that is compromised in the ability to form appressoria identified proteins whose developmental regulation is dependent on cAMP signaling. Selected reaction monitoring was used for absolute quantification of four regulated proteins to validate the global proteomics data and confirmed the germination or appressorium specific regulation of these proteins. Finally, a comparison of the proteome and transcriptome was performed and revealed little correlation between transcript and protein regulation. A subset of regulated proteins were identified whose transcripts show similar regulation patterns and include many of the most strongly regulated proteins indicating a central role in appressorium formation. A temporal quantitative RT-PCR analysis confirmed a strong correlation between transcript and protein abundance for some but not all genes. Collectively, the data presented here provide the first comprehensive view of the M. oryzae proteome during early infection-related development and

  18. Beta-Adrenergic Receptor Population is Up-Regulated by Increased Cyclic Amp Concentration in Chicken Skeletal Muscle Cells in Culture

    NASA Technical Reports Server (NTRS)

    Young, Ronald B.; Bridge, Kristin Y.; Vaughn, Jeffrey R.

    1999-01-01

    Skeletal muscle hypertrophy is promoted in vivo by administration of beta-drenergic receptor (bAR) agonists. Chicken skeletal muscle cells were treated with 1 (mu)M isoproterenol, a strong bAR agonist, between days 7 and 10 in culture. bAR population increased by approximately 40% during this treatment; however, the ability of the cells to synthesize cyclic AMP (cAMP) was diminished by two-fold. The quantity of myosin heavy chain (MHC) was not affected. To understand further the relationship between intracellular cAMP levels, bAR population, and muscle protein accumulation, intracellular cAMP levels were artificially elevated by treatment with 0-10 uM forskolin for up to three days. The basal concentration of CAMP in forskolin-treated cells increased up to 7-fold in a dose dependent manner. Increasing concentrations of forskolin also led to an increase in bAR population, with a maximum increase of approximately 40-60% at 10 uM forskolin. A maximum increase of 40-50% in the quantity of MHC was observed at 0.2 uM forskolin, but higher concentrations of forskolin reduced the quantity of MHC back to control levels. At 0.2 uM forskolin, intracellular levels of cAMP were higher by approximately 35%, and the (beta)AR population was higher by approximately 30%. Neither the number of muscle nuclei fused into myotubes nor the percentage of nuclei in myotubes were affected by forskolin at any of the concentrations studied.

  19. The effects of calcium buffering and cyclic AMP on mechano-electrical transduction in turtle auditory hair cells.

    PubMed Central

    Ricci, A J; Fettiplace, R

    1997-01-01

    1. The effects of intracellular Ca2+ buffering on hair cell mechanotransduction were studied in an intact cochlear epithelium where the endolymphatic and perilymphatic surfaces could be separately perfused with different Ca2+ solutions. 2. The speed and extent of transducer adaptation increased as the concentration in the patch electrode of the Ca2+ buffer BAPTA was lowered. In 0.1 mM BAPTA or less, the transducer adapted almost completely, with a mean time constant of 0.8 ms. 3. For a fixed internal BAPTA concentration, the transducer conductance varied with hair cell location, increasing towards the high-frequency end of the cochlea, and the time constant of adaptation decreased proportionally. At a given cochlear location, hair cells with larger transducer conductances displayed faster adaptation. We suggest that transducer adaptation accounts for a variable high-pass filter observed in the acoustic tuning curve. 4. The effects of perfusion of 50 microM Ca2+ endolymph depended on the BAPTA concentration of the electrode: with 3 mM BAPTA, adaptation was abolished, but in most recordings with 0.01 or 0.1 mM BAPTA, rapid adaptation was retained. The current-displacement curve was also shifted less the lower the intracellular BAPTA concentration. Cells in the high-frequency half of the papilla retained adaptation at a higher BAPTA concentration. 5. Treatment with the cAMP agonist, 8-bromo-cAMP, or with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, caused a rightward shift in the current-displacement curve which was independent of the internal BAPTA concentration. 6. We conclude that the free Ca2+ and cyclic nucleotide concentrations of the hair bundle modulate the position of the activation curve of the transducer. The factors which may be important for the correct functioning of adaptation in vivo are discussed. Images Figure 1 PMID:9174998

  20. Functional Similarities between the Listeria monocytogenes Virulence Regulator PrfA and Cyclic AMP Receptor Protein: the PrfA* (Gly145Ser) Mutation Increases Binding Affinity for Target DNA

    PubMed Central

    Vega, Yolanda; Dickneite, Carmen; Ripio, María-Teresa; Böckmann, Regine; González-Zorn, Bruno; Novella, Susana; Domínguez-Bernal, Gustavo; Goebel, Werner; Vázquez-Boland, José A.

    1998-01-01

    Most Listeria monocytogenes virulence genes are positively regulated by the PrfA protein, a transcription factor sharing sequence similarities with cyclic AMP (cAMP) receptor protein (CRP). Its coding gene, prfA, is regulated by PrfA itself via an autoregulatory loop mediated by the upstream PrfA-dependent plcA promoter. We have recently characterized prfA* mutants from L. monocytogenes which, as a result of a single amino acid substitution in PrfA, Gly145Ser, constitutively overexpress prfA and the genes of the PrfA virulence regulon. Here, we show that about 10 times more PrfA protein is produced in a prfA* strain than in the wild type. Thus, the phenotype of prfA* mutants is presumably due to the synthesis of a PrfA protein with higher promoter-activating activity (PrfA*), which keeps its intracellular levels constantly elevated by positive feedback. We investigated the interaction of PrfA and PrfA* (Gly145Ser) with target DNA. Gel retardation assays performed with a DNA fragment carrying the PrfA binding site of the plcA promoter demonstrated that the PrfA* mutant form is much more efficient than wild-type PrfA at forming specific DNA-protein complexes. In footprinting experiments, the two purified PrfA forms interacted with the same nucleotides at the target site, although the minimum amount required for protection was 6 to 7 times lower with PrfA*. These results show that the primary functional consequence of the Gly145Ser mutation is an increase in the affinity of PrfA for its target sequence. Interestingly, similar mutations at the equivalent position in CRP result in a transcriptionally active, CRP* mutant form which binds with high affinity to target DNA in the absence of the activating cofactor, cAMP. Our observations suggest that the structural similarities between PrfA and CRP are also functionally relevant and support a model in which the PrfA protein, like CRP, shifts from transcriptionally inactive to active conformations by interaction with a

  1. 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

    PubMed Central

    Xie, Xiang-Zhu; Huang, Xin; Zhao, Shui-Ping; Yu, Bi-Lian; Zhong, Qiao-Qing; Cao, Jian

    2016-01-01

    Background: Adipocytes behave like a rich source of pro-inflammatory cytokines including monocyte chemoattractant protein-1 (MCP-1). Oxidized low-density lipoprotein (oxLDL) participates in the local chronic inflammatory response, and high-density lipoprotein could counterbalance the proinflammatory function of oxLDL, but the underlying mechanism is not completely understood. This study aimed to evaluate the effect of apolipoprotein A-I mimetic peptide L-4F on the secretion and expression of MCP-1 in fully differentiated 3T3-L1 adipocytes induced by oxLDL and to elucidate the possible mechanisms. Methods: Fully differentiated 3T3-L1 adipocytes were incubated in the medium containing various concentration of L-4F (0–50 μg/ml) with oxLDL (50 μg/ml) stimulated, with/without protein kinase A (PKA) inhibitor H-89 (10 μmol/L) preincubated. The concentrations of MCP-1 in the supernatant, the mRNA expression of MCP-1, the levels of CCAAT/enhancer binding protein α (C/EBPα), and CCAAT/enhancer binding protein β (C/EBPβ) were evaluated. The monocyte chemotaxis assay was performed by micropore filter method using a modified Boyden chamber. Results: OxLDL stimulation induced a significant increase of MCP-1 expression and secretion in 3T3-L1 adipocytes, which were inhibited by L-4F preincubation in a dose-dependent manner. PKA inhibitor H-89 markedly reduced the oxLDL-induced MCP-1 expression, but no further decrease was observed when H-89 was used in combination with L-4F (50 μg/ml) (P > 0.05). OxLDL stimulation showed no significant effect on C/EBPα protein level but increased C/EBPβ protein level in a time-dependent manner. H-89 and L-4F both attenuated C/EBPβ protein level in oxLDL-induced 3T3-L1 adipocytes. 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

  2. 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

    PubMed Central

    Hofer, Gaby; Mullis, Primus E.; Flück, Christa E.

    2013-01-01

    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. PMID:23874725

  3. Analysis of a novel cyclic Amp inducible prespore gene in Dictyostelium discoideum: evidence for different patterns of cAMP regulation.

    PubMed

    Agarwal, A; Sloger, M S; Oyama, M; Blumberg, D D

    1994-09-01

    The D7 cDNA clone hybridizes to a 2.8 kb mRNA which first appears at the mound stage of development in the cellular slime mold Dictyostelium discoideum. This gene which is cyclic AMP (cAMP) inducible and is expressed specifically in the prespore cells contains an open reading frame interrupted by only one intron. The predicted amino acid sequence indicates a novel prespore protein which differs from all of the previously described prespore proteins in that it contains no internal repeats and does not share any homology with any of the other prespore genes. The amino acid sequence predicts a protein of 850 amino acids with a molecular weight of 95,343 daltons and an isoelectric point of 4.25. The protein is very rich in glutamine (13.8%), asparagine (10.6%) and glutamic acid (10.4%) with one potential glycosylation site and 28 possible sites for phosphorylation. The amino terminus is hydrophobic with characteristics of a signal sequence while the entire carboxyl half of the protein is notable for its hydrophilicity. Comparison of cAMP regulation of the D7 gene with the regulation of two other cAMP regulated prespore genes, the PL3(SP87) gene and the Psa(D19), reveals some striking differences. Disaggregation in the presence of cAMP results in transient degradation of mRNA for all three genes. The transcription rate for the D7 and PsA(D19) genes remains relatively unaffected by disaggregation but there is a rapid although transient decline in the transcription rate of the PL3(SP87) gene. Although the accumulation of all three mRNAs is first detectable at mound stage, transcription of the D7 and PsA(D19) genes is detected earlier in development, at rippling aggregate stage several hours prior to the earliest time when transcription of the PL3(SP87) gene is detected. Analysis of the promoter region of the D7 gene reveals three CA like boxes flanked by direct repeats as well as four G rich regions that may serve as regulatory elements. PMID:7988791

  4. Endogenous phosphorylation of microsomal proteins in bovine corpus luteum. Tenfold activation by adenosine 3′:5′-cyclic monophosphate

    PubMed Central

    Hardie, D. Grahame; Stansfield, David A.

    1977-01-01

    Free ribosomes and a smooth-microsomal fraction were prepared from bovine corpus luteum. Both preparations will self-phosphorylate when incubated with Mg2+ and ATP, but at low concentrations of Mg2+ and ATP the self-phosphorylation of the smooth-microsomal fraction was much more dependent on cyclic AMP than was that of free ribosomes, stimulation by the nucleotide being up to 10-fold in the former case. The self-phosphorylation of the smooth-microsomal fraction was studied further. The reaction bears similarities to that brought about by soluble cyclic AMP-dependent protein kinase, being inhibited by Ca2+ and the heat-stable inhibitor protein from skeletal muscle. Cyclic GMP will activate the reaction at concentrations higher than those required for full activation by cyclic AMP. In the presence of cyclic AMP, phosphate bound to protein is found almost exclusively as phosphoserine. Several proteins are phosphorylated, as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and the phosphorylation of all of them is markedly stimulated by cyclic AMP. If the reaction is carried out at high concentrations of Mg2+ and ATP, a distinct cyclic AMP-independent phosphorylation is observed. This activity is not inhibited by the heat-stable inhibitor protein, and phosphate is found esterified with both threonine and serine residues. PMID:195580

  5. Phorbol ester stimulates secretory activity while inhibiting receptor-activated aminopyrine uptake by gastric glands

    SciTech Connect

    Brown, M.R.; Chew, C.S.

    1986-03-05

    Both cyclic AMP-dependent and -independent secretagogues stimulate pepsinogen release, respiration and H/sup +/ secretory activity (AP uptake) in rabbit gastric glands. 12-O-tetradecanoylphorbol-13-acetate (T), a diacyglycerol analog, activates protein kinase C (PKC) and stimulates secretion in many systems. T stimulated respiration and pepsinogen release by glands and increased AP uptake by both glands and purified parietal cells. However, T reduced AP uptake by glands stimulated with carbachol (C) or histamine (H) with an apparent IC/sub 50/ of 1 nM. Preincubation with T for 30 min produced maximum inhibition which was not reversed by removal of T. T accelerated the decline of the transient C peak while the late steady state response to H was most inhibited. H-stimulated AP uptake was also inhibited by 50 ..mu..g/ml 1-oleoyl-2-acetyl-glycerol, a reported PKC activator, but not by the inactive phorbol, 4..cap alpha..-phorbol-12,13-didecanoate. In contrast, T potentiated AP uptake by glands stimulated with submaximal doses of dibutyryl cyclic AMP. These results suggest inhibition by T is a specific effect of PKC activators. The differing effects of T on secretion indicators may result from a dual action of T on receptor and post-receptor intracellular events.

  6. Role of calcium in the modulation of ornithine decarboxylase activity in isolated pig granulosa cells in vitro

    PubMed Central

    Veldhuis, Johannes D.; Hammond, James M.

    1981-01-01

    We examined the role of Ca2+ in the control of basal and hormone-stimulated ornithine decarboxylase activity in isolated pig granulosa cells maintained under chemically defined conditions in vitro. Omission of Ca2+ from the incubation medium (measured Ca2+ concentration 5μm) decreased basal enzymic activity, and significantly (P<0.01) impaired the response to maximally stimulating doses of either lutropin or follitropin. No significant alteration occurred in the concentration of either gonadotropin required to elicit half-maximal effects. The addition of EGTA (1.27–2.0mm) to chelate residual extracellular Ca2+ further decreased hormone-induced rises in ornithine decarboxylase activity. Despite the presence of 1.27mm concentrations of extracellular Ca2+, the administration of presumptive Ca2+ antagonists, believed to impair trans-membrane Ca2+ influx [verapamil (10–100μm), nifedipine (1–100μm) or CoCl2 (1mm)] suppressed hormone-stimulated ornithine decarboxylase activity. The inhibitory effects of verapamil or of Ca2+ omission from the medium were not overcome by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.25mm), or by cholera toxin, or by an exogenously supplied cyclic AMP analogue, 8-bromo cyclic AMP. Conversely, micromolar concentrations of a putative bivalent-cation ionophore, A23187, increased significantly the stimulation of ornithine decarboxylase activity by saturating concentrations of lutropin or 8-bromo cyclic AMP. Thus the present observations implicate Ca2+ ions in the modulation of hormone action and cellular function in normal ovarian cells. PMID:6172119

  7. PAC1hop, null and hip receptors mediate differential signaling through cyclic AMP and calcium leading to splice variant-specific gene induction in neural cells

    PubMed Central

    Holighaus, Yvonne; Mustafa, Tomris; Eiden, Lee E.

    2011-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP)-mediated activation of its G protein-coupled receptor PAC1 results in activation of the two G proteins Gs and Gq to alter second messenger generation and gene transcription in the nervous system, important for homeostatic responses to stress and injury. Heterologous expression of the three major splice variants of the rat PAC1 receptor, PAC1hop, null and hip, in neural NG108-15 cells conferred PACAP-mediated intracellular cAMP generation, while elevation of [Ca2+]i occurred only in PAC1hop-, and to a lesser extent in PAC1null-expressing cells. Induction of vasoactive intestinal polypeptide (VIP) and stanniocalcin 1 (STC1), two genes potentially involved in PACAP’s homeostatic responses, was examined as a function of the expressed PAC1 variant. VIP induction was greatest in PAC1hop-expressing cells, suggesting that a maximal transcriptional response requires combinatorial signaling through both cAMP and Ca2+. STC1 induction was similar for all three receptor splice variants and was mimicked by the adenylate cyclase activator forskolin, indicating that cAMP elevation is sufficient to induce STC1. The degree of activation of two different second messenger pathways appears to determine the transcriptional response, suggesting that cellular responses to stressors are fine-tuned through differential receptor isoform expression. Signaling to the VIP gene proceeded through cAMP and protein kinase A (PKA) in these cells, independently of the MAP kinase ERK1/2. STC1 gene induction by PACAP was dependent on cAMP and ERK1/2, independently of PKA. Differential gene induction via different cAMP dependent signaling pathways potentially provides further targets for the design of treatments for stress-associated disorders. PMID:21693142

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

    SciTech Connect

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

    1987-04-01

    cAMP regulates transcription of the gene encoding the ..cap alpha..-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 ..cap alpha..-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 ..cap alpha..-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 ..cap alpha..-subunit gene.

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed

    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. PMID:25344550

  11. Heterozygous mutations in cyclic AMP phosphodiesterase-4D (PDE4D) and protein kinase A (PKA) provide new insights into the molecular pathology of acrodysostosis.

    PubMed

    Kaname, Tadashi; Ki, Chang-Seok; Niikawa, Norio; Baillie, George S; Day, Jonathan P; Yamamura, Ken-Ichi; Ohta, Tohru; Nishimura, Gen; Mastuura, Nobuo; Kim, Ok-Hwa; Sohn, Young Bae; Kim, Hyun Woo; Cho, Sung Yoon; Ko, Ah-Ra; Lee, Jin Young; Kim, Hyun Wook; Ryu, Sung Ho; Rhee, Hwanseok; Yang, Kap-Seok; Joo, Keehyoung; Lee, Jooyoung; Kim, Chi Hwa; Cho, Kwang-Hyun; Kim, Dongsan; Yanagi, Kumiko; Naritomi, Kenji; Yoshiura, Ko-Ichiro; Kondoh, Tatsuro; Nii, Eiji; Tonoki, Hidefumi; Houslay, Miles D; Jin, Dong-Kyu

    2014-11-01

    Acrodysostosis without hormone resistance is a rare skeletal disorder characterized by brachydactyly, nasal hypoplasia, mental retardation and occasionally developmental delay. Recently, loss-of-function mutations in the gene encoding cAMP-hydrolyzing phosphodiesterase-4D (PDE4D) have been reported to cause this rare condition but the pathomechanism has not been fully elucidated. To understand the pathogenetic mechanism of PDE4D mutations, we conducted 3D modeling studies to predict changes in the binding efficacy of cAMP to the catalytic pocket in PDE4D mutants. Our results indicated diminished enzyme activity in the two mutants we analyzed (Gly673Asp and Ile678Thr; based on PDE4D4 residue numbering). Ectopic expression of PDE4D mutants in HEK293 cells demonstrated this reduction in activity, which was identified by increased cAMP levels. However, the cells from an acrodysostosis patient showed low cAMP accumulation, which resulted in a decrease in the phosphorylated cAMP Response Element-Binding Protein (pCREB)/CREB ratio. The reason for this discrepancy was due to a compensatory increase in expression levels of PDE4A and PDE4B isoforms, which accounted for the paradoxical decrease in cAMP levels in the patient cells expressing mutant isoforms with a lowered PDE4D activity. Skeletal radiographs of 10-week-old knockout (KO) rats showed that the distal part of the forelimb was shorter than in wild-type (WT) rats and that all the metacarpals and phalanges were also shorter in KO, as the name acrodysostosis implies. Like the G-protein α-stimulatory subunit and PRKAR1A, PDE4D critically regulates the cAMP signal transduction pathway and influences bone formation in a way that activity-compromising PDE4D mutations can result in skeletal dysplasia. We propose that specific inhibitory PDE4D mutations can lead to the molecular pathology of acrodysostosis without hormone resistance but that the pathological phenotype may well be dependent on an over-compensatory induction

  12. Studies of mice with cyclic AMP-dependent protein kinase (PKA) defects reveal the critical role of PKA's catalytic subunits in anxiety.

    PubMed

    Briassoulis, George; Keil, Margaret F; Naved, Bilal; Liu, Sophie; Starost, Matthew F; Nesterova, Maria; Gokarn, Nirmal; Batistatos, Anna; Wu, T John; Stratakis, Constantine A

    2016-07-01

    Cyclic adenosine mono-phosphate-dependent protein kinase (PKA) is critically involved in the regulation of behavioral responses. Previous studies showed that PKA's main regulatory subunit, R1α, is involved in anxiety-like behaviors. The purpose of this study was to determine how the catalytic subunit, Cα, might affect R1α's function and determine its effects on anxiety-related behaviors. The marble bury (MB) and elevated plus maze (EPM) tests were used to assess anxiety-like behavior and the hotplate test to assess nociception in wild type (WT) mouse, a Prkar1a heterozygote (Prkar1a(+/-)) mouse with haploinsufficiency for the regulatory subunit (R1α), a Prkaca heterozygote (Prkaca(+/-)) mouse with haploinsufficiency for the catalytic subunit (Cα), and a double heterozygote mouse (Prkar1a(+/-)/Prkaca(+/-)) with haploinsufficiency for both R1α and Cα. We then examined specific brain nuclei involved in anxiety. Results of MB test showed a genotype effect, with increased anxiety-like behavior in Prkar1a(+/-) and Prkar1a(+/-)/Prkaca(+/-) compared to WT mice. In the EPM, Prkar1a(+/-) spent significantly less time in the open arms, while Prkaca(+/-) and Prkar1a(+/-)/Prkaca(+/-) mice displayed less exploratory behavior compared to WT mice. The loss of one Prkar1a allele was associated with a significant increase in PKA activity in the basolateral (BLA) and central (CeA) amygdala and ventromedial hypothalamus (VMH) in both Prkar1a(+/-) and Prkar1a(+/-)/Prkaca(+/-) mice. Alterations of PKA activity induced by haploinsufficiency of its main regulatory or most important catalytic subunits result in anxiety-like behaviors. The BLA, CeA, and VMH are implicated in mediating these PKA effects in brain. PMID:26992826

  13. Widespread receptivity to neuropeptide PDF throughout the neuronal circadian clock network of Drosophila revealed by real-time cyclic AMP imaging

    PubMed Central

    Shafer, Orie T.; Kim, Dong Jo; Dunbar-Yaffe, Richard; Nikolaev, Viacheslav O.; Lohse, Martin J.; Taghert, Paul H.

    2008-01-01

    Summary The neuropeptide PDF is released by sixteen clock neurons in Drosophila and helps maintain circadian activity rhythms by coordinating a network of ~150 neuronal clocks. Whether PDF acts directly on elements of this neural network remains unknown. We address this question by adapting Epac1-camps, a genetically encoded cAMP FRET sensor, for use in the living brain. We find that a subset of the PDF-expressing neurons respond to PDF with long-lasting cAMP increases, and confirm that such responses require the PDF receptor. In contrast, an unrelated Drosophila neuropeptide, DH 31, stimulates large cAMP increases in all PDF-expressing clock neurons. Thus the network of ~150 clock neurons displays widespread, though not uniform, PDF receptivity. This work introduces a sensitive means of measuring cAMP changes in a living brain with sub-cellular resolution. Specifically, it experimentally confirms the longstanding hypothesis that PDF is a direct modulator of most neurons in the Drosophila clock network. PMID:18439407

  14. Cyclic AMP formation in chicken brain: effect of vasoactive intestinal peptide, peptide histidine-isoleucine (PHI), and some PHI-related peptides.

    PubMed

    Dejda, Agnieszka; Matczak, Izabela; Wiktorowska-Owczarek, Anna; Nowak, Jerzy Z

    2003-01-01

    Vasoactive intestinal peptide (chicken form; chVIP), peptide histidine-isoleucine (porcine and rat forms; pPHI and rPHI), D-Phe(4) derivative of porcine PHI (D-Phe(4)-pPHI), peptide histidine-methionine (PHM; human PHI), and helodermin, were tested for their ability to stimulate cAMP production in [(3)H]adenine-prelabeled slices of chick cerebral cortex (CCx) and hypothalamus (HTh). The chVIP (0.1-3 microM) concentration-dependently and potently stimulated cAMP production in HTh and CCx; the responses observed after 3 microM of chVIP were comparable to those produced by 0.1 microM PACAP38. Helodermin (5 microM) moderately but significantly stimulated cAMP formation in both HTh and CCx, whereas pPHI, rPHI, PHM at 5 microM concentration only weakly affected cAMP production in CCx, and were inactive in HTh; D-Phe(4)-pPHI was inactive in both tissues. These data demonstrate that chVIP, PACAP, and to a lesser extent helodermin were capable of potently stimulating cAMP generation in the avian central nervous system. PHI-related peptides showed only weak or no activity, depending on the tissue. PMID:14704471

  15. Regulation by intracellular Ca sup 2+ and cyclic AMP of the growth factor-induced ruffling membrane formation and stimulation of fluid-phase endocytosis and exocytosis

    SciTech Connect

    Miyata, Yoshihiko Tokyo Metropolitan Inst. of Medical Science ); Nishida, Eisuke; Sakai, Hikoichi ); Koyasu, Shigeo; Yahara, Ichiro )

    1989-04-01

    Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) induce formation of ruffling membranes and stimulate the fluid-phase endocytosis and exocytosis in human epidermoid carcinoma KB cells. An increase in intracellular Ca{sup 2+} concentration by treatment with A23187, a calcium ionophore, or an increase in intracellular cAMP level by treatment with dibutyryl cAMP or forskolin almost completely inhibited the insulin-, IGF-I-, or EGF-induced formation of ruffling membranes. Increases in Ca{sup 2+} or cAMP concentration also inhibited almost completely the stimulation of fluid-phase endocytosis and exocytosis elicited by these growth factors. These results suggest that the growth factor-induced ruffling membrane formation and the stimulation of fluid-phase endocytosis and exocytosis have a common regulatory mechanism involving intracellular concentrations of Ca{sup 2+} and cAMP. {sup 125}I-EGF binding assays and immunoprecipitation experiments with anti-phosphotyrosine antibody revealed that treatment of KB cells with A23187, dibutyryl cAMP, or forskolin did not inhibit the EGF binding to the cells nor subsequent tyrosine autophosphorylation of its receptors. These results indicate that Ca{sup 2+}- and/or cAMP-sensitive intracellular reactions exist downstream from the receptor kinase activation in the process of these early cellular responses.

  16. CYCLIC AMP-DEPENDENT PROTEIN KINASE INDUCTION BY POLYCHLORINATED BIPHENYLS (PCBS) STIMULATES CREB PHOSPHORYLATION VIA A CALCIUM-DEPENDENT, PKC-INDEPENDENT PATHWAY IN CORTICAL NEURONS.

    EPA Science Inventory

    We have previously demonstrated that the PCB mixture, Aroclor 1254 (A1254), increases the phosphorylated form of CREB (pCREB), the cAMP-responsive element binding protein. This transcription factor is important in nervous system development and plasticity. Phosphorylation
    of C...

  17. Enzyme Changes in the Offspring of Female Rats due to Long-Term Administration of Cyclic AMP and Insulin before Pregnancy.

    PubMed

    Strumilo, S A; Czyzewska, U; Siemieniuk, M; Strumilo, J; Tylicki, A

    2016-07-01

    We studied the effects of insulin and cAMP on the offspring of female rats after daily treatment with these substances over 4 weeks. In adult offspring from cAMP-treated females, activities of pyruvate kinase and glucose-6-phosphate dehydrogenase decreased in the liver and brain and activities of NADP-dependent malate dehydrogenase and 6-phosphogluconate dehydrogenase decreased in the liver. In the offspring of insulin-treated females, we observed only activation of glucose-6-phosphate dehydrogenase and malate dehydrogenase in the liver and only in females. Enzyme activity probably correlates with their content, as no changes in their kinetic properties were observed under these conditions. Long-term hormone treatment before pregnancy can affect the expression of genes for some enzymes in the offspring due to transmission of epigenetic signals by the ovum. However, further studies are required to confirm this mechanism. PMID:27502537

  18. In Vitro Activation of the Transcription of araBAD Operon by araC Activator

    PubMed Central

    Lee, Nancy; Wilcox, Gary; Gielow, William; Arnold, John; Cleary, Paul; Englesberg, Ellis

    1974-01-01

    The transcription of araBAD operon requires araC activator and cyclic AMP. D-Fucose inhibits ara mRNA synthesis. Our results indicate that the positive control by araC activator is exerted at the level of transcription. PMID:4362626

  19. Thyrotropin but not epidermal growth factor down-regulates the isozyme I (PKa I) of cyclic AMP-dependent protein kinases in dog thyroid cells in primary cultures.

    PubMed

    Breton, M F; Roger, P P; Omri, B; Dumont, J E; Pavlovic-Hournac, M

    1989-01-01

    The activity of the two cAMP-dependent protein kinases (PKa I and PKa II) was evaluated in dog thyroid cells in primary cultures after a 6-day growth period induced by either thyrotropin (TSH) or epidermal growth factor (EGF). Although the total PKa activity was not affected in cells cultured in the presence of TSH or EGF, their actions on the PKa I and PKa II expressions were significantly different. The activity of PKa I was strongly inhibited by TSH (70-80%) while with EGF it was either stimulated or unaffected with respect to controls. The two mitogens did not have a significant effect on the activity of PKa II. Forskolin (Fk) mimicked the effect of TSH. The expression of the two regulatory subunits (R I and R II), evaluated by the covalent binding of 8-azido-cAMP, was similar to the expression of the corresponding catalytic activities, suggesting a coregulation of the catalytic and regulatory subunits from the same isozyme. After chronic stimulation by TSH, differentiated dog thyroid cells are almost completely deprived of PKa I. PMID:2545480

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

    PubMed Central

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

    1994-01-01

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

  1. Differences in the control of basal L-type Ca(2+) current by the cyclic AMP signaling cascade in frog, rat, and human cardiac myocytes.

    PubMed

    Treinys, Rimantas; Bogdelis, Andrius; Rimkutė, Lina; Jurevičius, Jonas; Skeberdis, Vytenis Arvydas

    2016-07-01

    β-adrenergic receptors (β-ARs) mediate the positive inotropic effects of catecholamines by cAMP-dependent phosphorylation of the L-type Ca(2+) channels (LTCCs), which provide Ca(2+) for the initiation and regulation of cell contraction. The overall effect of cAMP-modulating agents on cardiac calcium current (I Ca,L) and contraction depends on the basal activity of LTCCs which, in turn, depends on the basal activities of key enzymes involved in the cAMP signaling cascade. Our current work is a comparative study demonstrating the differences in the basal activities of β-ARs, adenylyl cyclase, phosphodiesterases, phosphatases, and LTCCs in the frog and rat ventricular and human atrial myocytes. The main conclusion is that the basal I Ca,L, and consequently the contractile function of the heart, is secured from unnecessary elevation of its activity and energy consumption at the several "checking-points" of cAMP-dependent signaling cascade and the loading of these "checking-points" may vary in different species and tissues. PMID:26676115

  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

    PubMed Central

    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. PMID:27493554

  3. Phosphorylation of McArdle phosphorylase induces activity.

    PubMed Central

    Cerri, C G; Willner, J H

    1981-01-01

    In McArdle disease, myophosphorylase deficiency, enzyme activity is absent but the presence of an altered enzyme protein can frequently be demonstrated. We have found that phosphorylation of this protein in vitro can result in catalytic activity. We studied muscle of four patients; all lacked myophosphorylase activity, but myophosphorylase protein was demonstrated by immunodiffusion or gel electrophoresis. Incubation of muscle homogenate supernatants with cyclic AMP-dependent protein kinase and ATP resulted in phosphorylase activity. The activated enzyme comigrated with normal human myophosphorylase in gel electrophoresis. Incubation with [gamma-32P]ATP resulted in incorporatin of 32P into the band possessing phosphorylase activity. Activation of phosphorylase by cyclic AMP-dependent protein kinase was inhibited by antibodies to normal human myophosphorylase or by inhibitory protein to cyclic AMP-dependent protein kinase. Incubation of muscle homogenates with phosphorylase b kinase and ATP also resulted in phosphorylase activity. After the action of cyclic AMP-dependent protein kinase, the resulting activity was similar to that of phosphorylase b. However, incubation with phosphorylase kinase resulted in activity similar to that of phosphorylase a. For several reasons, it is not likely that McArdle disease is due to lack of normal phosphorylation, but restoration of activity to the mutant protein by phosphorylation may provide a clue to understanding the mechanism of this genetic defect. Images PMID:6265901

  4. Intracellular localization of 7-benzylamino-6-chloro-2-piperazino-4-pyrrolidino-pteridine in membrane structures impeding the inhibition of cytosolic cyclic AMP-specific phosphodiesterase.

    PubMed

    Marko, Doris; Merz, Karl Heinz; Kunz, Claudia; Müller, Anja; Tarasova, Nadya; Eisenbrand, Gerhard

    2002-02-15

    7-Benzylamino-6-chloro-2-piperazino-4-pyrrolidino-pteridine (DC-TA-46) is a potent inhibitor of the rolipram-sensitive cAMP-specific phosphodiesterase isoenzyme family PDE4. DC-TA-46 inhibits cAMP-hydrolysis of PDE4 isolated from solid tumors of the human large cell lung tumor xenograft LXFL529 in the nanomolar range (IC(50)=16+/-5nM). Tumor cells, however, are growth inhibited only in the lower micromolar range as shown for the human large cell lung carcinoma cell line LXFL529L. To investigate reasons for the discrepancy between IC(50) values for target inhibition and inhibition of cell growth, uptake, subcellular distribution and elimination of the compound were measured. DC-TA-46 was rapidly taken up by the cells, predominantly localized in intracellular membranes. Elimination was slow, with 70% of the compound still persisting in the membranes 50hr after withdrawal. Confocal laser scanning microscopy showed a clear colocalization with a fluorescent marker for the endoplasmatic reticulum (ER). As a result of the subcellular localization, the membrane-bound PDE activity of LXFL529L cells was effectively inhibited by DC-TA-46 (IC(50)=0.06+/-0.02 microM). In contrast, inhibition of the cytosolic PDE activity was only achieved at concentrations >1 microM (IC(50)=2.0+/-0.5 microM), in the concentration range where also growth inhibition was observed. Thus, the inhibition of the intracellular PDE activity in the different cellular compartments appears to represent an important parameter for the evaluation of the inhibitory properties at least of this class of compounds. PMID:11992634

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

    NASA Astrophysics Data System (ADS)

    Habara, Yoshiaki

    1989-06-01

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

  6. Rescue of Cyclic AMP Mediated Long Term Potentiation Impairment in the Hippocampus of Mecp2 Knockout (Mecp2-/y) Mice by Rolipram

    PubMed Central

    Balakrishnan, Saju; Niebert, Marcus; Richter, Diethelm W.

    2016-01-01

    Rett syndrome (RTT) patients experience learning difficulties and memory loss. Analogous deficits of hippocampal plasticity are reported in mouse models of RTT. To elucidate the underlying pathophysiology, we studied long term potentiation (LTP) at the CA3 to CA1 synapses in the hippocampus in acute brain slices from WT and Mecp2-/y mice, by either activating cAMP dependent pathway or using high frequency stimulation, by means of patch clamp. We have observed that, the NMDA channel current characteristics remain unchanged in the Mecp2-/y mice. The adenylyl cyclase (AC) agonist forskolin evoked a long lasting potentiation of evoked EPSCs in WT CA1 neurons, but only minimally enhanced the EPSCs in the Mecp2-/y mice. This weaker potentiation in Mecp2-/y mice was ameliorated by application of phosphodiesterase 4 inhibitor rolipram. The hyperpolarization activated cyclic nucleotide gated channel current (Ih) was potentiated to similar extent by forskolin in both phenotypes. Multiple tetanus induced cAMP-dependent plasticity was also impaired in the Mecp2-/y mice, and was also partially rescued by rolipram. Western blot analysis of CA region of Mecp2-/y mice hippocampus revealed more than twofold up-regulation of protein kinase A (PKA) regulatory subunits, while the expression of the catalytic subunit remained unchanged. We hypothesize that the overexpressed PKA regulatory subunits buffer cAMP and restrict the PKA mediated phosphorylation of target proteins necessary for LTP. Blocking the degradation of cAMP, thereby saturating the regulatory subunits alleviated this defect. PMID:26869885

  7. Rescue of Cyclic AMP Mediated Long Term Potentiation Impairment in the Hippocampus of Mecp2 Knockout (Mecp2(-/y) ) Mice by Rolipram.

    PubMed

    Balakrishnan, Saju; Niebert, Marcus; Richter, Diethelm W

    2016-01-01

    Rett syndrome (RTT) patients experience learning difficulties and memory loss. Analogous deficits of hippocampal plasticity are reported in mouse models of RTT. To elucidate the underlying pathophysiology, we studied long term potentiation (LTP) at the CA3 to CA1 synapses in the hippocampus in acute brain slices from WT and Mecp2(-/y) mice, by either activating cAMP dependent pathway or using high frequency stimulation, by means of patch clamp. We have observed that, the NMDA channel current characteristics remain unchanged in the Mecp2(-/y) mice. The adenylyl cyclase (AC) agonist forskolin evoked a long lasting potentiation of evoked EPSCs in WT CA1 neurons, but only minimally enhanced the EPSCs in the Mecp2(-/y) mice. This weaker potentiation in Mecp2 (-/) (y) mice was ameliorated by application of phosphodiesterase 4 inhibitor rolipram. The hyperpolarization activated cyclic nucleotide gated channel current (I h) was potentiated to similar extent by forskolin in both phenotypes. Multiple tetanus induced cAMP-dependent plasticity was also impaired in the Mecp2 (-/) (y) mice, and was also partially rescued by rolipram. Western blot analysis of CA region of Mecp2 (-/) (y) mice hippocampus revealed more than twofold up-regulation of protein kinase A (PKA) regulatory subunits, while the expression of the catalytic subunit remained unchanged. We hypothesize that the overexpressed PKA regulatory subunits buffer cAMP and restrict the PKA mediated phosphorylation of target proteins necessary for LTP. Blocking the degradation of cAMP, thereby saturating the regulatory subunits alleviated this defect. PMID:26869885

  8. Modulatory effects of steroid hormones, oxytocin, arachidonic acid, forskolin and cyclic AMP on the expression of aquaporin 1 and aquaporin 5 in the porcine uterus during placentation.

    PubMed

    Skowronska, A; Mlotkowska, P; Okrasa, S; Nielsen, S; Skowronski, M T

    2016-04-01

    Aquaporins (AQPs) are proteins forming trans-membrane channels responsible for water transport. AQP1 and AQP5 are present in structures of the female reproductive system. In the uterus, these AQPs are involved in water movement between the intraluminal, interstitial and capillary compartments and their uterine expression is essential throughout the pregnancy, including its early stages. Thus, the study aimed to assess the influence of P4 (progesterone), E2 (estradiol), OT (oxytocin), AA (arachidonic acid), cAMP and FSK (forskolin) on the AQP1 and AQP5 mRNA and protein expression in the uterine tissue of gilts on Days 30 - 32 of gestation (the placentation period), following short (3 h) and long (24 h) incubations. Steroid hormones influenced the expression of AQP1 and AQP5; E2 up-regulated, but P4 down-regulated mRNAs of these AQPs, whereas the protein level of studied AQPs was increased by both steroids. OT treatment decreased AQP1 (after 24 h), but increased AQP5 (after 3 h) mRNA expression. Treatment with AA significantly reduced the AQP1 expression at the mRNA level, but stimulated at the protein level. The expression of AQP5 mRNA and protein was stimulated by AA. FSK markedly decreased AQP1 mRNA, but increased of AQP5 after 3-h incubation. In turn, cAMP stimulated and inhibited transcription of AQP5 after 3- and 24-h incubations, respectively. Immunohistochemical analysis confirmed the uterine localization of AQP1 in the apical and basal membranes of endothelial cells and AQP5 in the apical membranes of epithelial cells under control condition. Treatments with P4, E2, AA, cAMP or FSK have caused additional appearance of AQP5 labeling in the basolateral membranes of epithelial cells. These results suggest a participation of steroid hormones (P4 and E2), AA derivatives and cAMP in controlling the expression of AQP1 and AQP5 as well as the distribution of AQP5 in the uterine tissue of pregnant gilts during placentation (Days 30 - 32 of gestation). PMID:27226190

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

    PubMed Central

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

    1981-01-01

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

  10. Direct regulation of the natural competence regulator gene tfoX by cyclic AMP (cAMP) and cAMP receptor protein (CRP) in Vibrios

    PubMed Central

    Wu, Rui; Zhao, Meng; Li, Jing; Gao, He; Kan, Biao; Liang, Weili

    2015-01-01

    TfoX (Sxy) and CRP are two important competence activators. The link between tfoX and CRP has been shown in H. influenza but lacking evidence of direct interaction. Recently a Sxy-dependent CRP (CRP-S) site autoregulating Sxy was reported in E. coli. Here, we show that the cAMP-CRP complex transcriptionally regulates tfoX expression through multiple canonical CRP (CRP-N) sites in Vibrios. This conclusion is supported by an analysis of the tfoX mRNA levels and tfoX transcriptional reporter fusions. The reduced expression of tfoXVC was restored by trans-complementation of crp in ∆crp and by exogenous cAMP in ∆cya. A promoter deletion analysis and the site-directed mutagenesis of the putative CRP-N sites revealed the presence of two functional CRP-N sites. The direct binding of cAMP-CRP to the tfoXVCpromoter was demonstrated by EMSA assays. Additionally, the transcriptional start site (TSS) of tfoXVF in V. fluvialis was determined, and −10/−35 regions were predicted. Further comparison of the tfoX promoter in Vibrios revealed the existence of similar −10 motifs and putative CRP-N sites, indicating the conserved mechanism of CRP regulation on tfoX. Our study demonstrates the direct binding of the cAMP-CRP complex to tfoX promoter, and broadens the understanding of the molecular mechanism regulating tfoX in Vibrios. PMID:26442598

  11. Cyclic AMP and the reverse transformation reaction.

    PubMed

    Puck, Theodore T; Webb, Patricia; Johnson, Robert

    2002-06-01

    Traditional methods for cancer treatment have been aimed at killing the cancer cells. Unfortunately this approach all too often is accompanied by harmful killing of normal cells. The present paper describes an experimental program in our laboratory in which cancer cells are treated so as to revert to normal cell behavior. This process, which we have named reverse transformation, appears to offer considerable hope in the treatment of a large number of malignancies. PMID:12119272

  12. Long-Term Memory for Place Learning Is Facilitated by Expression of cAMP Response Element-Binding Protein in the Dorsal Hippocampus

    ERIC Educational Resources Information Center

    Brightwell, Jennifer J.; Smith, Clayton A.; Neve, Rachael L.; Colombo, Paul J.

    2007-01-01

    Extensive research has shown that the hippocampus is necessary for consolidation of long-term spatial memory in rodents. We reported previously that rats using a place strategy to solve a cross maze task showed sustained phosphorylation of hippocampus cyclic AMP response element-binding protein (CREB), a transcription factor implicated in…

  13. Inhibition of histone deacetylase 6 activity reduces cyst growth in polycystic kidney disease.

    PubMed

    Cebotaru, Liudmila; Liu, Qiangni; Yanda, Murali K; Boinot, Clement; Outeda, Patricia; Huso, David L; Watnick, Terry; Guggino, William B; Cebotaru, Valeriu

    2016-07-01

    Abnormal proliferation of cyst-lining epithelium and increased intracystic fluid secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) are thought to contribute to cyst growth in autosomal dominant polycystic kidney disease (ADPKD). Histone deacetylase 6 (HDAC6) expression and activity are increased in certain cancers, neurodegenerative diseases, and in Pkd1-mutant renal epithelial cells. Inhibition of HDAC6 activity with specific inhibitors slows cancer growth. Here we studied the effect of tubacin, a specific HDAC6 inhibitor, on cyst growth in polycystic kidney disease. Treatment with tubacin prevented cyst formation in MDCK cells, an in vitro model of cystogenesis. Cyclic AMP stimulates cell proliferation and activates intracystic CFTR-mediated chloride secretion in ADPKD. Treatment with tubacin downregulated cyclic AMP levels, inhibited cell proliferation, and inhibited cyclic AMP-activated CFTR chloride currents in MDCK cells. We also found that tubacin reduced cyst growth by inhibiting proliferation of cyst-lining epithelial cells, downregulated cyclic AMP levels, and improved renal function in a Pkd1-conditional mouse model of ADPKD. Thus, HDAC6 could play a role in cyst formation and could serve as a potential therapeutic target in ADPKD. PMID:27165822

  14. Activation response of martensitic steels

    SciTech Connect

    Forty, C.B.A.

    1997-09-01

    A hypothetical martensitic steel has been compositionally designed in order to optimize both metallurgical and reduced activation properties. When compared with two other martensitic steels, its activation characteristics are shown to be superior for all activation indices examined. However, these excellent properties are found to be due to the assumed absence of deleterious tramp impurities. When limiting impurity concentrations are determined for the hypothetical steel, they are found to be extremely stringent, and wholly unachievable using industrial scale production methods. It is concluded that only slight improvements can be made to currently available low activation martensitic steels to reduce residual activity responses further. 26 refs., 1 fig., 2 tabs.

  15. Activation Response of Martensitic Steels

    NASA Astrophysics Data System (ADS)

    Forty, C. B. A.

    1997-09-01

    A hypothetical martensitic steel has been compositionally designed in order to optimize both metallurgical and reduced activation properties. When compared with two other martensitic steels, its activation characteristics are shown to be superior for all activation indices examined. However, these excellent properties are found to be due to the assumed absence of deleterious tramp impurities. When limiting impurity concentrations are determined for the hypothetical steel, they are found to be extremely stringent, and wholly unachievable using industrial scale production methods. It is concluded that only slight improvements can be made to currently available low activation martensitic steels to reduce residual activity responses further.

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

    SciTech Connect

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

    1985-06-01

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

  17. Neural activation during response competition

    NASA Technical Reports Server (NTRS)

    Hazeltine, E.; Poldrack, R.; Gabrieli, J. D.

    2000-01-01

    The flanker task, introduced by Eriksen and Eriksen [Eriksen, B. A., & Eriksen, C. W. (1974). Effects of noise letters upon the identification of a target letter in a nonsearch task. Perception & Psychophysics, 16, 143--149], provides a means to selectively manipulate the presence or absence of response competition while keeping other task demands constant. We measured brain activity using functional magnetic resonance imaging (fMRI) during performance of the flanker task. In accordance with previous behavioral studies, trials in which the flanking stimuli indicated a different response than the central stimulus were performed significantly more slowly than trials in which all the stimuli indicated the same response. This reaction time effect was accompanied by increases in activity in four regions: the right ventrolateral prefrontal cortex, the supplementary motor area, the left superior parietal lobe, and the left anterior parietal cortex. The increases were not due to changes in stimulus complexity or the need to overcome previously learned associations between stimuli and responses. Correspondences between this study and other experiments manipulating response interference suggest that the frontal foci may be related to response inhibition processes whereas the posterior foci may be related to the activation of representations of the inappropriate responses.

  18. Bronchodilator and anti-inflammatory activities of glaucine: In vitro studies in human airway smooth muscle and polymorphonuclear leukocytes.

    PubMed

    Cortijo, J; Villagrasa, V; Pons, R; Berto, L; Martí-Cabrera, M; Martinez-Losa, M; Domenech, T; Beleta, J; Morcillo, E J

    1999-08-01

    1. Selective phosphodiesterase 4 (PDE4) inhibitors are of potential interest in the treatment of asthma. We examined the effects of the alkaloid S-(+)-glaucine, a PDE4 inhibitor, on human isolated bronchus and granulocyte function. 2. Glaucine selectively inhibited PDE4 from human bronchus and polymorphonuclear leukocytes (PMN) in a non-competitive manner (Ki=3.4 microM). Glaucine displaced [3H]-rolipram from its high-affinity binding sites in rat brain cortex membranes (IC50 approximately 100 microM). 3. Glaucine inhibited the spontaneous and histamine-induced tone in human isolated bronchus (pD2 approximately 4.5). Glaucine (10 microM) did not potentiate the isoprenaline-induced relaxation but augmented cyclic AMP accumulation by isoprenaline. The glaucine-induced relaxation was resistant to H-89, a protein kinase A inhibitor. Glaucine depressed the contractile responses to Ca2+ (pD'2 approximately 3.62) and reduced the sustained rise of [Ca2+]i produced by histamine in cultured human airway smooth muscle cells (-log IC50 approximately 4.3). 4. Glaucine augmented cyclic AMP levels in human polymorphonuclear leukocytes challenged with N-formyl-Met-Leu-Phe (FMLP) or isoprenaline, and inhibited FMLP-induced superoxide generation, elastase release, leukotriene B4 production, [Ca2+]i signal and platelet aggregation as well as opsonized zymosan-, phorbol myristate acetate-, and A23187-induced superoxide release. The inhibitory effect of glaucine on superoxide generation by FMLP was reduced by H-89. 5. In conclusion, Ca2+ channel antagonism by glaucine appears mainly responsible for the relaxant effect of glaucine in human isolated bronchus while PDE4 inhibition contributes to the inhibitory effects of glaucine in human granulocytes. The very low PDE4/binding site ratio found for glaucine makes this compound attractive for further structure-activity studies. PMID:10455321

  19. Bronchodilator and anti-inflammatory activities of glaucine: In vitro studies in human airway smooth muscle and polymorphonuclear leukocytes

    PubMed Central

    Cortijo, J; Villagrasa, V; Pons, R; Berto, L; Martí-Cabrera, M; Martinez-Losa, M; Domenech, T; Beleta, J; Morcillo, E J

    1999-01-01

    Selective phosphodiesterase 4 (PDE4) inhibitors are of potential interest in the treatment of asthma. We examined the effects of the alkaloid S-(+)-glaucine, a PDE4 inhibitor, on human isolated bronchus and granulocyte function.Glaucine selectively inhibited PDE4 from human bronchus and polymorphonuclear leukocytes (PMN) in a non-competitive manner (Ki=3.4 μM). Glaucine displaced [3H]-rolipram from its high-affinity binding sites in rat brain cortex membranes (IC50∼100 μM).Glaucine inhibited the spontaneous and histamine-induced tone in human isolated bronchus (pD2∼4.5). Glaucine (10 μM) did not potentiate the isoprenaline-induced relaxation but augmented cyclic AMP accumulation by isoprenaline. The glaucine-induced relaxation was resistant to H-89, a protein kinase A inhibitor. Glaucine depressed the contractile responses to Ca2+ (pD'2∼3.62) and reduced the sustained rise of [Ca2+]i produced by histamine in cultured human airway smooth muscle cells (−log IC50∼4.3).Glaucine augmented cyclic AMP levels in human polymorphonuclear leukocytes challenged with N-formyl-Met-Leu-Phe (FMLP) or isoprenaline, and inhibited FMLP-induced superoxide generation, elastase release, leukotriene B4 production, [Ca2+]i signal and platelet aggregation as well as opsonized zymosan-, phorbol myristate acetate-, and A23187-induced superoxide release. The inhibitory effect of glaucine on superoxide generation by FMLP was reduced by H-89.In conclusion, Ca2+ channel antagonism by glaucine appears mainly responsible for the relaxant effect of glaucine in human isolated bronchus while PDE4 inhibition contributes to the inhibitory effects of glaucine in human granulocytes. The very low PDE4/binding site ratio found for glaucine makes this compound attractive for further structure-activity studies. PMID:10455321

  20. Evidence that cyclic nucleotides are not mediators of fever in rabbits.

    PubMed Central

    Dascombe, M. J.

    1984-01-01

    The N6-2'-O-dibutyryl derivative of adenosine 3',5'-monophosphate (db cyclic AMP) and related compounds have been micro-injected into the preoptic/anterior hypothalamic nuclei (PO/AH) of the unanaesthetized, restrained rabbit and the effects on deep body temperature observed. Db cyclic AMP (100-400 micrograms) produced hypothermia of rapid onset in rabbits at an ambient temperature of 20-23 degrees C. Hypothermia was also produced by N2-2'-O-dibutyryl guanosine 3',5'-monophosphate (db cyclic GMP), but not by saline, sodium n-butyrate, adenosine 3',5'-monophosphate (cyclic AMP), guanosine 3',5'-monophosphate, adenosine 5'-mono-, di- or triphosphate. The initial hypothermic response to db cyclic AMP and db cyclic GMP was followed by a sustained rise in temperature. However, all compounds injected into the PO/AH produced a similar hyperthermia which was attenuated by paracetamol. Development of this tissue-damage fever abolished the hypothermic response to db cyclic AMP in some rabbits. The effects of db cyclic AMP on body temperature and behaviour were not reproduced by the adenylate cyclase activators, cholera toxin (0.125-5 micrograms) and guanyl imidodiphosphate (5-400 micrograms). It is concluded that hypothermia is the principal effect of db cyclic AMP on body temperature when injected into the PO/AH in rabbits. These data do not support the proposal that endogenous cyclic AMP in the rabbit brain mediates pyrexia. PMID:6326920

  1. Protein kinase C activity is not involved in N-formylmethionyl-leucyl-phenylalanine-induced phospholipase D activation in human neutrophils, but is essential for concomitant NADPH oxidase activation: studies with a staurosporine analogue with improved selectivity for protein kinase C.

    PubMed

    Kessels, G C; Krause, K H; Verhoeven, A J

    1993-06-15

    Stimulation of human neutrophils by the receptor agonist N-formylmethionyl-leucyl-phenylalanine (fMLP) results in a respiratory burst, catalysed by an NADPH oxidase. Concomitantly, phospholipase D (PLD) is activated. To investigate the role of protein kinase C (PKC) in these neutrophil responses, we have compared the effects of staurosporine and a structural analogue of staurosporine (cgp41251), that reflects a higher selectivity towards PKC [Meyer, Regenass, Fabbro, Alteri, Rösel, Müller, Caravatti and Matter (1989) Int. J. Cancer 43, 851-856]. Both staurosporine and cgp41251 dose-dependently inhibited the production of superoxide induced by phorbol 12-myristate 13-acetate (PMA). Both compounds also caused inhibition of the fMLP-induced respiratory burst, but with a lower efficacy during the initiation phase of this response. This latter observation cannot be taken as evidence against PKC involvement in the activation of the respiratory burst, because pretreatment of neutrophils with ionomycin before PMA stimulation also results in a lower efficacy of inhibition. Activation of PLD by fMLP was enhanced in the presence of staurosporine, but not in the presence of cgp41251. Enhancement of PLD activation was also observed in the presence of H-89, an inhibitor of cyclic-AMP-dependent protein kinase (PKA). Both staurosporine and H-89 reversed the dibutyryl-cyclic-AMP-induced inhibition of PLD activation, whereas cgp41251 was without effect. These results indicate that the potentiating effect of staurosporine on PLD activation induced by fMLP does not reflect a feedback inhibition by PKC activation, but instead a feedback inhibition by PKC activation. Taken together, our results indicate that in human neutrophils: (i) PKC activity is not essential for fMLP-induced activation of PLD; (ii) PKC activity does play an essential role in the activation of the respiratory burst by fMLP, other than mediating or modulating PLD activation; (iii) there exists a negative

  2. Active Response Gravity Offload System

    NASA Technical Reports Server (NTRS)

    Valle, Paul; Dungan, Larry; Cunningham, Thomas; Lieberman, Asher; Poncia, Dina

    2011-01-01

    The Active Response Gravity Offload System (ARGOS) provides the ability to simulate with one system the gravity effect of planets, moons, comets, asteroids, and microgravity, where the gravity is less than Earth fs gravity. The system works by providing a constant force offload through an overhead hoist system and horizontal motion through a rail and trolley system. The facility covers a 20 by 40-ft (approximately equals 6.1 by 12.2m) horizontal area with 15 ft (approximately equals4.6 m) of lifting vertical range.

  3. Sleep deprivation impairs contextual fear conditioning and attenuates subsequent behavioural, endocrine and neuronal responses.

    PubMed

    Hagewoud, Roelina; Bultsma, Lillian J; Barf, R Paulien; Koolhaas, Jaap M; Meerlo, Peter

    2011-06-01

    Sleep deprivation (SD) affects hippocampus-dependent memory formation. Several studies in rodents have shown that brief SD immediately following a mild foot shock impairs consolidation of contextual fear memory as reflected in a reduced behavioural freezing response during re-exposure to the shock context later. In the first part of this study, we examined whether this reduced freezing response is accompanied by an attenuated fear-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis. Results show that 6h of SD immediately following the initial shock results in a diminished adrenal corticosterone (CORT) response upon re-exposure to the shock context the next day. In the second part, we established whether the attenuated freezing response in SD animals is associated with reduced activation of relevant brain areas known to be involved in the retrieval and expression of fear memory. Immunohistochemical analysis of brain slices showed that the normal increase in phosphorylation of the transcription factor 3',5'-cyclic AMP response-element binding protein (CREB) upon re-exposure to the shock context was reduced in SD animals in the CA1 region of the hippocampus and in the amygdala. In conclusion, brief SD impairs the consolidation of contextual fear memory. Upon re-exposure to the context, this is reflected in a diminished behavioural freezing response, an attenuated HPA axis response and a reduction of the normal increase of phosphorylated CREB expression in the hippocampus and amygdala. PMID:20946438

  4. Protein kinase activity associated with pancreatic zymogen granules.

    PubMed

    Burnham, D B; Munowitz, P; Thorn, N; Williams, J A

    1985-05-01

    Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues. PMID:4004796

  5. Protein kinase activity associated with pancreatic zymogen granules.

    PubMed Central

    Burnham, D B; Munowitz, P; Thorn, N; Williams, J A

    1985-01-01

    Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues. Images Fig. 1. Fig. 2. Fig. 7. Fig. 8. PMID:4004796

  6. Thyrotropin via cyclic AMP induces insulin receptor expression and insulin Co-stimulation of growth and amplifies insulin and insulin-like growth factor signaling pathways in dog thyroid epithelial cells.

    PubMed

    Burikhanov, R; Coulonval, K; Pirson, I; Lamy, F; Dumont, J E; Roger, P P

    1996-11-15

    Despite the similarity of their receptors and signal transduction pathways, insulin is regarded as a regulator of glucose, protein, and lipid metabolism, whereas insulin-like growth factors (IGF-I and IGF-II) mainly act as mitogenic hormones. In the dog thyroid primary culture model, the triggering of DNA synthesis by thyrotropin (TSH) through cAMP, or by cAMP-independent factors including epidermal growth factor, hepatocyte growth factor and phorbol esters, requires insulin or IGFs as comitogenic factors. In the present study, in TSH-treated cells, IGF-I receptors and insulin receptors were paradoxically equivalent in their capacity to elicit the comitogenic pathway, which, however, was mediated only by IGF-I receptors in dog thyroid cells stimulated by cAMP-independent mitogens. Moreover, prior cell exposure to TSH or forskolin increased their responsiveness to insulin, IGF-I, and IGF-II, as seen on DNA synthesis and activation of a common insulin/IGF signaling pathway. To understand these observations, binding characteristics and expression of insulin and IGF-I receptors were examined. To analyze IGF-I receptor characteristics, the unexpected interference of a huge presence of IGF-binding proteins at the cell membrane was avoided using labeled Long R3 IGF-I instead of IGF-I. Strikingly, TSH, through cAMP, time-dependently induced insulin binding and insulin receptor mRNA and protein accumulation without any effect on IGF-I receptors. These findings constitute a first example of an induction of insulin receptor gene expression by a cAMP-mediated hormone. In dog thyroid cells, this allows low physiological insulin concentrations to act as a comitogenic factor and might explain in part the enhanced responsiveness to IGFs in response to TSH. This raises the possibility that TSH-insulin interactions may play a role in the regulation of thyroid growth and function in vivo. PMID:8910605

  7. On the induction of cyclooxygenase-2, inducible nitric oxide synthase and soluble phospholipase A2 in rat mesangial cells by a nonsteroidal anti-inflammatory drug: the role of cyclic AMP.

    PubMed

    Klein, T; Ullrich, V; Pfeilschifter, J; Nüsing, R

    1998-03-01

    One of the challenges in the therapy with anti-inflammatory drugs is the avoidance of gastrointestinal side effects, which may be achieved by selective inhibition of cyclooxygenase (COX) -2. CGP 28238 is reported with these characteristics inhibiting selectively the COX-2 activity at nanomolar concentrations. However, we report here on a novel action of this compound uncovered during the application of higher concentrations. In rat mesangial cells, CGP 28238 induced the mRNA and the protein of COX-2 as well as those of inducible nitric oxide synthase and soluble phospholipase A2. In the case of COX-2, this stimulation had no effect on the production of COX-2 metabolites because of the effective blockade of the enzyme. In contrast, the level of NO produced by the cells increased in a concentration-dependent manner from 1.2 to 12.5 nmol of nitrite/3 x 10(5) cells. Furthermore, in combination with low doses of IL-1 CGP 28238 superinduced the formation of nitrite. The observed effects were independent of the inhibition of prostaglandin formation, as suggested by the failure of the potent COX inhibitor diclofenac to cause similar effects. Furthermore, the activity and expression of enzymes downstream of the COX step, such as prostacyclin synthase, were unaffected by CGP 28238. The inductive action of CGP 28238 could be blocked by inhibitors for tyrosine kinases and protein kinase A, such as genistein and KT5720, respectively. The increase in intracellular cAMP concentration in rat mesangial cells and the inhibition by CGP 28238 of phosphodiesterase 4 activity with an IC50 value of 23 muM gave a rationale to explain the underlying mechanisms for the induction of the inflammatory response genes COX-2, soluble phospholipase A2 and inducible NO synthase in rat mesangial cells. PMID:9495802

  8. Allergic manifestations and cutaneous histamine responses in patients with McCune Albright syndrome

    PubMed Central

    2013-01-01

    Background McCune Albright syndrome (MAS) is a rare disorder characterized by precocious puberty, café-au-lait spots, and fibrous dysplasia. Its cause is an activating mutation in the GNAS gene, encoding a subunit of the stimulatory G protein, Gsalpha (Gsα). The action of any mediator that signals via Gsα and cyclic AMP can be up regulated in MAS. We had observed gastritis, gastroesophageal reflux, and anaphylaxis in McCune Albright patients. Objective As histamine is known to signal via histamine 1 (H1) and histamine 2 (H2) receptors, which couple with stimulatory G proteins, we attempted to mechanistically link histamine responsiveness to the activating GNAS mutation. We hypothesized that responsiveness to histamine skin testing would differ between MAS patients and healthy controls. Patients and methods After obtaining informed consent, we performed a systematic review of histamine responsiveness and allergic manifestations in 11 MAS patients and 11 sex-matched, Tanner-stage matched controls. We performed skin prick testing, quantifying the orthogonal diameters of wheals and erythema. We also quantitated G protein mRNA expression. Results The peak wheal and flare responses to histamine were significantly higher in MAS patients compared to controls. Conclusions This study suggests that MAS patients may be at risk for exaggerated histamine responsiveness compared to unaffected controls. PMID:23663565

  9. Resveratrol Inhibits LPS-Induced MAPKs Activation via Activation of the Phosphatidylinositol 3-Kinase Pathway in Murine RAW 264.7 Macrophage Cells

    PubMed Central

    Liu, Bin; Deng, Yi-Shu; Zhan, Dong; Chen, Yuan-Li; He, Ying; Liu, Jing; Zhang, Zong-Ji; Sun, Jun; Lu, Di

    2012-01-01

    Background Resveratrol is a natural polyphenolic compound that has cardioprotective, anticancer and anti-inflammatory properties. We investigated the capacity of resveratrol to protect RAW 264.7 cells from inflammatory insults and explored mechanisms underlying inhibitory effects of resveratrol on RAW 264.7 cells. Methodology/Principal Findings Murine RAW 264.7 cells were treated with resveratrol (1, 5, and 10 µM) and/or LPS (5 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by ELISA, RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of Akt, cyclic AMP-responsive element-binding protein (CREB), mitogen-activated protein kinases (MAPKs) cascades, AMP-activated protein kinase (AMPK) and expression of SIRT1(Silent information regulator T1) were measured by western blot. Wortmannin (1 µM), a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, was used to determine if PI3-K/Akt signaling pathway might be involved in resveratrol’s action on RAW 264.7 cells. Resveratrol significantly attenuated the LPS-induced expression of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW 264.7 cells. Resveratrol increased Akt phosphorylation in a time-dependent manner. Wortmannin, a specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, blocked the effects of resveratrol on LPS-induced RAW 264.7 cells activation. In addition, PI3-K inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of cyclic AMP-responsive element-binding protein (CREB) and mitogen-activated protein kinases (MAPKs) cascades. Meanwhile, PI3-K is essential for resveratrol-mediated phosphorylation of AMPK and expression of SIRT1. Conclusion and Implications This investigation

  10. Nod2-mediated recognition of the microbiota is critical for mucosal adjuvant activity of cholera toxin.

    PubMed

    Kim, Donghyun; Kim, Yun-Gi; Seo, Sang-Uk; Kim, Dong-Jae; Kamada, Nobuhiko; Prescott, Dave; Philpott, Dana J; Rosenstiel, Philip; Inohara, Naohiro; Núñez, Gabriel

    2016-05-01

    Cholera toxin (CT) is a potent adjuvant for inducing mucosal immune responses. However, the mechanism by which CT induces adjuvant activity remains unclear. Here we show that the microbiota is critical for inducing antigen-specific IgG production after intranasal immunization. After mucosal vaccination with CT, both antibiotic-treated and germ-free (GF) mice had reduced amounts of antigen-specific IgG, smaller recall-stimulated cytokine responses, impaired follicular helper T (TFH) cell responses and reduced numbers of plasma cells. Recognition of symbiotic bacteria via the nucleotide-binding oligomerization domain containing 2 (Nod2) sensor in cells that express the integrin CD11c (encoded by Itgax) was required for the adjuvanticity of CT. Reconstitution of GF mice with a Nod2 agonist or monocolonization with Staphylococcus sciuri, which has high Nod2-stimulatory activity, was sufficient to promote robust CT adjuvant activity, whereas bacteria with low Nod2-stimulatory activity did not. Mechanistically, CT enhanced Nod2-mediated cytokine production in dendritic cells via intracellular cyclic AMP. These results show a role for the microbiota and the intracellular receptor Nod2 in promoting the mucosal adjuvant activity of CT. PMID:27064448

  11. Sex differences in feeding behavior in rats: the relationship with neuronal activation in the hypothalamus.

    PubMed

    Fukushima, Atsushi; Hagiwara, Hiroko; Fujioka, Hitomi; Kimura, Fukuko; Akema, Tatsuo; Funabashi, Toshiya

    2015-01-01

    There is general agreement that the central nervous system in rodents differs between sexes due to the presence of gonadal steroid hormone during differentiation. Sex differences in feeding seem to occur among species, and responses to fasting (i.e., starvation), gonadal steroids (i.e., testosterone and estradiol), and diet (i.e., western-style diet) vary significantly between sexes. The hypothalamus is the center for controlling feeding behavior. We examined the activation of feeding-related peptides in neurons in the hypothalamus. Phosphorylation of cyclic AMP response element-binding protein (CREB) is a good marker for neural activation, as is the Fos antigen. Therefore, we predicted that sex differences in the activity of melanin-concentrating hormone (MCH) neurons would be associated with feeding behavior. We determined the response of MCH neurons to glucose in the lateral hypothalamic area (LHA) and our results suggested MCH neurons play an important role in sex differences in feeding behavior. In addition, fasting increased the number of orexin neurons harboring phosphorylated CREB in female rats (regardless of the estrous day), but not male rats. Glucose injection decreased the number of these neurons with phosphorylated CREB in fasted female rats. Finally, under normal spontaneous food intake, MCH neurons, but not orexin neurons, expressed phosphorylated CREB. These sex differences in response to fasting and glucose, as well as under normal conditions, suggest a vulnerability to metabolic challenges in females. PMID:25870535

  12. THE MOLECULAR PHYSIOLOGY OF CRH NEURONS

    PubMed Central

    Aguilera, Greti; Liu, Ying

    2012-01-01

    Corticotropin releasing hormone (CRH) is essential for stress adaptation by mediating hypothalamic-pituitary adrenal (HPA) axis, behavioral and autonomic responses to stress. Activation of CRH neurons depends on neural afferents from the brain stem and limbic system, leading to sequential CRH release and synthesis. CRH transcription is required to restore mRNA and peptide levels, but termination of the response is essential to prevent pathology associated with chronic elevations of CRH and HPA axis activity. Inhibitory feedback mediated by glucocorticoids and intracellular production of the repressor, Inducible Cyclic AMP Early Repressor (ICER), limit the magnitude and duration of CRH neuronal activation. Induction of CRH transcription is mediated by the cyclic AMP/protein kinase A/cyclic AMP responsive element binding protein (CREB)-dependent pathways, and requires cyclic AMP-dependent nuclear translocation of the CREB co-activator, Transducer of Regulated CREB activity (TORC). This article reviews current knowledge on the mechanisms regulating CRH neuron activity. PMID:21871477

  13. The molecular physiology of CRH neurons.

    PubMed

    Aguilera, Greti; Liu, Ying

    2012-01-01

    Corticotropin releasing hormone (CRH) is essential for stress adaptation by mediating hypothalamic-pituitary-adrenal (HPA) axis, behavioral and autonomic responses to stress. Activation of CRH neurons depends on neural afferents from the brain stem and limbic system, leading to sequential CRH release and synthesis. CRH transcription is required to restore mRNA and peptide levels, but termination of the response is essential to prevent pathology associated with chronic elevations of CRH and HPA axis activity. Inhibitory feedback mediated by glucocorticoids and intracellular production of the repressor, Inducible Cyclic AMP Early Repressor (ICER), limit the magnitude and duration of CRH neuronal activation. Induction of CRH transcription is mediated by the cyclic AMP/protein kinase A/cyclic AMP responsive element binding protein (CREB)-dependent pathways, and requires cyclic AMP-dependent nuclear translocation of the CREB co-activator, Transducer of Regulated CREB activity (TORC). This article reviews current knowledge on the mechanisms regulating CRH neuron activity. PMID:21871477

  14. Comparison of cellular responses induced by low level light in different cell types

    NASA Astrophysics Data System (ADS)

    Huang, Ying-Ying; Chen, Aaron C.-H.; Sharma, Sulbha K.; Wu, Qiuhe; Hamblin, Michael R.

    2010-02-01

    Discoveries are rapidly being made in multiple laboratories that shed "light" on the fundamental molecular and cellular mechanisms underlying the use of low level light therapy (LLLT) in vitro, in animal models and in clinical practice. Increases in cellular levels of respiration, in cytochrome c oxidase activity, in ATP levels and in cyclic AMP have been found. Increased expression of reactive oxygen species and release of nitric oxide have also been shown. In order for these molecular changes to have a major effect on cell behavior, it is likely that various transcription factors will be activated, possibly via different signal transduction pathways. In this report we compare and contrast the effects of LLLT in vitro on murine embryonic fibroblasts, primary cortical neurons, cardiomyocytes and bone-marrow derived dendritic cells. We also examined two human cell lines, HeLa cancer cells and HaCaT keratinocytes. The effects of 810-nm near-infra-red light delivered at low and high fluences were addressed. Reactive oxygen species generation, transcription factor activation and ATP increases are reported. The data has led to the hypothesis that cells with a high level of mitochondrial activity (mitochondrial membrane potential) have a higher response to light than cells with low mitochondrial activity.

  15. Role of hippocampal activity-induced transcription in memory consolidation.

    PubMed

    Eagle, Andrew L; Gajewski, Paula A; Robison, Alfred J

    2016-08-01

    Experience-dependent changes in the strength of connections between neurons in the hippocampus (HPC) are critical for normal learning and memory consolidation, and disruption of this process drives a variety of neurological and psychiatric diseases. Proper HPC function relies upon discrete changes in gene expression driven by transcription factors (TFs) induced by neuronal activity. Here, we describe the induction and function of many of the most well-studied HPC TFs, including cyclic-AMP response element binding protein, serum-response factor, AP-1, and others, and describe their role in the learning process. We also discuss the known target genes of many of these TFs and the purported mechanisms by which they regulate long-term changes in HPC synaptic strength. Moreover, we propose that future research in this field will depend upon unbiased identification of additional gene targets for these activity-dependent TFs and subsequent meta-analyses that identify common genes or pathways regulated by multiple TFs in the HPC during learning or disease. PMID:27180338

  16. Effect of Global Regulators RpoS and Cyclic-AMP/CRP on the Catabolome and Transcriptome of Escherichia coli K12 during Carbon- and Energy-Limited Growth.

    PubMed

    Franchini, Alessandro G; Ihssen, Julian; Egli, Thomas

    2015-01-01

    For heterotrophic microbes, limited availability of carbon and energy sources is one of the major nutritional factors restricting the rate of growth in most ecosystems. Physiological adaptation to this hunger state requires metabolic versatility which usually involves expression of a wide range of different catabolic pathways and of high-affinity carbon transporters; together, this allows for simultaneous utilization of mixtures of carbonaceous compounds at low concentrations. In Escherichia coli the stationary phase sigma factor RpoS and the signal molecule cAMP are the major players in the regulation of transcription under such conditions; however, their interaction is still not fully understood. Therefore, during growth of E. coli in carbon-limited chemostat culture at different dilution rates, the transcriptomes, expression of periplasmic proteins and catabolomes of strains lacking one of these global regulators, either rpoS or adenylate cyclase (cya), were compared to those of the wild-type strain. The inability to synthesize cAMP exerted a strong negative influence on the expression of alternative carbon source uptake and degradation systems. In contrast, absence of RpoS increased the transcription of genes belonging to high-affinity uptake systems and central metabolism, presumably due to reduced competition of σ(D) with σ(S). Phenotypical analysis confirmed this observation: The ability to respire alternative carbon substrates and to express periplasmic high-affinity binding proteins was eliminated in cya and crp mutants, while these properties were not affected in the rpoS mutant. As expected, transcription of numerous stress defence genes was negatively affected by the rpoS knock-out mutation. Interestingly, several genes of the RpoS stress response regulon were also down-regulated in the cAMP-negative strain indicating a coordinated global regulation. The results demonstrate that cAMP is crucial for catabolic flexibility during slow, carbon

  17. Effect of Global Regulators RpoS and Cyclic-AMP/CRP on the Catabolome and Transcriptome of Escherichia coli K12 during Carbon- and Energy-Limited Growth

    PubMed Central

    Egli, Thomas

    2015-01-01

    For heterotrophic microbes, limited availability of carbon and energy sources is one of the major nutritional factors restricting the rate of growth in most ecosystems. Physiological adaptation to this hunger state requires metabolic versatility which usually involves expression of a wide range of different catabolic pathways and of high-affinity carbon transporters; together, this allows for simultaneous utilization of mixtures of carbonaceous compounds at low concentrations. In Escherichia coli the stationary phase sigma factor RpoS and the signal molecule cAMP are the major players in the regulation of transcription under such conditions; however, their interaction is still not fully understood. Therefore, during growth of E. coli in carbon-limited chemostat culture at different dilution rates, the transcriptomes, expression of periplasmic proteins and catabolomes of strains lacking one of these global regulators, either rpoS or adenylate cyclase (cya), were compared to those of the wild-type strain. The inability to synthesize cAMP exerted a strong negative influence on the expression of alternative carbon source uptake and degradation systems. In contrast, absence of RpoS increased the transcription of genes belonging to high-affinity uptake systems and central metabolism, presumably due to reduced competition of σD with σS. Phenotypical analysis confirmed this observation: The ability to respire alternative carbon substrates and to express periplasmic high-affinity binding proteins was eliminated in cya and crp mutants, while these properties were not affected in the rpoS mutant. As expected, transcription of numerous stress defence genes was negatively affected by the rpoS knock-out mutation. Interestingly, several genes of the RpoS stress response regulon were also down-regulated in the cAMP-negative strain indicating a coordinated global regulation. The results demonstrate that cAMP is crucial for catabolic flexibility during slow, carbon-limited growth

  18. Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion*S⃞

    PubMed Central

    Sabbatini, Maria E.; Chen, Xuequn; Ernst, Stephen A.; Williams, John A.

    2008-01-01

    Rap1 is a member of the Ras superfamily of small GTP-binding proteins and is localized on pancreatic zymogen granules. The current study was designed to determine whether GTP-Rap1 is involved in the regulation of amylase secretion. Rap1A/B and the two Rap1 guanine nucleotide exchange factors, Epac1 and CalDAG-GEF III, were identified in mouse pancreatic acini. A fraction of both Rap1 and Epac1 colocalized with amylase in zymogen granules, but only Rap1 was integral to the zymogen granule membranes. Stimulation with cholecystokinin (CCK), carbachol, and vasoactive intestinal peptide all induced Rap1 activation, as did calcium ionophore A23187, phorbol ester, forskolin, 8-bromo-cyclic AMP, and the Epac-specific cAMP analog 8-pCPT-2′-O-Me-cAMP. The phospholipase C inhibitor U-73122 abolished carbachol- but not forskolin-induced Rap1 activation. Co-stimulation with carbachol and 8-pCPT-2′-O-Me-cAMP led to an additive effect on Rap1 activation, whereas a synergistic effect was seen on amylase release. Although the protein kinase A inhibitor H-89 abolished forskolin-stimulated CREB phosphorylation, it did not modify forskolin-induced GTP-Rap1 levels, excluding PKA participation. Overexpression of Rap1 GTPase-activating protein, which blocked Rap1 activation, reduced the effect of 8-bromo-cyclic AMP, 8-pCPT-2′-O-Me-cAMP, and vasoactive intestinal peptide on amylase release by 60% and reduced CCK- as well as carbachol-stimulated pancreatic amylase release by 40%. These findings indicate that GTP-Rap1 is required for pancreatic amylase release. Rap1 activation not only mediates the cAMP-evoked response via Epac1 but is also involved in CCK- and carbachol-induced amylase release, with their action most likely mediated by CalDAG-GEF III. PMID:18577515

  19. β-Adrenergic stimulation does not activate p38 MAP kinase or induce PGC-1α in skeletal muscle.

    PubMed

    Kim, Sang Hyun; Asaka, Meiko; Higashida, Kazuhiko; Takahashi, Yumiko; Holloszy, John O; Han, Dong-Ho

    2013-04-15

    There are reports that the β-adrenergic agonist clenbuterol induces a large increase in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in skeletal muscle. This has led to the hypothesis that the increases in PGC-1α and mitochondrial biogenesis induced in muscle by endurance exercise are mediated by catecholamines. In the present study, we evaluated this possibility and found that injecting rats with clenbuterol or norepinephrine induced large increases in PGC-1α and mitochondrial proteins in brown adipose tissue but had no effect on PGC-1α expression or mitochondrial biogenesis in skeletal muscle. In brown adipocytes, the increase in PGC-1α expression induced by β-adrenergic stimulation is mediated by activation of p38 mitogen-activated protein kinase (p38 MAPK), which phosphorylates and activates the cAMP response element binding protein (CREB) family member activating transcription factor 2 (ATF2), which binds to a cyclic AMP response element (CRE) in the PGC-1α promoter and mediates the increase in PGC-1α transcription. Phospho-CREB does not have this effect. Our results show that the reason for the lack of effect of β-adrenergic stimulation on PGC-1α expression in muscle is that catecholamines do not activate p38 or increase ATF2 phosphorylation in muscle. PMID:23443926

  20. The Neuroprotective Effect of Lithium in cannabinoid Dependence is Mediated through Modulation of Cyclic AMP, ERK1/2 and GSK-3β Phosphorylation in Cerebellar Granular Neurons of Rat

    PubMed Central

    Rahimi, Hamid Reza; Ghahremani, Mohammad Hossein; Dehpour, Ahmad Reza; Sharifzadeh, Mohammad; Ejtemaei-Mehr, Shahram; Razmi, Ali; Ostad, Seyed Nasser

    2015-01-01

    Lithium (Li), a glycogen synthase kinase-3β (GSK-3β) inhibitor, has used to attenuate the cannabinoid-induced dependence/withdrawal signs, but molecular mechanisms related to this are unclear. Recent studies indicate the involvement of upstream extracellular signal kinase1/2 (ERK1/2) and downstream GSK-3β pathways in the development of cannabinoid-induced dependence. This is mediated through cannabinoid receptor 1 (CB1) enriched in cerebellar granular neurons (CGNs). Accordingly, the present study aimed to investigate the mechanism of modulatory/neuroprotective effects of Li on a cannabinoid agonist (WIN 55,212-2 (WIN))-induced dependence, through quantitative analysis of some involved proteins such as ERK1/2, GSK-3β and related signaling pathways including their phosphorylated forms; and cAMP level as the other molecular mechanisms leading to dependence, in CGNs model. The CGNs were prepared from 7-day-old Wistar rat pup in a 12-well plate, pretreated with Li (1mM) and an ERK1/2 inhibitor SL327 (SL, 10 µM). The WIN (1 µM) was added 30 minutes prior to treatment and AM251 (AM, 1 µM), as a cannabinoid antagonist was co-treated with WIN. The cAMP level, as an indicator of cannabinoid-induced dependence, was measured by ELISA following forskolin (FSK) stimulation. Western blot analyses determined the phosphorylated forms of ERK1/2 (p-ERK1/2), GSK-3β (p-GSK-3β) as well as their total expressions in various treatment times and doses in CGNs. WIN alone could down regulate the cAMP/p-ERK1/2 cascade compared to AM treatment. However, P-GSK-3β was up-regulated with Li and WIN or with SL and Li pretreatment to AM-induced cellular response, which was the highest 60 minutes after CGNs exposure. Results further suggested the potential role of Li pretreatment to diminish the development of cannabinoid-induced dependence/neuronal injury through possible mechanisms of modulating the cAMP/p-ERK1/2 cascade independent of p-GSK-3β signaling pathway in-vitro. PMID:26664379

  1. Age-related changes in AMPK activation: Role for AMPK phosphatases and inhibitory phosphorylation by upstream signaling pathways.

    PubMed

    Salminen, Antero; Kaarniranta, Kai; Kauppinen, Anu

    2016-07-01

    AMP-activated protein kinase (AMPK) is a fundamental regulator of energy metabolism, stress resistance, and cellular proteostasis. AMPK signaling controls an integrated signaling network which is involved in the regulation of healthspan and lifespan e.g. via FoxO, mTOR/ULK1, CRCT-1/CREB, and SIRT1 signaling pathways. Several studies have demonstrated that the activation capacity of AMPK signaling declines with aging, which impairs the maintenance of efficient cellular homeostasis and enhances the aging process. However, it seems that the aging process affects AMPK activation in a context-dependent manner since occasionally, it can also augment AMPK activation, possibly attributable to the type of insult and tissue homeostasis. Three protein phosphatases, PP1, PP2A, and PP2C, inhibit AMPK activation by dephosphorylating the Thr172 residue of AMPKα, required for AMPK activation. In addition, several upstream signaling pathways can phosphorylate Ser/Thr residues in the β/γ interaction domain of the AMPKα subunit that subsequently blocks the activation of AMPK. These inhibitory pathways include the insulin/AKT, cyclic AMP/PKA, and RAS/MEK/ERK pathways. We will examine the evidence whether the efficiency of AMPK responsiveness declines during the aging process. Next, we will review the mechanisms involved in curtailing the activation of AMPK. Finally, we will elucidate the potential age-related changes in the inhibitory regulation of AMPK signaling that might be a part of the aging process. PMID:27060201

  2. The impact of social status on the erythrocyte beta-adrenergic response in rainbow trout, Oncorhynchus mykiss.

    PubMed

    Thomas, J B; Gilmour, K M

    2006-02-01

    The objective of the present investigation was to determine whether chronic increases in circulating cortisol concentrations, resulting from the occupation of subordinate status in rainbow trout social hierarchies, resulted in an enhancement of the erythrocyte adrenergic response. Rainbow trout (Oncorhynchus mykiss) were confined in fork length matched pairs for 6 h, 18 h, 48 h or 5-7 days, and social status was assigned through observations of behaviour. Erythrocyte adrenergic responsiveness, determined in vitro as changes in water content following incubation with the beta-adrenoreceptor agonist isoproterenol, was significantly greater in subordinate than dominant fish at 48 h of social interactions but not after 5-7 days, nor when assessed as changes in extracellular pH (pHe). However, the activity of the Na+/H+ exchanger (beta-NHE), assessed in vitro as the pHe change following incubation with the permeable cyclic AMP analogue 8-bromo-cyclic AMP, was significantly lower in subordinate fish. The number of erythrocyte membrane-bound adrenergic receptors (Bmax) was significantly higher in subordinate than dominant fish at 48 h, but had decreased by 5-7 days to a value that was not significantly different from that for dominant fish. The apparent dissociation constant (KD) of these receptors was not significantly impacted by either social status or interaction time. Finally, the relative expressions of beta-3b adrenergic receptor (AR) and beta-NHE mRNA were determined using real-time PCR and were found to be minimally affected by social rank. Relative to a control group, beta-3b AR mRNA was significantly up-regulated in both dominant and subordinate trout at all time periods, whereas the expression of beta-NHE was in general significantly down-regulated. Unlike the situation in rainbow trout treated with exogenous cortisol, elevations in circulating cortisol resulting from low social status did not "pre-adapt" the erythrocyte adrenergic response, but rather may

  3. Identification and Functional Characterization of Rca1, a Transcription Factor Involved in both Antifungal Susceptibility and Host Response in Candida albicans

    PubMed Central

    Vandeputte, Patrick; Pradervand, Sylvain; Ischer, Françoise; Coste, Alix T.; Ferrari, Sélène; Harshman, Keith

    2012-01-01

    The identification of novel transcription factors associated with antifungal response may allow the discovery of fungus-specific targets for new therapeutic strategies. A collection of 241 Candida albicans transcriptional regulator mutants was screened for altered susceptibility to fluconazole, caspofungin, amphotericin B, and 5-fluorocytosine. Thirteen of these mutants not yet identified in terms of their role in antifungal response were further investigated, and the function of one of them, a mutant of orf19.6102 (RCA1), was characterized by transcriptome analysis. Strand-specific RNA sequencing and phenotypic tests assigned Rca1 as the regulator of hyphal formation through the cyclic AMP/protein kinase A (cAMP/PKA) signaling pathway and the transcription factor Efg1, but also probably through its interaction with a transcriptional repressor, most likely Tup1. The mechanisms responsible for the high level of resistance to caspofungin and fluconazole observed resulting from RCA1 deletion were investigated. From our observations, we propose that caspofungin resistance was the consequence of the deregulation of cell wall gene expression and that fluconazole resistance was linked to the modulation of the cAMP/PKA signaling pathway activity. In conclusion, our large-scale screening of a C. albicans transcription factor mutant collection allowed the identification of new effectors of the response to antifungals. The functional characterization of Rca1 assigned this transcription factor and its downstream targets as promising candidates for the development of new therapeutic strategies, as Rca1 influences host sensing, hyphal development, and antifungal response. PMID:22581526

  4. Strain activation of bovine aortic smooth muscle cell proliferation and alignment: study of strain dependency and the role of protein kinase A and C signaling pathways

    NASA Technical Reports Server (NTRS)

    Mills, I.; Cohen, C. R.; Kamal, K.; Li, G.; Shin, T.; Du, W.; Sumpio, B. E.

    1997-01-01

    Smooth muscle cell (SMC) phenotype can be altered by physical forces as demonstrated by cyclic strain-induced changes in proliferation, orientation, and secretion of macromolecules. However, the magnitude of strain required and the intracellular coupling pathways remain ill defined. To examine the strain requirements for SMC proliferation, we selectively seeded bovine aortic SMC either on the center or periphery of silastic membranes which were deformed with 150 mm Hg vacuum (0-7% center; 7-24% periphery). SMC located in either the center or peripheral regions showed enhanced proliferation compared to cells grown under the absence of cyclic strain. Moreover, SMC located in the center region demonstrated significantly (P < 0.005) greater proliferation as compared to those in the periphery. In contrast, SMC exposed to high strain (7-24%) demonstrated alignment perpendicular to the strain gradient, whereas SMC in the center (0-7%) remained aligned randomly. To determine the mechanisms of these phenomena, we examined the effect of cyclic strain on bovine aortic SMC signaling pathways. We observed strain-induced stimulation of the cyclic AMP pathway including adenylate cyclase activity and cyclic AMP accumulation. In addition, exposure of SMC to cyclic strain caused a significant increase in protein kinase C (PKC) activity and enzyme translocation from the cytosol to a particulate fraction. Further study was conducted to examine the effect of strain magnitude on signaling, particularly protein kinase A (PKA) activity as well as cAMP response element (CRE) binding protein levels. We observed significantly (P < 0.05) greater PKA activity and CRE binding protein levels in SMC located in the center as compared to the peripheral region. However, inhibition of PKA (with 10 microM Rp-cAMP) or PKC (with 5-20 ng/ml staurosporine) failed to alter either the strain-induced increase in SMC proliferation or alignment. These data characterize the strain determinants for activation of

  5. Sucralose, an activator of the glucose-sensing receptor, increases ATP by calcium-dependent and -independent mechanisms.

    PubMed

    Li, Longfei; Ohtsu, Yoshiaki; Nakagawa, Yuko; Masuda, Katsuyoshi; Kojima, Itaru

    2016-08-31

    Sucralose is an artificial sweetener and activates the glucose-sensing receptor expressed in pancreatic β-cells. Although sucralose does not enter β-cells nor acts as a substrate for glucokinase, it induces a marked elevation of intracellular ATP ([ATP]c). The present study was conducted to identify the signaling pathway responsible for the elevation of [ATP]c induced by sucralose. Previous studies have shown that sucralose elevates cyclic AMP (cAMP), activates phospholipase C (PLC) and stimulates Ca(2+) entry by a Na(+)-dependent mechanism in MIN6 cells. The addition of forskolin induced a marked elevation of cAMP, whereas it did not affect [ATP]c. Carbachol, an activator of PLC, did not increase [ATP]c. In addition, activation of protein kinase C by dioctanoylglycerol did not affect [ATP]c. In contrast, nifedipine, an inhibitor of the voltage-dependent Ca(2+) channel, significantly reduced [ATP]c response to sucralose. Removal of extracellular Na(+) nearly completely blocked sucralose-induced elevation of [ATP]c. Stimulation of Na(+) entry by adding a Na(+) ionophore monensin elevated [ATP]c. The monensin-induced elevation of [ATP]c was only partially inhibited by nifedipine and loading of BAPTA, both of which completely abolished elevation of [Ca(2+)]c. These results suggest that Na(+) entry is critical for the sucralose-induced elevation of [ATP]c. Both calcium-dependent and -independent mechanisms are involved in the action of sucralose. PMID:27250218

  6. Physiological Response to Physical Activity in Children.

    ERIC Educational Resources Information Center

    Gilliam, Thomas B.

    This is a report on research in the field of physical responses of children to strenuous activity. The paper is divided into three subtopics: (1) peak performance measure in children; (2) training effects on children; and (3) importance of physical activity for children. Measurements used are oxygen consumption, ventilation, heart rate, cardiac…

  7. Ibudilast, a Pharmacologic Phosphodiesterase Inhibitor, Prevents Human Immunodeficiency Virus-1 Tat-Mediated Activation of Microglial Cells

    PubMed Central

    Kiebala, Michelle; Maggirwar, Sanjay B.

    2011-01-01

    Human Immunodeficiency Virus-1 (HIV-1)-associated neurocognitive disorders (HAND) occur, in part, due to the inflammatory response to viral proteins, such as the HIV-1 transactivator of transcription (Tat), in the central nervous system (CNS). Given the need for novel adjunctive therapies for HAND, we hypothesized that ibudilast would inhibit Tat-induced excess production of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNFα) in microglial cells. Ibudilast is a non-selective cyclic AMP phosphodiesterase inhibitor that has recently shown promise as a treatment for neuropathic pain via its ability to attenuate glial cell activation. Accordingly, here we demonstrate that pre-treatment of both human and mouse microglial cells with increasing doses of ibudilast inhibited Tat-induced synthesis of TNFα by microglial cells in a manner dependent on serine/threonine protein phosphatase activity. Ibudilast had no effect on Tat-induced p38 MAP kinase activation, and blockade of adenosine A2A receptor activation did not reverse ibudilast's inhibition of Tat-induced TNFα production. Interestingly, ibudilast reduced Tat-mediated transcription of TNFα, via modulation of nuclear factor-kappa B (NF-κB) signaling, as shown by transcriptional activity of NF-κB and analysis of inhibitor of kappa B alpha (IκBα) stability. Together, our findings shed light on the mechanism of ibudilast's inhibition of Tat-induced TNFα production in microglial cells and may implicate ibudilast as a potential novel adjunctive therapy for the management of HAND. PMID:21494611

  8. Stress Reorganization and Response in Active Solids

    NASA Astrophysics Data System (ADS)

    Hawkins, Rhoda J.; Liverpool, Tanniemola B.

    2014-07-01

    We present a microscopic model of a disordered viscoelastic active solid, i.e., an active material whose long time behavior is elastic as opposed to viscous. It is composed of filaments, passive cross-links, and molecular motors powered by stored chemical energy, e.g., actomyosin powered by adenosine triphosphate. Our model allows us to study the collective behavior of contractile active elements and how their interaction with each other and the passive elastic elements determines the macroscopic mechanical properties of the active material. As a result of the (un)binding dynamics of the active elements, we find that this system provides a highly responsive material with a dynamic mechanical response strongly dependent on the amount of deformation.

  9. Receptor-mediated gonadotropin action in the ovary. Regulatory role of cyclic nucleotide phosphodiesterase(s) in intracellular adenosine 3′:5′-cyclic monophosphate turnover and gonadotropin-stimulated progesterone production by rat ovarian cells

    PubMed Central

    Azhar, Salman; Menon, K. M. Jairam

    1979-01-01

    The regulatory role of cyclic nucleotide phosphodiesterase(s) and cyclic AMP metabolism in relation to progesterone production by gonadotropins has been studied in isolated rat ovarian cells. Low concentrations of choriogonadotropin (0.4–5ng/ml) increased steroid production without any detectable increase in cyclic AMP, when experiments were carried out in the absence of phosphodiesterase inhibitors. The concentration of choriogonadotropin (10ng/ml) that stimulated progesterone synthesis maximally resulted in a minimal increase in cyclic AMP accumulation and choriogonadotropin binding. Choriogonadotropin at a concentration of 10ng/ml and higher, however, significantly stimulated protein kinase activity and reached a maximum between 250 and 1000ng of hormone/ml. Higher concentrations (50–2500ng/ml) of choriogonadotropin caused an increase in endogenous cyclic AMP, and this increase preceded the increase in steroid synthesis. Analysis of dose–response relationships of gonadotropin-stimulated cyclic AMP accumulation, progesterone production and protein kinase activity revealed a correlation between these responses over a wide concentration range when experiments were performed in the presence of 3-isobutyl-1-methylxanthine. The phosphodiesterase inhibitors papaverine, theophylline and 3-isobutyl-1-methylxanthine each stimulated steroid production in a dose-dependent manner. Incubation of ovarian cells with dibutyryl cyclic AMP or 8-bromo cyclic AMP mimicked the steroidogenic action of gonadotropins and this effect was dependent on both incubation time and nucleotide concentration. Maximum stimulation was obtained with 2mm-dibutyryl cyclic AMP and 8-bromo cyclic AMP, and this increase was close to that produced by a maximally stimulating dose of choriogonadotropin. Other 8-substituted derivatives such as 8-hydroxy cyclic AMP and 8-isopropylthio cyclic AMP, which were less susceptible to phosphodiesterase action, also effectively stimulated steroidogenesis. The

  10. Active thermal isolation for temperature responsive sensors

    NASA Technical Reports Server (NTRS)

    Martinson, Scott D. (Inventor); Gray, David L. (Inventor); Carraway, Debra L. (Inventor); Reda, Daniel C. (Inventor)

    1994-01-01

    A temperature responsive sensor is located in the airflow over the specified surface of a body and is maintained at a constant temperature. An active thermal isolator is located between this temperature responsive sensor and the specified surface of the body. The temperature of this isolator is controlled to reduce conductive heat flow from the temperature responsive sensor to the body. This temperature control includes: (1) operating the isolator at the same temperature as the constant temperature of the sensor and (2) establishing a fixed boundary temperature which is either less than or equal to or slightly greater than the sensor constant temperature.

  11. Cellular Stress Responses and Monitored Cellular Activities.

    PubMed

    Sawa, Teiji; Naito, Yoshifumi; Kato, Hideya; Amaya, Fumimasa

    2016-08-01

    To survive, organisms require mechanisms that enable them to sense changes in the outside environment, introduce necessary responses, and resist unfavorable distortion. Consequently, through evolutionary adaptation, cells have become equipped with the apparatus required to monitor their fundamental intracellular processes and the mechanisms needed to try to offset malfunction without receiving any direct signals from the outside environment. It has been shown recently that eukaryotic cells are equipped with a special mechanism that monitors their fundamental cellular functions and that some pathogenic proteobacteria can override this monitoring mechanism to cause harm. The monitored cellular activities involved in the stressed intracellular response have been researched extensively in Caenorhabditis elegans, where discovery of an association between key mitochondrial activities and innate immune responses was named "cellular associated detoxification and defenses (cSADD)." This cellular surveillance pathway (cSADD) oversees core cellular activities such as mitochondrial respiration and protein transport into mitochondria, detects xenobiotics and invading pathogens, and activates the endocrine pathways controlling behavior, detoxification, and immunity. The cSADD pathway is probably associated with cellular responses to stress in human inflammatory diseases. In the critical care field, the pathogenesis of lethal inflammatory syndromes (e.g., respiratory distress syndromes and sepsis) involves the disturbance of mitochondrial respiration leading to cell death. Up-to-date knowledge about monitored cellular activities and cSADD, especially focusing on mitochondrial involvement, can probably help fill a knowledge gap regarding the pathogenesis of lethal inflammatory syndromes in the critical care field. PMID:26954943

  12. Characterization of quinolone antibacterial-induced convulsions and increases in nuclear AP-1 DNA- and CRE-binding activities in mouse brain.

    PubMed

    Ito, Y; Ishige, K; Aizawa, M; Fukuda, H

    1999-05-01

    The quinolone antibacterials enoxacin and norfloxacin (2.5 mg/kg, i.v.) provoked clonic convulsions in mice treated concomitantly with biphenylacetic acid (BPAA, 100 mg/kg, i.p.), a major metabolite of the nonsteroidal anti-inflammatory drug fenbufen. Gel-shift assays showed that enoxacin-induced convulsions resulted in increases in nuclear activator protein 1 (AP-1) DNA- and cyclic AMP responsive element (CRE)-binding activities in the cerebral cortex and hippocampus, but not in other regions, such as the cerebellum and thalamus. In contrast, ofloxacin and levofloxacin, at the same doses, in the presence of BPAA did not evoke convulsions or increase these DNA-binding activities. Administration of these quinolones and BPAA alone elicited neither convulsions nor increases in these DNA-binding activities. These results suggest that the increased nuclear AP-1 DNA- and CRE-binding activities in the cerebral cortex and hippocampus induced by quinolones with BPAA correlated with seizure activities and that these brain regions play pivotal roles in quinolone-induced convulsions. PMID:10340309

  13. Acetaldehyde Induces Cytotoxicity of SH-SY5Y Cells via Inhibition of Akt Activation and Induction of Oxidative Stress

    PubMed Central

    Yan, Tingting; Zhao, Yan; Zhang, Xia

    2016-01-01

    Excessive alcohol consumption can lead to brain tissue damage and cognitive dysfunction. It has been shown that heavy drinking is associated with an earlier onset of neurodegenerative diseases such as Alzheimer's disease. Acetaldehyde, the most toxic metabolite of ethanol, is speculated to mediate the brain tissue damage and cognitive dysfunction induced by the chronic excessive consumption of alcohol. However, the exact mechanisms by which acetaldehyde induces neurotoxicity are not totally understood. In this study, we investigated the cytotoxic effects of acetaldehyde in SH-SY5Y cells and found that acetaldehyde induced apoptosis of SH-SY5Y cells by downregulating the expression of antiapoptotic Bcl-2 and Bcl-xL and upregulating the expression of proapoptotic Bax. Acetaldehyde treatment led to a significant decrease in the levels of activated Akt and cyclic AMP-responsive element binding protein (CREB). In addition, acetaldehyde induced the activation of p38 mitogen-activated protein kinase (MAPK) while inhibiting the activation of extracellular signal-regulated kinases (ERKs, p44/p42MAPK). Meanwhile, acetaldehyde treatment caused an increase in the production of reactive oxygen species and elevated the oxidative stress in SH-SY5Y cells. Therefore, acetaldehyde induces cytotoxicity of SH-SY5Y cells via promotion of apoptotic signaling, inhibition of cell survival pathway, and induction of oxidative stress. PMID:26649137

  14. Chronic mild stress inhibits BDNF protein expression and CREB activation in the dentate gyrus but not in the hippocampus proper.

    PubMed

    Grønli, Janne; Bramham, Clive; Murison, Robert; Kanhema, Tambudzai; Fiske, Eldbjørg; Bjorvatn, Bjørn; Ursin, Reidun; Portas, Chiara M

    2006-12-01

    Chronic stress is linked to development of depression and may trigger neurobiological changes underlying the disease. Downregulation of the secretory peptide brain-derived neurotrophic factor (BDNF) and the transcriptional regulator calcium/cyclic-AMP responsive binding protein (CREB) have been implicated in stress and depression-related pathology in animal studies. When animals are exposed to the chronic mild stress (CMS) protocol, multiple depression-like symptoms are observed. Here we investigated the effect of CMS on BDNF protein expression and CREB activation in the dentate gyrus and hippocampus proper. Rats exposed for 5 weeks to repeated, unpredictable, mild stressors showed reduced BDNF expression and inhibited phosphorylation of CREB (Ser-133) in the dentate gyrus (-25.0%+/-3.5% and -29.7+/-7.3%, respectively), whereas no significant effects were observed in the hippocampus proper. CMS-treated rats consumed less sucrose compared to control rats, indicating a state of anhedonia. Moreover, phospho-CREB levels in the dentate gyrus were positively correlated with the animals' sucrose intake at the end of the CMS protocol. These results couple chronic mild stress to a downregulation of CREB activity and BDNF protein expression specifically within the dentate gyrus and support the possibility that the BDNF-CREB system plays an important role in the response to environmental challenges. PMID:17204313

  15. Granulosa cell apoptosis, aromatase activity, cyclic adenosine 3',5'-monophosphate response to gonadotropins, and follicular fluid steroid levels during spontaneous and induced follicular atresia in ewes.

    PubMed

    Jolly, P D; Tisdall, D J; De'ath, G; Heath, D A; Lun, S; Hudson, N L; McNatty, K P

    1997-04-01

    The aims of the present study in ewes were 1) to test the hypothesis that apoptosis in granulosa cells is one of the processes involved in the structural demise of follicles and 2) to define the temporal relationships among the occurrence and degree of apoptosis in granulosa cells, aromatase activity, production of cyclic AMP (cAMP) by granulosa cells in response to FSH or LH, concentrations of estradiol 17 beta (E2) and progesterone in follicular fluid, and the characteristic morphometric changes associated with the process of follicular atresia. To address these aims, ewes were treated with either saline or steroid-free bovine follicular fluid (bFF) at 60 h after estrus, and ovarian follicles > or = 3 mm diameter were recovered at 0, 12, 18, or 24 h later. Apoptotic granulosa cells were identified by the presence of oligonucleosomes after 3'-end labeling of extracted DNA with [32P]alpha dideoxy ATP (ddATP). The degree of oligonucleosome formation, based on the intensity of radiolabeling, was given an apoptosis score (AP) of 0 (nondetectable), 1 (slight), 2 (moderate), or 3 (marked). Moreover, a labeling index (LI) was calculated from the amount of radiolabeled ddATP incorporated into low-molecular weight (< 4.2 kb) DNA fragments. On the basis of morphometric criteria, 73% (141 of 194) of the follicles classified as healthy had apoptotic granulosa cells compared to 86% (18 of 21) of the follicles classified as atretic. In the bFF-but not saline-treated ewes, the concentrations of plasma FSH had declined to basal values at 12 h after treatment. At the beginning of the treatment period, the degree of granulosa cell apoptosis was either undetectable (AP = 0, 47% of follicles) or slight (AP = 1, 44% of follicles) in the majority of follicles. After 12 h from the bFF but not the saline injection, there was a significant increase in the proportion of follicles (> or = 3 mm diameter) per ewe containing apoptotic granulosa cells (p < 0.001) and a significant decrease in

  16. A CaMK cascade activates CRE-mediated transcription in neurons of Caenorhabditis elegans

    PubMed Central

    Kimura, Yoshishige; Corcoran, Ethan E.; Eto, Koh; Gengyo-Ando, Keiko; Muramatsu, Masa-aki; Kobayashi, Ryoji; Freedman, Jonathan H.; Mitani, Shohei; Hagiwara, Masatoshi; Means, Anthony R.; Tokumitsu, Hiroshi

    2002-01-01

    Calcium (Ca2+) signals regulate a diverse set of cellular responses, from proliferation to muscular contraction and neuro-endocrine secretion. The ubiquitous Ca2+ sensor, calmodulin (CaM), translates changes in local intracellular Ca2+ concentrations into changes in enzyme activities. Among its targets, the Ca2+/CaM-dependent protein kinases I and IV (CaMKs) are capable of transducing intraneuronal signals, and these kinases are implicated in neuronal gene regulation that mediates synaptic plasticity in mammals. Recently, the cyclic AMP response element binding protein (CREB) has been proposed as a target for a CaMK cascade involving not only CaMKI or CaMKIV, but also an upstream kinase kinase that is also CaM regulated (CaMKK). Here, we report that all components of this pathway are coexpressed in head neurons of Caenorhabditis elegans. Utilizing a transgenic approach to visualize CREB-dependent transcription in vivo, we show that this CaMK cascade regulates CRE-mediated transcription in a subset of head neurons in living nematodes. PMID:12231504

  17. Teaching Responsibility through Physical Activity. Second Edition.

    ERIC Educational Resources Information Center

    Hellison, Don

    This book guides teachers in using physical activity to foster personal and social responsibility. Focusing on teaching in school settings, the book features comments from real students to motivate teachers to apply the concept; take-aways that summarize each chapter and help teachers consider their own situations; new chapters on the lesson plan…

  18. Rigor and Responsiveness in Classroom Activity

    ERIC Educational Resources Information Center

    Thomspon, Jessica; Hagenah, Sara; Kang, Hosun; Stroupe, David; Braaten, Melissa; Colley, Carolyn; Windschitl, Mark

    2016-01-01

    Background/Context: There are few examples from classrooms or the literature that provide a clear vision of teaching that simultaneously promotes rigorous disciplinary activity and is responsive to all students. Maintaining rigorous and equitable classroom discourse is a worthy goal, yet there is no clear consensus of how this actually works in a…

  19. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  20. Activation state of the hyperpolarization-activated current modulates temperature-sensitivity of firing in locus coeruleus neurons from bullfrogs.

    PubMed

    Santin, Joseph M; Hartzler, Lynn K

    2015-06-15

    Locus coeruleus neurons of anuran amphibians contribute to breathing control and have spontaneous firing frequencies that, paradoxically, increase with cooling. We previously showed that cooling inhibits a depolarizing membrane current, the hyperpolarization-activated current (I h) in locus coeruleus neurons from bullfrogs, Lithobates catesbeianus (Santin JM, Watters KC, Putnam RW, Hartzler LK. Am J Physiol Regul Integr Comp Physiol 305: R1451-R1464, 2013). This suggests an unlikely role for I h in generating cold activation, but led us to hypothesize that inhibition of I h by cooling functions as a physiological brake to limit the cold-activated response. Using whole cell electrophysiology in brain slices, we employed 2 mM Cs(+) (an I h antagonist) to isolate the role of I h in spontaneous firing and cold activation in neurons recorded with either control or I h agonist (cyclic AMP)-containing artificial intracellular fluid. I h did not contribute to the membrane potential (V m) and spontaneous firing at 20°C. Although voltage-clamp analysis confirmed that cooling inhibits I h, its lack of involvement in setting baseline firing and V m precluded its ability to regulate cold activation as hypothesized. In contrast, neurons dialyzed with cAMP exhibited greater baseline firing frequencies at 20°C due to I h activation. Our hypothesis was supported when the starting level of I h was enhanced by elevating cAMP because cold activation was converted to more ordinary cold inhibition. These findings indicate that situations leading to enhancement of I h facilitate firing at 20°C, yet the hyperpolarization associated with inhibiting a depolarizing cation current by cooling blunts the net V m response to cooling to oppose normal cold-depolarizing factors. This suggests that the influence of I h activation state on neuronal firing varies in the poikilothermic neuronal environment. PMID:25833936

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

    PubMed Central

    Trevillyan, J M; Pall, M L

    1979-01-01

    It has been reported that diverse treatments which depolarize the plasma membrane of Neurospora crassa produce rapid increases in cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels. In the current study, membrane active antibiotics, which are known or putative depolarizing agents, were found to produce similar cyclic AMP increases, not only in N. crassa, but also in the distantly related fungi Saccharomyces cerevisiae and Mucor racemosus. Uncouplers of oxidative phosphorylation, which have been found to depolarize Neurospora, also produced cyclic AMP increases in all three fungi. The time course of the cyclic AMP response to these various treatments was similar in all three fungi. The fungal studies and studies on depolarized central nervous tissue suggest that cyclic AMP increases may be produced in response to plasma membrane depolarization in diverse eucaryotic cells. A model is proposed for eucaryotic microorganisms in which membrane depolarization serves as a signal of breakdown of the plasma membrane integrity. The subsequent cyclic AMP increase, in turn, may mediate cellular response to help protect the plasma membrane from chemical and mechanical threats to its integrity. PMID:220213

  2. Multiple mRNA isoforms of the transcription activator protein CREB: generation by alternative splicing and specific expression in primary spermatocytes.

    PubMed Central

    Ruppert, S; Cole, T J; Boshart, M; Schmid, E; Schütz, G

    1992-01-01

    We have characterized cDNA clones representing mouse CREB (cyclic AMP responsive element binding protein) mRNA isoforms. These include CREB delta and CREB alpha, of which the rat and human homologues have been previously identified. Both encode proteins with CRE-binding activity and identical transactivation potential. The additional CREB mRNA isoforms potentially encode CREB related proteins. From the structural organization of the mouse CREB gene we conclude that the multiple transcripts are generated by alternative splicing. Furthermore we show that specific CREB mRNA isoforms are expressed at a high level in the adult testis. Expression of these isoforms is induced after commencement of spermatogenesis. In situ hybridization suggests that this expression occurs predominantly in the primary spermatocytes. Comparison of the CREB gene with the recently isolated CREM (cAMP responsive element modulator) cDNAs illustrates that the two genes have arisen by gene duplication and have diverged to encode transcriptional activators and repressors of the cAMP signal transduction pathway. Images PMID:1532935

  3. Measurements of thermospheric response to auroral activities

    NASA Technical Reports Server (NTRS)

    Okano, S.; Kim, J. S.

    1986-01-01

    The Joule heating produced by auroral electrojets and its thermospheric response can be studied by monitoring the thermospheric temperatures by optical methods; simultaneously, the concurrent auroral electrojet activities can be investigated by using geomagnetic records obtained from stations along a meridian close to the observation site of optical measurements. The measurements are reported of thermospheric response to auroral activities which were made at Albany (42.68 deg N, 73.82 deg W), New York on September 2, 1978 (UT) when an isolated substorm occured. The thermospheric temperatures were measured by using a high resolution Fabry-Perot interferometer that determines the line profiles of the (OI) 6300A line emission. The intensities and latitudinal positions of auroral electrojets were obtained by the analysis of magnetograms from the IMS Fort Churchill meridian chain stations.

  4. Monoclonal antibodies to purified muscarinic receptor display agonist-like activity.

    PubMed Central

    Leiber, D; Harbon, S; Guillet, J G; André, C; Strosberg, A D

    1984-01-01

    Monoclonal antibody M-35, which immunoprecipitates native calf brain acetylcholine muscarinic receptor, mimics agonist stimulation of the intact guinea pig myometrium: the antibody, just like carbamoylcholine hydrochloride, causes a rise in intracellular cyclic GMP content, an inhibition of cyclic AMP accumulation due to prostacyclin, and induces uterine contractions. Another antibody, M-23, which reacts with the denatured muscarinic receptor, is devoid of agonist-like activity at the cyclic nucleotide level but is still able to induce contractions of both rat and guinea pig myometrium. The cyclic nucleotide changes caused by both carbamoylcholine and antibody M-35 are inhibited by atropine; this antagonist, which blocks carbamoylcholine-mediated contractions, fails however, to prevent contractions induced by antibodies M-35 and M-23. These results suggest that the information necessary to transmit muscarinic signals is entirely contained in the receptor and that ligands only act to trigger the biological response. The data also imply that the muscarinic receptors of the myometrium are coupled to multiple effector systems. PMID:6087318

  5. Diverse signaling systems activated by the sweet taste receptor in human GLP-1-secreting cells.

    PubMed

    Ohtsu, Yoshiaki; Nakagawa, Yuko; Nagasawa, Masahiro; Takeda, Shigeki; Arakawa, Hirokazu; Kojima, Itaru

    2014-08-25

    Sweet taste receptor regulates GLP-1 secretion in enteroendocrine L-cells. We investigated the signaling system activated by this receptor using Hutu-80 cells. We stimulated them with sucralose, saccharin, acesulfame K and glycyrrhizin. These sweeteners stimulated GLP-1 secretion, which was attenuated by lactisole. All these sweeteners elevated cytoplasmic cyclic AMP ([cAMP]c) whereas only sucralose and saccharin induced a monophasic increase in cytoplasmic Ca(2+) ([Ca(2+)]c). Removal of extracellular calcium or sodium and addition of a Gq/11 inhibitor greatly reduced the [Ca(2+)]c responses to two sweeteners. In contrast, acesulfame K induced rapid and sustained reduction of [Ca(2+)]c. In addition, glycyrrhizin first reduced [Ca(2+)]c which was followed by an elevation of [Ca(2+)]c. Reductions of [Ca(2+)]c induced by acesulfame K and glycyrrhizin were attenuated by a calmodulin inhibitor or by knockdown of the plasma membrane calcium pump. These results indicate that various sweet molecules act as biased agonists and evoke strikingly different patterns of intracellular signals. PMID:25017733

  6. Active thermal isolation for temperature responsive sensors

    NASA Technical Reports Server (NTRS)

    Martinson, Scott D. (Inventor); Gray, David L. (Inventor); Carraway, Debra L. (Inventor); Reda, Daniel C. (Inventor)

    1994-01-01

    The detection of flow transition between laminar and turbulent flow and of shear stress or skin friction of airfoils is important in basic research for validation of airfoil theory and design. These values are conventionally measured using hot film nickel sensors deposited on a polyimide substrate. The substrate electrically insulates the sensor and underlying airfoil but is prevented from thermally isolating the sensor by thickness constraints necessary to avoid flow contamination. Proposed heating of the model surface is difficult to control, requires significant energy expenditures, and may alter the basic flow state of the airfoil. A temperature responsive sensor is located in the airflow over the specified surface of a body and is maintained at a constant temperature. An active thermal isolator is located between this temperature responsive sensor and the specific surface of the body. The total thickness of the isolator and sensor avoid any contamination of the flow. The temperature of this isolator is controlled to reduce conductive heat flow from the temperature responsive sensor to the body. This temperature control includes (1) operating the isolator at the same temperature as the constant temperature of the sensor; and (2) establishing a fixed boundary temperature which is either less than or equal to, or slightly greater than the sensor constant temperature. The present invention accordingly thermally isolates a temperature responsive sensor in an energy efficient, controllable manner while avoiding any contamination of the flow.

  7. Dynamic response of active twist rotor blades

    NASA Astrophysics Data System (ADS)

    Cesnik, Carlos E. S.; Shin, Sang Joon; Wilbur, Matthew L.

    2001-02-01

    Dynamic characteristics of active twist rotor (ATR) blades are investigated analytically and experimentally in this paper. The ATR system is intended for vibration and potentially for noise reductions in helicopters through individual blade control. An aeroelastic model is developed to identify frequency response characteristics of the ATR blade with integral, generally anisotropic, strain actuators embedded in its composite construction. An ATR prototype blade was designed and manufactured to experimentally study the vibration reduction capabilities of such systems. Several bench and hover tests were conducted and those results are presented and discussed here. Selected results on sensitivity of the ATR system to collective setting (i.e. blade loading), blade rpm (i.e. centrifugal force and blade station velocity), and media density (i.e. altitude) are presented. They indicated that the twist actuation authority of the ATR blade is independent of the collective setting up to approximately 10P, and dependent on rotational speed and altitude near the torsional resonance frequency due to its dependency on the aerodynamic damping. The proposed model captures very well the physics and sensitivities to selected test parameters of the ATR system. The numerical result of the blade torsional loads show an average error of 20% in magnitude and virtually no difference in phase for the blade frequency response. Overall, the active blade model is in very good agreement with the experiments and can be used to analyze and design future active helicopter blade systems.

  8. Propionate induces the bovine cytosolic phosphoenolpyruvate carboxykinase promoter activity.

    PubMed

    Zhang, Qian; Koser, Stephanie L; Donkin, Shawn S

    2016-08-01

    Cytosolic phosphoenolpyruvate carboxykinase (PCK1) is a critical enzyme within the metabolic networks for gluconeogenesis, hepatic energy metabolism, and tricarboxylic acid cycle function, and is controlled by several transcription factors including hepatic nuclear factor 4α (HNF4α). The primary objective of the present study was to determine whether propionate regulates bovine PCK1 transcription. The second objective was to determine the action of cyclic AMP (cAMP), glucocorticoids, and insulin, hormonal cues known to modulate glucose metabolism, on bovine PCK1 transcriptional activity. The proximal promoter of the bovine PCK1 gene was ligated to a Firefly luciferase reporter and transfected into H4IIE hepatoma cells. Cells were exposed to treatments for 23 h and luciferase activity was determined in cell lysates. Activity of the PCK1 promoter was linearly induced by propionate, and maximally increased 7-fold with 2.5 mM propionate, which was not muted by 100 nM insulin. Activity of the PCK1 promoter was increased 1-fold by either 1.0 mM cAMP or 5.0µM dexamethasone, and 2.2-fold by their combination. Induction by cAMP and dexamethasone was repressed 50% by 100 nM insulin. Propionate, cAMP, and dexamethasone acted synergistically to induce the PCK1 promoter activity. Propionate-responsive regions, identified by 5' deletion analysis, were located between -1,238 and -409 bp and between -85 and +221 bp. Deletions of the core sequences of the 2 putative HNF4α sites decreased the responsiveness to propionate by approximately 40%. These data indicate that propionate regulates its own metabolism through transcriptional stimulation of the bovine PCK1 gene. This induction is mediated, in part, by the 2 putative HNF4α binding sites in the bovine PCK1 promoter. PMID:27289145

  9. Protein Kinase A and Mitogen-Activated Protein Kinase Pathways Antagonistically Regulate Fission Yeast fbp1 Transcription by Employing Different Modes of Action at Two Upstream Activation Sites

    PubMed Central

    Neely, Lori A.; Hoffman, Charles S.

    2000-01-01

    A significant challenge to our understanding of eukaryotic transcriptional regulation is to determine how multiple signal transduction pathways converge on a single promoter to regulate transcription in divergent fashions. To study this, we have investigated the transcriptional regulation of the Schizosaccharomyces pombe fbp1 gene that is repressed by a cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway and is activated by a stress-activated mitogen-activated protein kinase (MAPK) pathway. In this study, we identified and characterized two cis-acting elements in the fbp1 promoter required for activation of fbp1 transcription. Upstream activation site 1 (UAS1), located approximately 900 bp from the transcriptional start site, resembles a cAMP response element (CRE) that is the binding site for the atf1-pcr1 heterodimeric transcriptional activator. Binding of this activator to UAS1 is positively regulated by the MAPK pathway and negatively regulated by PKA. UAS2, located approximately 250 bp from the transcriptional start site, resembles a Saccharomyces cerevisiae stress response element. UAS2 is bound by transcriptional activators and repressors regulated by both the PKA and MAPK pathways, although atf1 itself is not present in these complexes. Transcriptional regulation of fbp1 promoter constructs containing only UAS1 or UAS2 confirms that the PKA and MAPK regulation is targeted to both sites. We conclude that the PKA and MAPK signal transduction pathways regulate fbp1 transcription at UAS1 and UAS2, but that the antagonistic interactions between these pathways involve different mechanisms at each site. PMID:10938120

  10. Specific activation of the thyrotropin receptor by trypsin.

    PubMed

    Van Sande, J; Massart, C; Costagliola, S; Allgeier, A; Cetani, F; Vassart, G; Dumont, J E

    1996-05-31

    The identification of 16 different activating mutations in the TSH receptor, found in patients suffering from toxic autonomous adenomas or congenital hyperthyroidism, leads to the concept that this receptor is in a constrained conformation in its wild-type form. We used mild trypsin treatment of CHO-K1 cells or COS-7 cells, stably or transiently transfected with the human TSH receptor, respectively, and measured its consequences on the TSH receptor coupled cascades, i.e. cyclic AMP and inositol-phosphates accumulation. A 2-min, 0.01% trypsin treatment increased stably cyclic AMP but not inositol-phosphates formation. This was not observed after chymotrypsin, thrombin and endoproteinase glu C treatment. The TSH action on cyclic AMP was decreased by only 25%. The effect was also observed in cells expressing the dog TSH receptor. It was not observed in MSH receptor, LH receptor expressing or mock transfected cells (vector alone). It is therefore specific for the TSH receptor, for its action on the Gs/adenylate cyclase cascade, and for the proteolytic cleavage caused by trypsin. Using monoclonal (A. Johnstone and P. Shepherd, personal communication) and polyclonal antibodies directed against the extracellular domain of the TSH receptor, it was shown that treatment by trypsin removes or destroys a VFFEEQ epitope (residues 354-359) from the receptor. The effect mimics the action of TSH as it activates Gs alpha and enhances the action of forskolin. It is not reversible in 1 h. The results support the concept that activation of the receptor (by hormone, autoantibodies, mutations or mild proteolysis) might involve the relief of a built-in negative constrain. They suggest that the C-terminal portion of the large extracellular domain plays a role in the maintenance of this constrain. PMID:8807635

  11. Trisomy 21 consistently activates the interferon response

    PubMed Central

    Sullivan, Kelly D; Lewis, Hannah C; Hill, Amanda A; Pandey, Ahwan; Jackson, Leisa P; Cabral, Joseph M; Smith, Keith P; Liggett, L Alexander; Gomez, Eliana B; Galbraith, Matthew D; DeGregori, James; Espinosa, Joaquín M

    2016-01-01

    Although it is clear that trisomy 21 causes Down syndrome, the molecular events acting downstream of the trisomy remain ill defined. Using complementary genomics analyses, we identified the interferon pathway as the major signaling cascade consistently activated by trisomy 21 in human cells. Transcriptome analysis revealed that trisomy 21 activates the interferon transcriptional response in fibroblast and lymphoblastoid cell lines, as well as circulating monocytes and T cells. Trisomy 21 cells show increased induction of interferon-stimulated genes and decreased expression of ribosomal proteins and translation factors. An shRNA screen determined that the interferon-activated kinases JAK1 and TYK2 suppress proliferation of trisomy 21 fibroblasts, and this defect is rescued by pharmacological JAK inhibition. Therefore, we propose that interferon activation, likely via increased gene dosage of the four interferon receptors encoded on chromosome 21, contributes to many of the clinical impacts of trisomy 21, and that interferon antagonists could have therapeutic benefits. DOI: http://dx.doi.org/10.7554/eLife.16220.001 PMID:27472900

  12. Dynamics of active cellular response under stress

    NASA Astrophysics Data System (ADS)

    de, Rumi; Zemel, Assaf; Safran, Samuel

    2008-03-01

    Forces exerted by and on adherent cells are important for many physiological processes such as wound healing and tissue formation. In addition, recent experiments have shown that stem cell differentiation is controlled, at least in part, by the elasticity of the surrounding matrix. Using a simple theoretical model that includes the forces due to both the mechanosensitive nature of cells and the elastic response of the matrix, we predict the dynamics of orientation of cells. The model predicts many features observed in measurements of cellular forces and orientation including the increase with time of the forces generated by cells in the absence of applied stress and the consequent decrease of the force in the presence of quasi-static stresses. We also explain the puzzling observation of parallel alignment of cells for static and quasi-static stresses and of nearly perpendicular alignment for dynamically varying stresses. In addition, we predict the response of the cellular orientation to a sinusoidally varying applied stress as a function of frequency. The dependence of the cell orientation angle on the Poisson ratio of the surrounding material can be used to distinguish systems in which cell activity is controlled by stress from those where cell activity is controlled by strain. Reference: Nature Physics, vol. 3, pp 655 (2007).

  13. Cyclin-dependent Kinase 1-dependent Phosphorylation of cAMP Response Element-binding Protein Decreases Chromatin Occupancy*

    PubMed Central

    Trinh, Anthony T.; Kim, Sang Hwa; Chang, Hae-yoon; Mastrocola, Adam S.; Tibbetts, Randal S.

    2013-01-01

    The cyclic AMP response element-binding protein (CREB) initiates transcriptional responses to a wide variety of stimuli. CREB activation involves its phosphorylation on Ser-133, which promotes interaction between the CREB kinase-inducible domain (KID) and the KID-interacting domain of the transcriptional coactivator, CREB-binding protein (CBP). The KID also contains a highly conserved phosphorylation cluster, termed the ATM/CK cluster, which is processively phosphorylated in response to DNA damage by the coordinated actions of ataxia-telangiectasia-mutated (ATM) and casein kinases (CKs) 1 and 2. The ATM/CK cluster phosphorylation attenuates CBP binding and CREB transcriptional activity. Paradoxically, it was recently reported that DNA damage activates CREB through homeodomain-interacting protein kinase 2-dependent phosphorylation of Ser-271 near the CREB bZIP DNA binding domain. In this study we sought to further clarify DNA damage-dependent CREB phosphorylation as well as to explore the possibility that the ATM/CK cluster and Ser-271 synergistically or antagonistically modulate CREB activity. We show that, rather than being induced by DNA damage, Ser-270 and Ser-271 of CREB cophosphorylated in a CDK1-dependent manner during G2/M phase. Functionally, we show that phosphorylation of CREB on Ser-270/Ser-271 during mitosis correlated with reduced CREB chromatin occupancy. Furthermore, CDK1-dependent phosphorylation of CREB in vitro inhibited its DNA binding activity. The combined results suggest that CDK1-dependent phosphorylation of CREB on Ser-270/Ser-271 facilitates its dissociation from chromatin during mitosis by reducing its intrinsic DNA binding potential. PMID:23814058

  14. Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity

    PubMed Central

    Londino, James D.; Lazrak, Ahmed; Jurkuvenaite, Asta; Collawn, James F.; Noah, James W.

    2013-01-01

    The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl−) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H+) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o−) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H+, did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection. PMID:23457187

  15. Cyclic adenosine 3', 5'-monophosphate in cerebrospinal fluid during thermoregulation and fever.

    PubMed Central

    Dascombe, M J; Milton, A S

    1976-01-01

    1. Samples of cerebrospinal fluid (c.s.f.) have been taken from the cisterna magna of unanaesthetized cats, whilst rectal temperature was recorded, during exposure of the animals to various ambient temperatures and during fever induced by pyrogen. The concentration of adenosine 3', 5'-monophosphate (cyclic AMP) in samples of c.s.f. has been assayed. 2. Cats exposed to low ambient temperatures (-2 to +2 degrees C) for 3 h maintained body temperature by both behavioural and autonomic heat gain activity. Exposure of cats to high ambient temperatures (44 - 45 degrees C) for 3.5 h caused a rise in body temperatures of about 2.5 degrees C, despite behavioural and autonomic heat loss activity. Neither cold nor heat stress had a significant effect on c.s.f. cyclic AMP. 3. Fever induced by intravenous Shigella dysenteriae (2 and 20 mug/kg) was associated with a dose-related increase in the concentration of cyclic AMP in c.s.f. Paracetamol (75 mg/kg) injected I.P. before the onset of fever, suppressed the increase in both temperature and c.s.f. cyclic AMP in response to pyrogen. Paracetamol (50 and 100 mg/kg), injected after the onset of fever, caused a fall in temperature, which was not associated with a decrease in the concentration of cyclic AMP in c.s.f. 4. Fever induced in cats by intravenous Shigella dysenteriae (20 mug/kg) was associated with an increase in the concentration of cyclic AMP in plasma as well as in c.s.f. 5. The sodium salt of cyclic AMP (0.1-10 mg/kg) injected I.V. into unanaesthetized cats caused a dose-related hypothermia, which was associated with autonomic heat loss activity and a dose-related increase in the concentration of cyclic AMP in cisternal c.s.f., which was not mimicked by adenosine. 6. It is concluded that the raised concentrations of cyclic AMP in c.s.f., in response to pyrogen I.V., do not mediate fever in the cat and that the concentration of cyclic AMP in cisternal c.s.f. may be affected by changes in the plasma concentration of the

  16. Chenodeoxycholic acid through a TGR5-dependent CREB signaling activation enhances cyclin D1 expression and promotes human endometrial cancer cell proliferation.

    PubMed

    Casaburi, Ivan; Avena, Paola; Lanzino, Marilena; Sisci, Diego; Giordano, Francesca; Maris, Pamela; Catalano, Stefania; Morelli, Catia; Andò, Sebastiano

    2012-07-15

    Endometrial cancer exhibits a strong incidence in western developed countries mainly due to fat-rich diet and obesity. Processing of dietary lipids is triggered by bile acids, amphipathic detergents that are synthesized in the liver and stored in the gallbladder. In addition to their well-recognized role in dietary lipid absorption and cholesterol homeostasis, bile acids can also act as signaling molecules with systemic endocrine functions. In the present study we investigated the biological effects of the primary bile chenodeoxycholic acid (CDCA) on a human endometrial cancer cell line, Ishikawa. Low concentrations of CDCA are able to stimulate Ishikawa cell growth by inducing a significant increase in Cyclin D1 protein and mRNA expression through the activation of the membrane G protein-coupled receptor (TGR5)-dependent pathway. Dissecting the molecular mechanism underlying this effect by mutagenesis, EMSA and ChIP analysis revealed that CDCA-induced Cyclin D1 expression requires the enhanced recruitment of the transcription factor CREB on the cyclic AMP-responsive element motif within the Cyclin D1 gene proximal promoter. Our results suggest a novel molecular mechanism explaining the potential contribution of high-fat diet and obesity to endometrial cancer growth and progression opening the rationale for strategies to prevent the risk of this obesity-related cancer in women. PMID:22751440

  17. Protein kinase activators alter glial cholesterol esterification

    SciTech Connect

    Jeng, I.; Dills, C.; Klemm, N.; Wu, C.

    1986-05-01

    Similar to nonneural tissues, the activity of glial acyl-CoA cholesterol acyltransferase is controlled by a phosphorylation and dephosphorylation mechanism. Manipulation of cyclic AMP content did not alter the cellular cholesterol esterification, suggesting that cyclic AMP is not a bioregulator in this case. Therefore, the authors tested the effect of phorbol-12-myristate 13-acetate (PMA) on cellular cholesterol esterification to determine the involvement of protein kinase C. PMA has a potent effect on cellular cholesterol esterification. PMA depresses cholesterol esterification initially, but cells recover from inhibition and the result was higher cholesterol esterification, suggesting dual effects of protein kinase C. Studies of other phorbol analogues and other protein kinase C activators such as merezein indicate the involvement of protein kinase C. Oleoyl-acetyl glycerol duplicates the effect of PMA. This observation is consistent with a diacyl-glycerol-protein kinase-dependent reaction. Calcium ionophore A23187 was ineffective in promoting the effect of PMA. They concluded that a calcium-independent and protein C-dependent pathway regulated glial cholesterol esterification.

  18. Correlation of protein kinase activation and testosterone production after stimulation of Leydig cells with luteinizing hormone.

    PubMed Central

    Cooke, B A; Lindh, M L; Janszen, F H

    1976-01-01

    The effect of different doses of luteinizing hormone on activation of protein kinases, cyclic AMP and testosterone production was studied in purified rat testis Leydig-cell preparations in the presence of 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor). In addition, the nature of the protein kinases present in these cells and other tissues was investigated. The following results were obtained. 1. With all the amounts of luteinizing hormone used (0.1-1000 ng/ml), both activation of protein kinase and stimulation of testosterone production were demonstrated. With the lowest amount of luteinizing hormone (0.1 ng/ml), an 8.4+/-0.9% (S.E.M.,n=6) stimulation of protein kinase activation occurred, increasing to 100% with 1000 ng/ml, compared with 3.2+/-1.0%(S.E.M.,n=7) and 100% stimulation of testosterone production with 0.1 and 100 ng/ml respectively. 2. With amounts of luteinizing hormone up to 1 ng/ml (which gave half-maximal stimulation of testosterone production) no detectable increases in net cyclic AMP production were obtained. With higher amounts of luteinizing hormone, cyclic AMP production increased, but maximal production was not reached with 1000 ng/ml. 3. Two isoenzymic forms of protein kinase were present in Leydig cells and seminiferous tubules; type I was eluted with 0.075 M-and type II with 0.22-0.25 m-NaCl from DEAE-cellulose columns. 4. The protein kinase activity was not affected by the presence of erythrocytes in the Leydig-cell preparation, but varied depending on the type of histone used as substrate (histone F2b greater than mixed greater than histone F1). PMID:189752

  19. Actions of adenosine A1 and A2 receptor antagonists on CFTR antibody-inhibited β-adrenergic mucin secretion response

    PubMed Central

    Pereira, M M C; Lloyd Mills, C; Dormer, R L; McPherson, M A

    1998-01-01

    The cystic fibrosis gene protein, the cystic fibrosis transmembrane conductance regulator (CFTR) acts as a chloride channel and is a key regulator of mucin secretion. The mechanism by which 3-isobutyl-1-methylxanthine (IBMX) corrects the defect in CFTR mediated β-adrenergic stimulation of mucin secretion has not been determined. The present study has investigated the actions of adenosine A1 and A2 receptor antagonists to determine whether ability to stimulate mucin secretion correlates with correction of CFTR antibody inhibited β-adrenergic response and whether excessive cyclic AMP rise is required.CFTR antibodies were introduced into living rat submandibular acini by hypotonic swelling. Following recovery, mucin secretion in response to isoproterenol was measured.The adenosine A1 receptor antagonist, 8 cyclopentyltheophylline (CPT) was a less potent stimulator of mucin secretion than was the A2 receptor antagonist dimethylpropargylxanthine (DMPX). A concentration of CPT close to the Ki for A1 receptor antagonism (10 nM) did not stimulate mucin secretion.DMPX, although a potent stimulator of mucin secretion, did not correct CFTR antibody inhibited mucin secretion.CPT corrected defective CFTR antibody inhibited mucin secretion at a high (1 mM) concentration, suggesting a mechanism other than adenosine receptor antagonism.DMPX potentiated the isoproterenol induced cyclic AMP rise, whereas CPT did not.Correction of the defective CFTR mucin secretion response did not correlate with ability to stimulate mucin secretion and did not require potentiation of β-adrenergic induced increases in cyclic AMP. This affords real promise for the development of a selective drug treatment for cystic fibrosis. PMID:9831904

  20. CREB, ATF, and AP-1 transcription factors regulate IFN-gamma secretion by human T cells in response to mycobacterial antigen.

    PubMed

    Samten, Buka; Townsend, James C; Weis, Steven E; Bhoumik, Anindita; Klucar, Peter; Shams, Homayoun; Barnes, Peter F

    2008-08-01

    IFN-gamma production by T cells is pivotal for defense against many pathogens, and the proximal promoter of IFN-gamma, -73 to -48 bp upstream of the transcription start site, is essential for its expression. However, transcriptional regulation mechanisms through this promoter in primary human cells remain unclear. We studied the effects of cAMP response element binding protein/activating transcription factor (CREB/ATF) and AP-1 transcription factors on the proximal promoter of IFN-gamma in human T cells stimulated with Mycobacterium tuberculosis. Using EMSA, supershift assays, and promoter pulldown assays, we demonstrated that CREB, ATF-2, and c-Jun, but not cyclic AMP response element modulator, ATF-1, or c-Fos, bind to the proximal promoter of IFN-gamma upon stimulation, and coimmunoprecipitation indicated the possibility of interaction among these transcription factors. Chromatin immunoprecipitation confirmed the recruitment of these transcription factors to the IFN-gamma proximal promoter in live Ag-activated T cells. Inhibition of ATF-2 activity in T cells with a dominant-negative ATF-2 peptide or with small interfering RNA markedly reduced the expression of IFN-gamma and decreased the expression of CREB and c-Jun. These findings suggest that CREB, ATF-2, and c-Jun are recruited to the IFN-gamma proximal promoter and that they up-regulate IFN-gamma transcription in response to microbial Ag. Additionally, ATF-2 controls expression of CREB and c-Jun during T cell activation. PMID:18641343

  1. Decreased hypothalamic growth hormone-releasing hormone content and pituitary responsiveness in hypothyroidism.

    PubMed Central

    Katakami, H; Downs, T R; Frohman, L A

    1986-01-01

    The effects of thyroidectomy (Tx) and thyroxine replacement (T4Rx) on pituitary growth hormone (GH) secretion and hypothalamic GH-releasing hormone (GRH) concentration were compared to define the mechanism of hypothyroid-associated GH deficiency. Thyroidectomized rats exhibited a complete loss of pulsatile GH secretion with extensive reduction in GRH responsiveness and pituitary GH content. Cultured pituitary cells from Tx rats exhibited reduced GRH sensitivity, maximal GH responsiveness, and intracellular cyclic AMP accumulation to GRH, while somatostatin (SRIF) suppressive effects on GH secretion were increased. Hypothalamic GRH content was also markedly reduced. T4Rx completely restored hypothalamic GRH content and spontaneous GH secretion despite only partial recovery of pituitary GH content, GRH and SRIF sensitivity, and intracellular cyclic AMP response to GRH. The results indicate multiple effects of hypothyroidism on GH secretion and suggest that a critical role of T4 in maintaining normal GH secretion, in addition to restoring GH synthesis, is related to its effect on hypothalamic GRH. Images PMID:2871046

  2. T3-induced liver AMP-activated protein kinase signaling: Redox dependency and upregulation of downstream targets

    PubMed Central

    Videla, Luis A; Fernández, Virginia; Cornejo, Pamela; Vargas, Romina; Morales, Paula; Ceballo, Juan; Fischer, Alvaro; Escudero, Nicolás; Escobar, Oscar

    2014-01-01

    AIM: To investigate the redox dependency and promotion of downstream targets in thyroid hormone (T3)-induced AMP-activated protein kinase (AMPK) signaling as cellular energy sensor to limit metabolic stresses in the liver. METHODS: Fed male Sprague-Dawley rats were given a single ip dose of 0.1 mg T3/kg or T3 vehicle (NaOH 0.1 N; controls) and studied at 8 or 24 h after treatment. Separate groups of animals received 500 mg N-acetylcysteine (NAC)/kg or saline ip 30 min prior T3. Measurements included plasma and liver 8-isoprostane and serum β-hydroxybutyrate levels (ELISA), hepatic levels of mRNAs (qPCR), proteins (Western blot), and phosphorylated AMPK (ELISA). RESULTS: T3 upregulates AMPK signaling, including the upstream kinases Ca2+-calmodulin-dependent protein kinase kinase-β and transforming growth factor-β-activated kinase-1, with T3-induced reactive oxygen species having a causal role due to its suppression by pretreatment with the antioxidant NAC. Accordingly, AMPK targets acetyl-CoA carboxylase and cyclic AMP response element binding protein are phosphorylated, with the concomitant carnitine palmitoyltransferase-1α (CPT-1α) activation and higher expression of peroxisome proliferator-activated receptor-γ co-activator-1α and that of the fatty acid oxidation (FAO)-related enzymes CPT-1α, acyl-CoA oxidase 1, and acyl-CoA thioesterase 2. Under these conditions, T3 induced a significant increase in the serum levels of β-hydroxybutyrate, a surrogate marker for hepatic FAO. CONCLUSION: T3 administration activates liver AMPK signaling in a redox-dependent manner, leading to FAO enhancement as evidenced by the consequent ketogenic response, which may constitute a key molecular mechanism regulating energy dynamics to support T3 preconditioning against ischemia-reperfusion injury. PMID:25516653

  3. Dopamine D2 receptor activates extracellular signal-regulated kinase through the specific region in the third cytoplasmic loop.

    PubMed

    Takeuchi, Yusuke; Fukunaga, Kohji

    2004-06-01

    To investigate whether the third cytoplasmic loop and the C-terminal cytoplasmic tail of dopamine D(2) receptor (D2R) are involved in extracellular signal-regulated kinase (ERK) activation and subsequent regulation of transcription factors, we established NG108-15 cells stably expressing D2LR and D2SR deleted 40 amino acid residues in the third cytoplasmic loop (NGD2LR-3rd-dele and NGD2SR-3rd-dele) or the C-terminal cytoplasmic tail (NGD2LR-C-dele and NGD2SR-C-dele) and evaluated these receptors' functions using luciferase reporter gene assay. Immunocytochemical studies showed similar intracellular distributions of D2LR-3rd-dele and D2SR-3rd-dele to D2LR and D2SR, respectively. Quinpirole-induced inhibition of forskolin-induced cyclic AMP responsive element (CRE) activation was not affected by the deletion of 40 amino acid residues. However, nuclear factor-kappa B (NF-kappaB) activation by D2R-3rd-dele was largely attenuated compared to that by D2R. Similarly, ERK or serum-responsive element (SRE) activation by quinpirole treatment was totally abolished in NGD2R-3rd-dele cells. Moreover, D2R-C-dele was diffusely distributed or clustered in the cell bodies and lost the receptor functions. Taken together, the 40 amino acid residues in the third cytoplasmic loop are essential for the ERK activation but not for inhibition of adenylyl cyclase through Gi/o proteins. In addition, the C-terminal cytoplasmic tail is essential for membrane association of D2Rs to elicit the receptor functions. PMID:15189353

  4. Inhibition of P2Y6 receptor-mediated phospholipase C activation and Ca(2+) signalling by prostaglandin E2 in J774 murine macrophages.

    PubMed

    Ito, Masaaki; Matsuoka, Isao

    2015-02-15

    Extracellular nucleotides act as inflammatory mediators through activation of multiple purinoceptors. Under inflammatory conditions, the purinergic signalling is affected by various inflammatory mediators. We previously showed that prostaglandin (PG) E2 suppressed the elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) stimulated by P2X4, P2Y2, and P2Y6 receptors in J774 murine macrophages. In this study, we examined the mechanism of PGE2 inhibitory effects on P2Y6 receptor-mediated function in J774 cells. The P2Y6 receptor agonist UDP induced a sustained elevation of [Ca(2+)]i by stimulating the phospholipase C (PLC) signalling pathway. PGE2 inhibited [Ca(2+)]i elevation and phosphatidylinositol (PI) hydrolysis in a concentration-dependent manner. J774 cells highly expressed the E-type prostanoid 2 (EP2) receptor subtype, a Gs-coupled receptor. PGE2 and a selective EP2 receptor agonist caused cyclic AMP (cAMP) accumulation in J774 cells. The inhibitory effects of PGE2 on P2Y6 receptor-mediated responses were mimicked by the selective EP2 receptor agonist. Although EP2 receptor is linked to adenylyl cyclase activation, PGE2-induced inhibition of Ca(2+) response and PI hydrolysis could not be mimicked by a lipophilic cAMP derivative, dibutyryl cAMP, or an adenylyl cyclase activator, forskolin. The inhibition of UDP-induced PLC activation by PGE2 was not affected by down-regulation of protein kinase C by phorbol-12-myristate-13-acetate treatment. PGE2 inhibited PLC activation induced by aluminium fluoride, but not by the Ca(2+)-ionophore, ionomycin. Finally, the inhibition of UDP-induced PLC activation by PGE2 was impaired by Gs knockdown using siRNA. These results suggest that EP2 receptor activation in macrophages negatively controls the Gq/11-PLC signalling through a Gs-mediated, but cAMP-independent signalling mechanism. PMID:25614334

  5. Evidence that M1 muscarinic receptors enhance noradrenaline release in mouse atria by activating protein kinase C.

    PubMed Central

    Costa, M.; Barrington, M.; Majewski, H.

    1993-01-01

    1. The M1 selective muscarinic agonist, McNeil A 343, enhanced the electrically evoked release of noradrenaline from postganglionic sympathetic nerves in mouse atria. This has been found previously to be due to activation of muscarinic receptors of the M1 subtype, probably located on sympathetic nerve terminals. The present study investigated the signal transduction mechanisms involved in the release-enhancing effects of McNeil A 343. The release of noradrenaline from mouse atria was assessed by measuring the electrically-induced (3 Hz, 60 s) outflow of radioactivity from atria which had been pre-incubated with [3H]-noradrenaline. 2. 8-Bromo cyclic AMP in the presence of IBMX was used to enhance maximally S-I noradrenaline release through cyclic AMP-dependent mechanisms. However, the facilitatory effect of McNeil A 343 (10 microM) was not different from the effect in the absence of these drugs, suggesting that McNeil A 343 enhances noradrenaline release independently of the cyclic AMP system. Furthermore, the release-enhancing effect of McNeil A 343 (10 microM) on noradrenaline release was also not altered by the 5-lipoxygenase inhibitor, BW A4C. 3. The facilitatory effect of McNeil A 343 was not altered in the presence of drugs (trifluoperazine, W7, and calmidazolium) which inhibit calmodulin-dependent processes, suggesting that the mechanisms of action of McNeil A 343 does not depend on calmodulin. 4. It was considered likely that the facilitatory effect of McNeil A 343 on noradrenaline release may be due to activation of protein kinase C, since activators of protein kinase C enhance noradrenaline release.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7694761

  6. A-kinase-anchoring proteins coordinate inflammatory responses to cigarette smoke in airway smooth muscle

    PubMed Central

    Heijink, Irene H.; Holtzer, Laura J.; Skroblin, Philipp; Klussmann, Enno; Halayko, Andrew J.; Timens, Wim; Maarsingh, Harm; Schmidt, Martina

    2015-01-01

    β2-Agonist inhibitors can relieve chronic obstructive pulmonary disease (COPD) symptoms by stimulating cyclic AMP (cAMP) signaling. A-kinase-anchoring proteins (AKAPs) compartmentalize cAMP signaling by establishing protein complexes. We previously reported that the β2-agonist fenoterol, direct activation of protein kinase A (PKA), and exchange factor directly activated by cAMP decrease cigarette smoke extract (CSE)-induced release of neutrophil attractant interleukin-8 (IL-8) from human airway smooth muscle (ASM) cells. In the present study, we tested the role of AKAPs in CSE-induced IL-8 release from ASM cells and assessed the effect of CSE on the expression levels of different AKAPs. We also studied mRNA and protein expression of AKAPs in lung tissue from patients with COPD. Our data show that CSE exposure of ASM cells decreases AKAP5 and AKAP12, both capable of interacting with β2-adrenoceptors. In lung tissue of patients with COPD, mRNA levels of AKAP5 and AKAP12 were decreased compared with lung tissue from controls. Using immunohistochemistry, we detected less AKAP5 protein in ASM of patients with COPD Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage II compared with control subjects. St-Ht31, which disrupts AKAP-PKA interactions, augmented CSE-induced IL-8 release from ASM cells and diminished its suppression by fenoterol, an effect mediated by disturbed ERK signaling. The modulatory role of AKAP-PKA interactions in the anti-inflammatory effects of fenoterol in ASM cells and the decrease in expression of AKAP5 and AKAP12 in response to cigarette smoke and in lungs of patients with COPD suggest that cigarette smoke-induced changes in AKAP5 and AKAP12 in patients with COPD may affect efficacy of pharmacotherapy. PMID:25637608

  7. A-kinase-anchoring proteins coordinate inflammatory responses to cigarette smoke in airway smooth muscle.

    PubMed

    Poppinga, Wilfred J; Heijink, Irene H; Holtzer, Laura J; Skroblin, Philipp; Klussmann, Enno; Halayko, Andrew J; Timens, Wim; Maarsingh, Harm; Schmidt, Martina

    2015-04-15

    β2-Agonist inhibitors can relieve chronic obstructive pulmonary disease (COPD) symptoms by stimulating cyclic AMP (cAMP) signaling. A-kinase-anchoring proteins (AKAPs) compartmentalize cAMP signaling by establishing protein complexes. We previously reported that the β2-agonist fenoterol, direct activation of protein kinase A (PKA), and exchange factor directly activated by cAMP decrease cigarette smoke extract (CSE)-induced release of neutrophil attractant interleukin-8 (IL-8) from human airway smooth muscle (ASM) cells. In the present study, we tested the role of AKAPs in CSE-induced IL-8 release from ASM cells and assessed the effect of CSE on the expression levels of different AKAPs. We also studied mRNA and protein expression of AKAPs in lung tissue from patients with COPD. Our data show that CSE exposure of ASM cells decreases AKAP5 and AKAP12, both capable of interacting with β2-adrenoceptors. In lung tissue of patients with COPD, mRNA levels of AKAP5 and AKAP12 were decreased compared with lung tissue from controls. Using immunohistochemistry, we detected less AKAP5 protein in ASM of patients with COPD Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage II compared with control subjects. St-Ht31, which disrupts AKAP-PKA interactions, augmented CSE-induced IL-8 release from ASM cells and diminished its suppression by fenoterol, an effect mediated by disturbed ERK signaling. The modulatory role of AKAP-PKA interactions in the anti-inflammatory effects of fenoterol in ASM cells and the decrease in expression of AKAP5 and AKAP12 in response to cigarette smoke and in lungs of patients with COPD suggest that cigarette smoke-induced changes in AKAP5 and AKAP12 in patients with COPD may affect efficacy of pharmacotherapy. PMID:25637608

  8. 76 FR 19766 - Agency Information Collection Activities OMB Responses

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-08

    ... From the Federal Register Online via the Government Publishing Office ENVIRONMENTAL PROTECTION AGENCY Agency Information Collection Activities OMB Responses AGENCY: Environmental Protection Agency... 1808.06; Environmental Impact Assessment of Nongovernmental Activities in Antarctica (Renewal); 40...

  9. Micro-electrode studies on the effects of exogenous cyclic adenosine monophosphate on active sodium transport in frog skin.

    PubMed Central

    Els, W J; Mahlangu, A F

    1987-01-01

    1. The electrical parameters of the sodium-transporting cells in frog skin of Rana angolensis were determined under control conditions by using the micro-electrode technique. The data were analysed in terms of an electrical model (Helman, 1979). 2. The control intracellular voltages averaged -84.7 mV while the electromotive force of the inner barrier, E'1, averaged 103.9 mV. The major portion (82%) of the transcellular resistance was situated at the outer, apical, barrier. 3. Exogenous cyclic AMP stimulated active sodium transport and the short-circuit current (Isc) increased by an average 88%. The change in Isc was mediated primarily by decreasing the resistance of the apical barrier (Ro) with little effect on the electromotive force or resistance (Ri) of the inner membranes. 4. Isoprenaline increased the Isc by an average of 165%. The major effect of isoprenaline was to decrease the apical resistance by an average 77%. 5. Forskolin (2.5 microM) stimulated the Isc by an average of 138%. Amiloride would not completely reduce the Isc, but with the low concentration of 0.2 microM-forskolin, the Isc was typically inhibited to values close to zero. The major effect of forskolin was also to reduce the resistance of the apical barrier, although it concurrently also caused the E'1 to decrease by about 13%. 6. Theophylline increased the Isc by reducing the resistance of the apical barrier by an average 61%, with little or no effect on the other parameters. Theophylline augmented the effect of cyclic AMP. 8. Our results are consistent with the theory that cyclic AMP is a second messenger in hormonal control of active sodium transport in frog skin. PMID:2821244

  10. IP receptor-dependent activation of PPAR{gamma} by stable prostacyclin analogues

    SciTech Connect

    Falcetti, Emilia; Flavell, David M.; Staels, Bart; Tinker, Andrew; Haworth, Sheila G.; Clapp, Lucie H. . E-mail: l.clapp@ucl.ac.uk

    2007-09-07

    Stable prostacyclin analogues can signal through cell surface IP receptors or by ligand binding to nuclear peroxisome proliferator-activated receptors (PPARs). So far these agents have been reported to activate PPAR{alpha} and PPAR{delta} but not PPAR{gamma}. Given PPAR{gamma} agonists and prostacyclin analogues both inhibit cell proliferation, we postulated that the IP receptor might elicit PPAR{gamma} activation. Using a dual luciferase reporter gene assay in HEK-293 cells stably expressing the IP receptor or empty vector, we found that prostacyclin analogues only activated PPAR{gamma} in the presence of the IP receptor. Moreover, the novel IP receptor antagonist, RO1138452, but not inhibitors of the cyclic AMP pathway, prevented activation. Likewise, the anti-proliferative effects of treprostinil observed in IP receptor expressing cells, were partially inhibited by the PPAR{gamma} antagonist, GW9662. We conclude that PPAR{gamma} is activated through the IP receptor via a cyclic AMP-independent mechanism and contributes to the anti-growth effects of prostacyclin analogues.

  11. Alpha-adrenoceptor mediated responses of the cauda epididymis of the guinea-pig.

    PubMed Central

    Haynes, J. M.; Hill, S. J.

    1996-01-01

    ). In the presence of both idazoxan (100 nM) and nifedipine (10 microM), and following the pre-incubation with chloroethylclonidine (50 microM, 30 min), 5-methylurapidil (30 to 300 nM) still shifted phenylephrine concentration-response curves to the right (pKB values 7.77 to 7.36, respectively). 4. Phenylephrine (1 microM to 1 mM) increased the accumulation of [3H]-inositol phosphates (10 fold) in preparations of cauda epididymis (EC50 12 microM). This effect was sensitive to chloroethylclonidine pretreatment (50 microM, 30 min), antagonized with low affinity by 5-methylurapidil (- log pKi 7.8), but not potentiated by xylazine (1 microM). Xylazine (10 nM - 100 microM) reversed the forskolin (10 or 30 microM) stimulated accumulation of [3H]-adenosine 3':5'-cylic monophosphate (cyclic AMP) in preparations of cauda epididymis (by approximately 45%). Incubation of tissues with both pertussis toxin (200 ng ml-1, 24 h) and pertussis toxin vehicle increased the basal activity of adenylate cyclase (3 fold) but did not increase the capacity of forskolin (30 microM) to stimulate the accumulation of [3H]-cyclic AMP in these tissues. Xylazine did not significantly inhibit the forskolin-stimulated accumulation of [3H]-cyclic AMP in either vehicle or pertussis toxin treated tissues. 5. These studies indicate that the epididymis of the guinea-pig contains alpha 1- and alpha 2-adrenoceptors. On the basis of the actions of chloroethylclonidine and 5-methylurapidil the alpha 1-adrenoceptors of this tissue may be of the alpha 1A- and alpha 1B-subtypes and are linked to both the influx of extracellular Ca2+ and to phospholipase C. The alpha 2-adrenoceptors of this tissue are negatively coupled to adenylate cyclase, sensitive to pertussis toxin, but do not amplify phenylephrine-stimulated [3H]-inositol phosphate accumulation. Stimulation of the alpha 2-adrenoceptors of this tissue may selectively potentiate the influx of extracellular Ca2+. PMID:8937724

  12. Role of trypsin-like cleavage at arginine 192 in the enzymatic and cytotonic activities of Escherichia coli heat-labile enterotoxin.

    PubMed Central

    Grant, C C; Messer, R J; Cieplak, W

    1994-01-01

    Previous studies of cholera toxin and Escherichia coli heat-labile enterotoxin have suggested that proteolytic cleavage plays an important role in the expression of ADP-ribosyltransferase activity and toxicity. Specifically, several studies have implicated a trypsin-like cleavage at arginine 192, which lies within an exposed region subtended by a disulfide bond in the intact A subunit, in toxicity. To investigate the role of this modification in the enzymatic and cytotonic properties of heat-labile enterotoxin, the response of purified, recombinant A subunit to tryptic activation and the effect of substituting arginine 192 with glycine on the activities of the holotoxin were examined. The recombinant A subunit of heat-labile enterotoxin exhibited significant levels of ADP-ribosyltransferase activity that were only nominally increased (approximately twofold) by prior limited trypsinolysis. The enzymatic activity also did not appear to be affected by auto-ADP-ribosylation that occurs during the high-level synthesis of the recombinant A subunit in E. coli. A mutant form of the holotoxin containing the arginine 192-to-glycine substitution exhibited levels of cytotonic activity for CHO cells that were similar to that of the untreated, wild-type holotoxin but exhibited a marked delay in the ability to increase intracellular levels of cyclic AMP in Caco-2 cells. The results indicate that trypsin-like cleavage of the A subunit of E. coli heat-labile enterotoxin at arginine 192 is not requisite to the expression of enzymatic activity by the A subunit and further reveal that this modification, although it enhances the biological and enzymatic activities of the toxin, is not absolutely required for the enterotoxin to elicit cytotonic effects. Images PMID:7927684

  13. Transplantation of Human Pericyte Progenitor Cells Improves the Repair of Infarcted Heart Through Activation of an Angiogenic Program Involving Micro-RNA-132

    PubMed Central

    Katare, Rajesh; Riu, Federica; Mitchell, Kathryn; Gubernator, Miriam; Campagnolo, Paola; Cui, Yuxin; Fortunato, Orazio; Avolio, Elisa; Cesselli, Daniela; Beltrami, Antonio Paolo; Angelini, Gianni; Emanueli, Costanza; Madeddu, Paolo

    2013-01-01

    Rationale Pericytes are key regulators of vascular maturation, but their value for cardiac repair remains unknown. Objective We investigated the therapeutic activity and mechanistic targets of saphenous vein-derived pericyte progenitor cells (SVPs) in a mouse myocardial infarction (MI) model. Methods and Results SVPs have a low immunogenic profile and are resistant to hypoxia/starvation (H/S). Transplantation of SVPs into the peri-infarct zone of immunodeficient CD1/Foxn-1nu/nu or immunocompetent CD1 mice attenuated left ventricular dilatation and improved ejection fraction compared to vehicle. Moreover, SVPs reduced myocardial scar, cardiomyocyte apoptosis and interstitial fibrosis, improved myocardial blood flow and neovascularization, and attenuated vascular permeability. SVPs secrete vascular endothelial growth factor A, angiopoietin-1, and chemokines and induce an endogenous angiocrine response by the host, through recruitment of vascular endothelial growth factor B expressing monocytes. The association of donor- and recipient-derived stimuli activates the proangiogenic and prosurvival Akt/eNOS/Bcl-2 signaling pathway. Moreover, microRNA-132 (miR-132) was constitutively expressed and secreted by SVPs and remarkably upregulated, together with its transcriptional activator cyclic AMP response element-binding protein, on stimulation by H/S or vascular endothelial growth factor B. We next investigated if SVP-secreted miR-132 acts as a paracrine activator of cardiac healing. In vitro studies showed that SVP conditioned medium stimulates endothelial tube formation and reduces myofibroblast differentiation, through inhibition of Ras-GTPase activating protein and methyl-CpG-binding protein 2, which are validated miR-132 targets. Furthermore, miR-132 inhibition by antimiR-132 decreased SVP capacity to improve contractility, reparative angiogenesis, and interstitial fibrosis in infarcted hearts. Conclusion SVP transplantation produces long-term improvement of cardiac

  14. Solar activity, the QBO, and tropospheric responses

    NASA Technical Reports Server (NTRS)

    Tinsley, Brian A.; Brown, Geoffrey M.; Scherrer, Philip H.

    1989-01-01

    The suggestion that galactic cosmic rays (GCR) as modulated by the solar wind are the carriers of the component of solar variability that affects weather and climate has been discussed in the literature for 30 years, and there is now a considerable body of evidence that supports it. Variations of GCR occur with the 11 year solar cycle, matching the time scale of recent results for atmospheric variations, as modulated by the quasibiennial oscillation of equatorial stratospheric winds (the QBO). Variations in GCR occur on the time scale of centuries with a well defined peak in the coldest decade of the little ice age. New evidence is presented on the meteorological responses to GCR variations on the time scale of a few days. These responses include changes in the vertical temperature profile in the troposphere and lower stratosphere in the two days following solar flare related high speed plasma streams and associated GCR decreases, and in decreases in Vorticity Area Index (VAI) following Forbush decreases of GCR. The occurrence of correlations of GCR and meteorological responses on all three time scales strengthens the hypothesis of GCR as carriers of solar variability to the lower atmosphere. Both short and long term tropospheric responses are understandable as changes in the intensity of cyclonic storms initiated by mechanisms involving cloud microphysical and cloud electrification processes, due to changes in local ion production from changes in GCR fluxes and other high energy particles in the MeV to low GeV range. The nature of these mechanisms remains undetermined. Possible stratospheric wind (particularly QBO) effects on the transport of HNO3 and other constituents incorporated in cluster ions and possible condensation and freezing nuclei are considered as relevant to the long term variations.

  15. Macroscopic response in active nonlinear photonic crystals.

    PubMed

    Alagappan, Gandhi; John, Sajeev; Li, Er Ping

    2013-09-15

    We derive macroscopic equations of motion for the slowly varying electric field amplitude in three-dimensional active nonlinear optical nanostructures. We show that the microscopic Maxwell equations and polarization dynamics can be simplified to a macroscopic one-dimensional problem in the direction of group velocity. For a three-level active material, we derive the steady-state equations for normal mode frequency, threshold pumping, nonlinear Bloch mode amplitude, and lasing in photonic crystals. Our analytical results accurately recapture the results of exact numerical methods. PMID:24104802

  16. Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation.

    PubMed

    Zhou, Wei-Jie; Wang, Sheng; Hu, Zhuang; Zhou, Zhen-Yu; Song, Cai-Juan

    2015-11-20

    Angelica sinensis polysaccharide (ASP) is purified from the fresh roots of Angelica sinensis (AS). This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases and used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. The antitumor activity of ASP is related to its biological activities, because it suppresses a variety of pro-proliferative or anti-apoptotic factors that are dramatically expressed in cancer cells of given types. In this study, we show that angelica sinensis polysaccharide induced apoptosis in breast cancer cells of T47D over-expressing the Cyclic AMP response element binding protein (CREB), inducing apoptosis-related signaling pathway activity. The result also found that ASP caused cell death was linked to caspase activity, accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. We found that ASP significantly affected the poly-ADP-ribose polymerase (PARP), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL and apoptotic protease activating facter-1 (Apaf1) protein expression in a dose- and time-dependent manner. DAPI staining and Flow cytometry were used to analyze apoptosis. The nude mice xenograft model was used to evaluate the antitumor effect of ASP in vivo. ASP has profound antitumor effect on T47D cells, probably by inducing apoptosis through CREB signaling pathway. Thus, these results suggest that ASP would be a promising therapeutic agent for breast cancer. PMID:26431878

  17. Rotor Flapping Response to Active Control

    NASA Technical Reports Server (NTRS)

    Nguyen, Khanh; Johnson, Wayne

    2004-01-01

    Rotor active control using higher harmonic blade pitch has been proposed as a means to reduce both rotor radiated noise and airframe vibration and to enhance rotor performance. The higher harmonic input, however, can affect rotor thrust and cyclic flapping - the basic trim characteristics of the rotor. Some of the trim changes can negate the active control benefits. For example, wind tunnel test results of a full scale BO-105 rotor with individual-blade control indicate some rotor performance improvements, accompanied with changes in rotor trim, using two-per-rev blade pitch input. The observed performance benefits could therefore be a simple manifestation of the trim change rather than an efficient redistribution of the rotor airloads. More recently, the flight test of the BO-105 helicopter equip,ped with individual-blade-control actuators also reported trim changes whenever the two-per-rev blade pitch for noise reduction was activated. The pilot had to adjust the trim control to maintain the aircraft under a constant flight path. These two cases highlight the, importance of trim considerations in the application of active control to rotorcraft.

  18. The unpredictability of prolonged activation of stress response pathways

    PubMed Central

    Lamech, Lilian T.

    2015-01-01

    In response to stress, cellular compartments activate signaling pathways that mediate transcriptional programs to promote survival and reestablish homeostasis. Manipulation of the magnitude and duration of the activation of stress responses has been proposed as a strategy to prevent or repair the damage associated with aging or degenerative diseases. However, as these pathways likely evolved to respond specifically to transient perturbations, the unpredictability of prolonged activation should be considered. PMID:26101215

  19. Tax abolishes histone H1 repression of p300 acetyltransferase activity at the human T-cell leukemia virus type 1 promoter.

    PubMed

    Konesky, Kasey L; Nyborg, Jennifer K; Laybourn, Paul J

    2006-11-01

    Upon infection of human T-cell leukemia virus type 1 (HTLV-1), the provirus is integrated into the host cell genome and subsequently packaged into chromatin that contains histone H1. Consequently, transcriptional activation of the virus requires overcoming the environment of chromatin and H1. To efficiently activate transcription, HTLV-1 requires the virally encoded protein Tax and cellular transcription factor CREB. Together Tax and CREB interact with three cis-acting promoter elements called viral cyclic-AMP response elements (vCREs). Binding of Tax and CREB to the vCREs promotes association of p300/CBP into the complex and leads to transcriptional activation. Therefore, to fully understand the mechanism of Tax transactivation, it is necessary to examine transcriptional activation from chromatin assembled with H1. Using a DNA template harboring the complete HTLV-1 promoter sequence and a highly defined recombinant assembly system, we demonstrate proper incorporation of histone H1 into chromatin. Addition of H1 to the chromatin template reduces HTLV-1 transcriptional activation through a novel mechanism. Specifically, H1 does not inhibit CREB or Tax binding to the vCREs or p300 recruitment to the promoter. Rather, H1 directly targets p300 acetyltransferase activity. Interestingly, in determining the mechanism of H1 repression, we have discovered a previously undefined function of Tax, overcoming the repressive effects of H1-chromatin. Tax specifically abrogates the H1 repression of p300 enzymatic activity in a manner independent of p300 recruitment and without displacement of H1 from the promoter. PMID:16943293

  20. Dynamics of telomerase activity in response to acute psychological stress

    PubMed Central

    Epel, Elissa S.; Lin, Jue; Dhabhar, Firdaus S.; Wolkowitz, Owen M.; Puterman, E; Karan, Lori; Blackburn, Elizabeth H.

    2010-01-01

    Telomerase activity plays an essential role in cel0l survival, by lengthening telomeres and promoting cell growth and longevity. It is now possible to quantify the low levels of telomerase activity in human leukocytes. Low basal telomerase activity has been related to chronic stress in people and to chronic glucocorticoid exposure in vitro. Here we test whether leukocyte telomerase activity changes under acute psychological stress. We exposed 44 elderly women, including 22 high stress dementia caregivers and 22 matched low stress controls, to a brief laboratory psychological stressor, while examining changes in telomerase activity of peripheral blood mononuclear cells (PBMC). At baseline, caregivers had lower telomerase activity levels than controls, but during stress telomerase activity increased similarly in both groups. Across the entire sample, subsequent telomerase activity increased by 18% one hour after the end of the stressor (p<0.01). The increase in telomerase activity was independent of changes in numbers or percentages of monocytes, lymphocytes, and specific T cell types, although we cannot fully rule out some potential contribution from immune cell redistribution in the change in telomerase activity. Telomerase activity increases were associated with greater cortisol increases in response to the stressor. Lastly, psychological response to the tasks (greater threat perception) was also related to greater telomerase activity increases in controls. These findings uncover novel relationships of dynamic telomerase activity with exposure to an acute stressor, and with two classic aspects of the stress response -- perceived psychological stress and neuroendocrine (cortisol) responses to the stressor. PMID:20018236

  1. Ionospheric Response Due to Seismic Activity

    NASA Astrophysics Data System (ADS)

    Sharma, Dinesh Kumar

    2016-07-01

    Signatures of the seismic activity in the ionospheric F2 region have been studied by analyzing the measurement of electron and ion temperatures during the occurrence of earthquake. The ionospheric electron and ion temperatures data recorded by the RPA payload aboard the Indian SROSS-C2 satellite during the period from January 1995 to December 2000 were used for the altitude range 430-630 km over Indian region. The normal day's electron and ion temperatures have been compared to the temperatures recorded during the seismic activity. The details of seismic events were obtained from USGS earthquake data information website. It has been found that the average electron temperature is enhanced during the occurrence of earthquakes by 1.2 to 1.5 times and this enhancement was for ion temperature ranging from 1.1to 1.3 times over the normal day's average temperatures. The above careful quantitative analysis of ionospheric electron and ion temperatures data shows the consistent enhancement in the ionospheric electron and ion temperatures. It is expected that the seismogenic vertical electrical field propagates up to the ionospheric heights and induces Joule heating that may cause the enhancement in ionospheric temperatures.

  2. P-glycoprotein activity and biological response

    SciTech Connect

    Vaalburg, W. . E-mail: w.vaalburg@pet.umcg.nl; Hendrikse, N.H.; Elsinga, P.H.; Bart, J.; Waarde, A. van

    2005-09-01

    P-glycoprotein (P-gp) is a transmembrane drug efflux pump encoded by the MDR-1 gene in humans. Most likely P-gp protects organs against endogenous and exogenous toxins by extruding toxic compounds such as chemotherapeutics and other drugs. Many drugs are substrates for P-gp. Since P-gp is also expressed in the blood-brain barrier, P-gp substrates reach lower concentrations in the brain than in P-gp-negative tissues. Failure of response to chemotherapy of malignancies can be due to intrinsic or acquired drug resistance. Many tumors are multidrug resistant (MDR); resistant to several structurally unrelated chemotherapeutic agents. Several mechanisms are involved in MDR of which P-gp is studied most extensively. P-gp extrudes drugs out of tumor cells resulting in decreased intracellular drug concentrations, leading to the MDR phenotype. Furthermore, the MDR-1 gene exhibits several single nucleotide polymorphisms, some of which result in different transport capabilities. P-gp functionality and the effect of P-gp modulation on the pharmacokinetics of novel and established drugs can be studied in vivo by positron emission tomography (PET) using carbon-11 and fluorine-18-labeled P-gp substrates and modulators. PET may demonstrate the consequences of genetic differences on tissue pharmacokinetics. Inhibitors such as calcium-channel blockers (verapamil), cyclosporin A, ONT-093, and XR9576 can modulate the P-gp functionality. With PET the effect of P-gp modulation on the bioavailability of drugs can be investigated in humans in vivo. PET also allows the measurement of the efficacy of newly developed P-gp modulators.

  3. Targeted Activation of Conventional and Novel Protein Kinases C through Differential Translocation Patterns

    PubMed Central

    Hui, Xin; Reither, Gregor; Kaestner, Lars

    2014-01-01

    Activation of the two ubiquitous families of protein kinases, protein kinase A (PKA) and protein kinase C (PKC), is thought to be independently coupled to stimulation of Gαs and Gαq, respectively. Live-cell confocal imaging of protein kinase C fluorescent protein fusion constructs revealed that simultaneous activation of Gαs and Gαq resulted in a differential translocation of the conventional PKCα to the plasma membrane while the novel PKCδ was recruited to the membrane of the endoplasmic reticulum (ER). We demonstrate that the PKCδ translocation was driven by a novel Gαs-cyclic AMP-EPAC-RAP-PLCε pathway resulting in specific diacylglycerol production at the membrane of the ER. Membrane-specific phosphorylation sensors revealed that directed translocation resulted in phosphorylation activity confined to the target membrane. Specific stimulation of PKCδ caused phosphorylation of the inositol-1,4,5-trisphosphate receptor and dampening of global Ca2+ signaling revealed by graded flash photolysis of caged inositol-1,4,5-trisphosphate. Our data demonstrate a novel signaling pathway enabling differential decoding of incoming stimuli into PKC isoform-specific membrane targeting, significantly enhancing the versatility of cyclic AMP signaling, thus demonstrating the possible interconnection between the PKA and PKC pathways traditionally treated independently. We thus provide novel and elementary understanding and insights into intracellular signaling events. PMID:24732802

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  9. Regulation of lysophosphatidic acid-stimulated tyrosine phosphorylation of mitogen-activated protein kinase by protein kinase C- and pertussis toxin-dependent pathways in the endothelial cell line EAhy 926.

    PubMed Central

    McLees, A; Graham, A; Malarkey, K; Gould, G W; Plevin, R

    1995-01-01

    In the endothelial cell line EAhy 926, 1-oleoyl-lysophosphatidic acid (LPA) stimulated the tyrosine phosphorylation of the pp42 isoform of mitogen-activated protein (MAP) kinase. Maximum phosphorylation was observed within 5 min of LPA addition, but the response was sustained for up to 120 min. Re-addition of LPA after 60 min stimulated a further sustained increase in the tyrosine phosphorylation of MAP kinase. In cells pretreated with phorbol 12-myristate 13-acetate (PMA; 24 h) or preincubated with the protein kinase C inhibitor Ro-318220, LPA-induced tyrosine phosphorylation of pp42 MAP kinase was substantially reduced at 2 min but potentiated at 60 min. Ro-318220 in combination with either PMA or pertussis toxin pretreatment abolished the LPA response at all time points, suggesting an involvement of protein kinase C in the pertussis toxin-sensitive part of the pathway. Agents which raised intracellular cyclic AMP levels did not affect the initial phase of LPA-stimulated MAP kinase activation, but abolished the late phase. However, this effect was prevented by Ro-318220, implicating a greater role for protein kinase C than protein kinase A in the regulation of sustained MAP kinase responses. LPA stimulated an increase in the tyrosine phosphorylation of focal adhesion kinase pp125 (pp125FAK) in EAhy 926 cells which was both protein kinase C- and pertussis toxin-independent. These results are discussed in terms of the pathways regulating both MAP kinase and pp125FAK in response to LPA in the EAhy 926 endothelial cells line. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7741705

  10. Regulation of lysophosphatidic acid-stimulated tyrosine phosphorylation of mitogen-activated protein kinase by protein kinase C- and pertussis toxin-dependent pathways in the endothelial cell line EAhy 926.

    PubMed

    McLees, A; Graham, A; Malarkey, K; Gould, G W; Plevin, R

    1995-05-01

    In the endothelial cell line EAhy 926, 1-oleoyl-lysophosphatidic acid (LPA) stimulated the tyrosine phosphorylation of the pp42 isoform of mitogen-activated protein (MAP) kinase. Maximum phosphorylation was observed within 5 min of LPA addition, but the response was sustained for up to 120 min. Re-addition of LPA after 60 min stimulated a further sustained increase in the tyrosine phosphorylation of MAP kinase. In cells pretreated with phorbol 12-myristate 13-acetate (PMA; 24 h) or preincubated with the protein kinase C inhibitor Ro-318220, LPA-induced tyrosine phosphorylation of pp42 MAP kinase was substantially reduced at 2 min but potentiated at 60 min. Ro-318220 in combination with either PMA or pertussis toxin pretreatment abolished the LPA response at all time points, suggesting an involvement of protein kinase C in the pertussis toxin-sensitive part of the pathway. Agents which raised intracellular cyclic AMP levels did not affect the initial phase of LPA-stimulated MAP kinase activation, but abolished the late phase. However, this effect was prevented by Ro-318220, implicating a greater role for protein kinase C than protein kinase A in the regulation of sustained MAP kinase responses. LPA stimulated an increase in the tyrosine phosphorylation of focal adhesion kinase pp125 (pp125FAK) in EAhy 926 cells which was both protein kinase C- and pertussis toxin-independent. These results are discussed in terms of the pathways regulating both MAP kinase and pp125FAK in response to LPA in the EAhy 926 endothelial cells line. PMID:7741705

  11. Brain-derived Neurotrophic Factor Prevents Phencyclidine-induced Apoptosis in Developing Brain by Parallel Activation of both the ERK and PI-3K/Akt Pathways

    PubMed Central

    Xia, Yan; Wang, Cheng Z.; Liu, Jie; Anastasio, Noelle C.; Johnson, Kenneth M.

    2009-01-01

    Summary Phencyclidine is an N-methyl D-aspartate receptor (NMDAR) blocker that has been reported to induce neuronal apoptosis during development and schizophrenia-like behaviors in rats later in life. Brain derived neurotrophic factor (BDNF) has been shown to prevent neuronal death caused by NMDAR blockade, but the precise mechanism is unknown. This study examined the role of the phosphatidylinositol-3 kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways in BDNF protection of PCP-induced apoptosis in corticostriatal organotypic cultures. It was observed that BDNF inhibited PCP-induced apoptosis in a concentration dependent fashion. BDNF effectively prevented PCP-induced inhibition of the ERK and PI-3K/Akt pathways and suppressed GSK-3β activation. Blockade of either PI-3K/Akt or ERK activation abolished BDNF protection. Western blot analysis revealed that the PI-3K inhibitor LY294002 prevented the stimulating effect of BDNF on the PI-3K/Akt pathway, but had no effect on the ERK pathway. Similarly, the ERK inhibitor PD98059 prevented the stimulating effect of BDNF on the ERK pathway, but not the PI-3K/Akt pathway. Co-application of LY294002 and PD98059 had no additional effect on BDNF-evoked activation of Akt or ERK. However, concurrent exposure to PD98059 and LY294002 caused much greater inhibition of BDNF-evoked phosphorylation of GSK-3β at serine 9 than did LY294002 alone. Finally, either BDNF or GSK-3β inhibition prevented PCP-induced suppression of cyclic-AMP response element binding protein (CREB) phosphorylation. These data demonstrate that the protective effect of BDNF against PCP-induced apoptosis is mediated by parallel activation of the PI-3K/Akt and ERK pathways, most likely involves inhibition of GSK-3β and activation of CREB. PMID:19887077

  12. Phosphorylation within the transactivation domain of adenovirus E1A protein by mitogen-activated protein kinase regulates expression of early region 4.

    PubMed Central

    Whalen, S G; Marcellus, R C; Whalen, A; Ahn, N G; Ricciardi, R P; Branton, P E

    1997-01-01

    A critical role of the 289-residue (289R) E1A protein of human adenovirus type 5 during productive infection is to transactivate expression of all early viral transcription. Sequences within and proximal to conserved region 3 (CR3) promote expression of these viral genes through interactions with a variety of transcription factors requiring the zinc binding motif in CR3 and in some cases a region at the carboxy-terminal end of CR3, including residues 183 to 188. It is known that 3',5' cyclic AMP (cAMP) reduces the level of phosphorylation of the 289R E1A protein through the activation of protein phosphatase 2A by the E4orf4 protein. This study was designed to identify the E1A phosphorylation sites affected by E4orf4 expression and to determine their importance in regulation of E1A activity. We report here that two previously unidentified sites at Ser-185 and Ser-188 are the targets for decreased phosphorylation in response to cAMP. At least one of these sites, presumably Ser-185, is phosphorylated in vitro by purified mitogen-activated protein kinase (MAPK), and both are hyperphosphorylated in cells which express a constitutively active form of MAPK kinase. Analysis of E1A-mediated transactivation activity indicated that elevated phosphorylation at these sites increased expression of the E4 promoter but not that of E3. We have recently shown that one or more E4 products induce cell death due to p53-independent apoptosis, and thus it seems likely that one role of the E4orf4 protein is to limit production of toxic E4 products by limiting expression of the E4 promoter. PMID:9094626

  13. Structure–activity relationship studies of naphthol AS-E and its derivatives as anticancer agents by inhibiting CREB-mediated gene transcription

    PubMed Central

    Li, Bingbing X.; Yamanaka, Kinrin; Xiao, Xiangshu

    2012-01-01

    CREB (cyclic AMP-response element binding protein) is a downstream transcription factor of a multitude of signaling pathways emanating from receptor tyrosine kinases or G-protein coupled receptors. CREB is not activated until it is phosphorylated at Ser133 and its subsequent binding to CREB-binding protein (CBP) through kinase-inducible domain (KID) in CREB and KID-interacting (KIX) domain in CBP. Tumor tissues from various organs present higher level of expression and activation of CREB. Thus CREB has been proposed as a promising cancer drug target. We previously described naphthol AS-E (1a) as a small molecule inhibitor of CREB-mediated gene transcription in living cells. Here we report the structure–activity relationship (SAR) studies of 1a by modifying the appendant phenyl ring. All the compounds were evaluated for in vitro inhibition of KIX–KID interaction, cellular inhibition of CREB-mediated gene transcription and inhibition of proliferation of four cancer cell lines (A549, MCF-7, MDA-MB-231 and MDA-MB-468). SAR indicated that a small and electron-withdrawing group was preferred at the para-position for KIX–KID interaction inhibition. Compound 1a was selected for further biological characterization and it was found that 1a down-regulated the expression of endogenous CREB target genes. Expression of a constitutively active CREB mutant, VP16-CREB in MCF-7 cells rendered the cells resistant to 1a, suggesting that CREB was critical in mediating its anticancer activity. Furthermore, 1a was not toxic to normal human cells. Collectively, these data support that 1a represents a structural template for further development into potential cancer therapeutics with a novel mechanism of action. PMID:23102993

  14. Pythium infection activates conserved plant defense responses in mosses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The moss Physcomitrella patens (P. patens) is a useful model to study abiotic stress responses since it is highly tolerant to drought, salt and osmotic stress. However, little is known about the defense mechanisms activated in this moss after pathogen assault. Here the induction of defense responses...

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  16. Magnetospheric impulse response for many levels of geomagnetic activity

    NASA Technical Reports Server (NTRS)

    Bargatze, L. F.; Baker, D. N.; Hones, E. W., Jr.; Mcpherron, R. L.

    1985-01-01

    The temporal relationship between the solar wind and magnetospheric activity has been studied using 34 intervals of high time resolution IMP 8 solar wind data and the corresponding AL auroral activity index. The median values of the AL index for each interval were utilized to rank the intervals according to geomagnetic activity level. The linear prediction filtering technique was then applied to model magnetospheric response as measured by the AL index to the solar wind input function VB(s). The linear prediction filtering routine produces a filter of time-lagged response coefficients which estimates the most general linear relationship between the chosen input and output parameters of the magnetospheric system. It is found that the filters are composed of two response pulses speaking at time lags of 20 and 60 min. The amplitude of the 60-min pulse is the larger for moderate activity levels, while the 20-min pulse is the larger for strong activity levels. A possible interpretation is that the 20-min pulse represents magnetospheric activity driven directly by solar wind coupling and that the 60-min pulse represents magnetospheric activity driven by the release of energy previously stored in the magnetotail. If this interpretation is correct, the linear filtering results suggest that both the driven and the unloading models of magnetospheric response are important facets of a more comprehensive response model.

  17. Overlapping CRE and E Box Motifs in the Enhancer Sequences of the Bovine Leukemia Virus 5′ Long Terminal Repeat Are Critical for Basal and Acetylation-Dependent Transcriptional Activity of the Viral Promoter: Implications for Viral Latency

    PubMed Central

    Calomme, Claire; Dekoninck, Ann; Nizet, Séverine; Adam, Emmanuelle; Nguyên, Thi Liên-Anh; Van Den Broeke, Anne; Willems, Luc; Kettmann, Richard; Burny, Arsène; Lint, Carine Van

    2004-01-01

    Bovine leukemia virus (BLV) infection is characterized by viral latency in a large proportion of cells containing an integrated provirus. In this study, we postulated that mechanisms directing the recruitment of deacetylases to the BLV 5′ long terminal repeat (LTR) could explain the transcriptional repression of viral expression in vivo. Accordingly, we showed that BLV promoter activity was induced by several deacetylase inhibitors (such as trichostatin A [TSA]) in the context of episomal LTR constructs and in the context of an integrated BLV provirus. Moreover, treatment of BLV-infected cells with TSA increased H4 acetylation at the viral promoter, showing a close correlation between the level of histone acetylation and transcriptional activation of the BLV LTR. Among the known cis-regulatory DNA elements located in the 5′ LTR, three E box motifs overlapping cyclic AMP responsive elements (CREs) in U3 were shown to be involved in transcriptional repression of BLV basal gene expression. Importantly, the combined mutations of these three E box motifs markedly reduced the inducibility of the BLV promoter by TSA. E boxes are susceptible to recognition by transcriptional repressors such as Max-Mad-mSin3 complexes that repress transcription by recruiting deacetylases. However, our in vitro binding studies failed to reveal the presence of Mad-Max proteins in the BLV LTR E box-specific complexes. Remarkably, TSA increased the occupancy of the CREs by CREB/ATF. Therefore, we postulated that the E box-specific complexes exerted their negative cooperative effect on BLV transcription by steric hindrance with the activators CREB/ATF and/or their transcriptional coactivators possessing acetyltransferase activities. Our results thus suggest that the overlapping CRE and E box elements in the BLV LTR were selected during evolution as a novel strategy for BLV to allow better silencing of viral transcription and to escape from the host immune response. PMID:15564493

  18. Acute effects of oral and intravenous ethanol on rat hepatic enzyme activities.

    PubMed

    Stifel, F B; Greene, H L; Lufkin, E G; Wrensch, M R; Hagler, L; Herman, R H

    1976-05-28

    1. Oral administration of ethanol (3 ml) of 95% in 12 ml total volume over a two day period) significantly decrease plasma glucose and insulin levels and the activities of two key gluconeogenic enzymes, pyruvate carboxylase (pyruvate: CO2 ligase (ADP), EC 6.4.1.1) and fructose diphosphatase, (D-Fru-1,6-P2 1-phosphohydrolase, EC 3.1.3.11), and one glycolytic enzyme, fructose-1,6-P2 aldolase (Fru-1,6-P2 D-glyceraldehyde-3-P lyase, EC 4.1.2.13). In each instance, the administration of 2400 mug daily of oral folate in conjuction with the ethanol prevented these alterations in carbohydrate metabolism. 2. Intravenous injection of ethanol produced a rapid decrease (within 10--15 min) in the activities of hepatic phosphofructokinase, (ATP:D-fructose-6-phosphate 6-phosphotransferase, EC 2.7.1.11), pyruvate kinase, (ATP:pyruvate phosphotransferase, EC 2.7.1.40), fructose diphosphatase and fructose-1,6-P2 aldolase. 3. Intravenous ethanol significantly increased hepatic cyclic AMP concentration approximately 60% within 10 min, while oral ethanol did not alter hepatic cyclic AMP concentrations. 4. These data confirm the known antagonism ethanol and folate and suggest that oral folate might offer a protective effect against hypoglycemia in rats receiving ethanol. PMID:179581

  19. The effects of vasopressin on electrical activity in the guinea-pig supraoptic nucleus in vitro.

    PubMed Central

    Abe, H; Inoue, M; Matsuo, T; Ogata, N

    1983-01-01

    Brain slices of the guinea-pig hypothalamus were used to determine the effects of vasopressin on intracellular potentials in neurones of the supraoptic nucleus. Vasopressin (0.05-1 i.u./ml.) depolarized the membrane without apparent change in the input resistance and decreased the spontaneous firing rate. This action of vasopressin was retained in the medium containing 0 mM-Ca2+, 12 mM-Mg2+ and 0.3 mM-EGTA. Amplitude of the vasopressin-induced depolarization was voltage-independent. Ion-substitution experiments showed that the changes in [K+]o, [Cl-]o and [Ca2+]o had little effect upon the amplitude of vasopressin-induced depolarization, whereas the depletion of [Na+]o slightly reduced the amplitude. The vasopressin-induced depolarization was blocked at a temperature of 15 degrees C and by ouabain in a dose of 10(-4) M. Dibutyryl cyclic AMP (2 mM) produced electrophysiological effects similar to those seen with vasopressin, and actions of both agents were potentiated by either papaverine (10(-4) M) or theophylline (10(-2) M). Contents of cyclic AMP in tissues incubated with vasopressin were significantly higher than in cases of incubation with normal Krebs solution. We conclude that vasopressin directly modulates the activity of supraoptic neurones, possibly through activation of adenylate cyclase. PMID:6308238

  20. Activated oxygen alters cerebral microvascular responses in newborn pigs

    SciTech Connect

    Leffler, C.W.; Busiia, D.W.; Armstead, W.M.; Mirro, R.; Thelin, O. )

    1990-02-26

    In piglets, cerebral ischemia/reperfusion blocks prostanoid dependent cerebral vasodilation to hypercapnia (CO{sub 2}) and hypotension but not prostanoid independent dilation to isoproterenol (Isu) or constriction to norepinephrine (NE). Ischemia/reperfusion increases activated-O{sub 2} production by piglet brains. Using cranial windows in piglets, the authors investigated the hypothesis that activated oxygen can block prostanoid dependent cerebral vasodilator responses to CO{sub 2} and hypotension without altering responses to Isu and NE. Exposure to an activated oxygen generating system of xanthine oxidase, hypoxanthine, and Fe that made about 3 times the activated-O{sub 2} on the brain surface as ischemia/reperfusion caused reversible pial arteriolar dilation. After exposure, pial arteriolar dilation was reduced to CO{sub 2} and hypotension but not to Isu. NE constrictor responses were also unaltered. H{sub 2}O{sub 2} or H{sub 2}O{sub 2} + Fe caused constriction followed by reversible dilation. After exposure, pial arteriolar dilation in response to CO{sub 2} and hypotension was not altered. However, addition of xanthine oxidase and hypoxanthine with H{sub 2}O{sub 2} and Fe totally eliminated pial arteriolar dilator responses to CO{sub 2} and hypotension but did not decrease dilation caused by Isu or constriction caused by NE. The authors conclude that activated oxygen could produce the altered prostanoid dependent pial arteriolar responses observed following ischemia in piglets.

  1. The effects of siguazodan, a selective phosphodiesterase inhibitor, on human platelet function.

    PubMed Central

    Murray, K. J.; England, P. J.; Hallam, T. J.; Maguire, J.; Moores, K.; Reeves, M. L.; Simpson, A. W.; Rink, T. J.

    1990-01-01

    1. The effects of siguazodan (SK&F 94836) a selective phosphodiesterase (PDE) inhibitor with inotropic and vasodilator activity, were studied on human platelets. 2. Siguazodan selectively inhibited the major cyclic AMP-hydrolysing PDE in human platelet supernatants. The inhibited enzyme has been variously termed cyclic GMP-inhibited PDE or PDE-III. 3. In platelet-rich plasma (PRP), siguazodan inhibited U46619-induced aggregation more potently than that induced by ADP and collagen. Treatment of the PRP with aspirin had no effect on the potency of siguazodan. 4. In washed platelets, siguazodan increased cyclic AMP levels and reduced cytoplasmic free calcium [( Ca2+]i). ADP decreased the ability of siguazodan to raise cyclic AMP and this may explain its lower potency in inhibiting responses to ADP. 5. Siguazodan has anti-platelet actions over the same concentration range that it is an inotrope and vasodilator. PMID:2158847

  2. Activation of the renin-angiotensin system, specifically in the subfornical organ is sufficient to induce fluid intake

    PubMed Central

    Coble, Jeffrey P.; Cassell, Martin D.; Davis, Deborah R.; Grobe, Justin L.

    2014-01-01

    Increased activity of the renin-angiotensin system within the brain elevates fluid intake, blood pressure, and resting metabolic rate. Renin and angiotensinogen are coexpressed within the same cells of the subfornical organ, and the production and action of ANG II through the ANG II type 1 receptor in the subfornical organ (SFO) are necessary for fluid intake due to increased activity of the brain renin-angiotensin system. We generated an inducible model of ANG II production by breeding transgenic mice expressing human renin in neurons controlled by the synapsin promoter with transgenic mice containing a Cre-recombinase-inducible human angiotensinogen construct. Adenoviral delivery of Cre-recombinase causes SFO-selective induction of human angiotensinogen expression. Selective production of ANG II in the SFO results in increased water intake but did not change blood pressure or resting metabolic rate. The increase in water intake was ANG II type 1 receptor-dependent. When given a choice between water and 0.15 M NaCl, these mice increased total fluid and sodium, but not water, because of an increased preference for NaCl. When provided a choice between water and 0.3 M NaCl, the mice exhibited increased fluid, water, and sodium intake, but no change in preference for NaCl. The increase in fluid intake was blocked by an inhibitor of PKC, but not ERK, and was correlated with increased phosphorylated cyclic AMP response element binding protein in the subfornical organ. Thus, increased production and action of ANG II specifically in the subfornical organ are sufficient on their own to mediate an increase in drinking through PKC. PMID:24965793

  3. Ginger improves cognitive function via NGF-induced ERK/CREB activation in the hippocampus of the mouse.

    PubMed

    Lim, Soonmin; Moon, Minho; Oh, Hyein; Kim, Hyo Geun; Kim, Sun Yeou; Oh, Myung Sook

    2014-10-01

    Ginger (the rhizome of Zingiber officinale Roscoe) has been used worldwide for many centuries in cooking and for treatment of several diseases. The main pharmacological properties of ginger include anti-inflammatory, antihyperglycemic, antiarthritic, antiemetic and neuroprotective actions. Recent studies demonstrated that ginger significantly enhances cognitive function in various cognitive disorders as well as in healthy brain. However, the biochemical mechanisms underlying the ginger-mediated enhancement of cognition have not yet been studied in normal or diseased brain. In the present study, we assessed the memory-enhancing effects of dried ginger extract (GE) in a model of scopolamine-induced memory deficits and in normal animals by performing a novel object recognition test. We found that GE administration significantly improved the ability of mice to recognize novel objects, indicating improvements in learning and memory. Furthermore, to elucidate the mechanisms of GE-mediated cognitive enhancement, we focused on nerve growth factor (NGF)-induced signaling pathways. NGF enzyme-linked immunosorbent assay analysis revealed that GE administration led to elevated NGF levels in both the mouse hippocampus and rat glioma C6 cells. GE administration also resulted in phosphorylation of extracellular-signal-regulated kinase (ERK) and cyclic AMP response element-binding protein (CREB), as revealed by Western blotting analysis. Neutralization of NGF with a specific NGF antibody inhibited GE-triggered activation of ERK and CREB in the hippocampus. Also, GE treatment significantly increased pre- and postsynaptic markers, synaptophysin and PSD-95, which are related to synapse formation in the brain. These data suggest that GE has a synaptogenic effect via NGF-induced ERK/CREB activation, resulting in memory enhancement. PMID:25049196

  4. Participation in Peer Response as Activity: An Examination of Peer Response Stances from an Activity Theory Perspective

    ERIC Educational Resources Information Center

    Zhu, Wei; Mitchell, Deborah A.

    2012-01-01

    This article reports a case study that examined English as a Second Language students' peer response stances from an activity theory perspective. More specifically, the study was guided by the constructs of activity and motive/object in Leont'ev's theory. Multiple sources of data were collected from two native Spanish-speaking students enrolled in…

  5. cAMP-responsive element binding protein mediates a cGMP/protein kinase G-dependent anti-apoptotic signal induced by nitric oxide in retinal neuro-glial progenitor cells.

    PubMed

    Nagai-Kusuhara, Azusa; Nakamura, Makoto; Mukuno, Hirokazu; Kanamori, Akiyasu; Negi, Akira; Seigel, Gail M

    2007-01-01

    Nitric oxide (NO) is cytoprotective to certain types of neuronal cells. The neuroprotective ability of NO in the retina was reportedly mediated by the cyclic GMP (cGMP) to protein kinase G (PKG) pathway. Cyclic AMP-responsive element binding protein (CREB) plays an essential role in the NO/cGMP/PKG-mediated survival of rat cerebellar granule cells. We tested whether CREB transduces the NO/cGMP/PKG anti-apoptotic cascade in R28 neuro-glial progenitor cells. Apoptosis was induced in R28 cells by serum deprivation for 24 h. Varying concentrations of two NO donors, sodium nitroprusside (SNP) and nipradilol, were added to medium with or without an NO scavenger, a soluble guanylyl cyclase inhibitor, or a PKG inhibitor. The cells were immunostained against activated caspase-3 and counterstained with Hoechst 33258. Apoptosis was quantified by counting activated caspase-3 positive or pyknotic cells. SNP and nipradilol rescued R28 cells from apoptosis in a dose-dependent manner, at an optimal concentration of 1.0 microM and 10 microM, respectively. Higher concentrations were cytotoxic. The NO scavenger and the inhibitors decreased the anti-apoptotic effect of the NO donors. Intracellular cGMP levels were increased after exposure to SNP and nipradilol. Western blotting showed that both NO donors increased CREB phosphorylation, which was blocked when pre-exposed to the inhibitors. Transfection with a dominant negative CREB construct defective of phosphorylation at Ser-133 interfered with the anti-apoptotic activity of SNP. These results indicate that CREB at least in part mediates the cGMP/PKG-dependent anti-apoptotic signal induced by NO in R28 cells. PMID:17081519

  6. Oct-1 Potentiates CREB-Driven Cyclin D1 Promoter Activation via a Phospho-CREB- and CREB Binding Protein-Independent Mechanism

    PubMed Central

    Boulon, Séverine; Dantonel, Jean-Christophe; Binet, Virginie; Vié, Annick; Blanchard, Jean-Marie; Hipskind, Robert A.; Philips, Alexandre

    2002-01-01

    Cyclin D1, the regulatory subunit for mid-G1 cyclin-dependent kinases, controls the expression of numerous cell cycle genes. A cyclic AMP-responsive element (CRE), located upstream of the cyclin D1 mRNA start site, integrates mitogenic signals that target the CRE-binding factor CREB, which can recruit the transcriptional coactivator CREB-binding protein (CBP). We describe an alternative mechanism for CREB-driven cyclin D1 induction that involves the ubiquitous POU domain protein Oct-1. In the breast cancer cell line MCF-7, overexpression of Oct-1 or its POU domain strongly increases transcriptional activation of cyclin D1 and GAL4 reporter genes that is specifically dependent upon CREB but independent of Oct-1 DNA binding. Gel retardation and chromatin immunoprecipitation assays confirm that POU forms a complex with CREB bound to the cyclin D1 CRE. In solution, CREB interaction with POU requires the CREB Q2 domain and, notably, occurs with CREB that is not phosphorylated on Ser 133. Accordingly, Oct-1 also potently enhances transcriptional activation mediated by a Ser133Ala CREB mutant. Oct-1/CREB synergy is not diminished by the adenovirus E1A 12S protein, a repressor of CBP coactivator function. In contrast, E1A strongly represses CBP-enhanced transactivation by CREB phosphorylated on Ser 133. Our observation that Oct-1 potentiates CREB-dependent cyclin D1 transcriptional activity independently of Ser 133 phosphorylation and E1A-sensitive coactivator function offers a new paradigm for the regulation of cyclin D1 induction by proliferative signals. PMID:12391146

  7. Rho5p is involved in mediating the osmotic stress response in Saccharomyces cerevisiae, and its activity is regulated via Msi1p and Npr1p by phosphorylation and ubiquitination.

    PubMed

    Annan, Robert B; Wu, Cunle; Waller, Daniel D; Whiteway, Malcolm; Thomas, David Y

    2008-09-01

    Small GTPases of the Rho family act as molecular switches, and modulation of the GTP-bound state of Rho proteins is a well-characterized means of regulating their signaling activity in vivo. In contrast, the regulation of Rho-type GTPases by posttranslational modifications is poorly understood. Here, we present evidence of the control of the Saccharomyces cerevisiae Rho-type GTPase Rho5p by phosphorylation and ubiquitination. Rho5p binds to Ste50p, and the expression of the activated RHO5(Q91H) allele in an Deltaste50 strain is lethal under conditions of osmotic stress. An overexpression screen identified RGD2 and MSI1 as being high-copy suppressors of the osmotic sensitivity of this lethality. Rgd2p had been identified as being a possible Rho5p GTPase-activating protein based on an in vitro assay; this result supports its function as a regulator of Rho5p activity in vivo. MSI1 was previously identified as being a suppressor of hyperactive Ras/cyclic AMP signaling, where it antagonizes Npr1p kinase activity and promotes ubiquitination. Here, we show that Msi1p also acts via Npr1p to suppress activated Rho5p signaling. Rho5p is ubiquitinated, and its expression is lethal in a strain that is compromised for proteasome activity. These data identify Rho5p as being a target of Msi1p/Npr1p regulation and describe a regulatory circuit involving phosphorylation and ubiquitination. PMID:18621925

  8. Baseline Brain Activity Predicts Response to Neuromodulatory Pain Treatment

    PubMed Central

    Jensen, Mark P.; Sherlin, Leslie H.; Fregni, Felipe; Gianas, Ann; Howe, Jon D.; Hakimian, Shahin

    2015-01-01

    Objectives The objective of this study was to examine the associations between baseline electroencephalogram (EEG)-assessed brain oscillations and subsequent response to four neuromodulatory treatments. Based on available research, we hypothesized that baseline theta oscillations would prospectively predict response to hypnotic analgesia. Analyses involving other oscillations and the other treatments (meditation, neurofeedback, and both active and sham transcranial direct current stimulation) were viewed as exploratory, given the lack of previous research examining brain oscillations as predictors of response to these other treatments. Design Randomized controlled study of single sessions of four neuromodulatory pain treatments and a control procedure. Methods Thirty individuals with spinal cord injury and chronic pain had their EEG recorded before each session of four active treatments (hypnosis, meditation, EEG biofeedback, transcranial direct current stimulation) and a control procedure (sham transcranial direct stimulation). Results As hypothesized, more presession theta power was associated with greater response to hypnotic analgesia. In exploratory analyses, we found that less baseline alpha power predicted pain reduction with meditation. Conclusions The findings support the idea that different patients respond to different pain treatments and that between-person treatment response differences are related to brain states as measured by EEG. The results have implications for the possibility of enhancing pain treatment response by either 1) better patient/treatment matching or 2) influencing brain activity before treatment is initiated in order to prepare patients to respond. Research is needed to replicate and confirm the findings in additional samples of individuals with chronic pain. PMID:25287554

  9. Patterning of sympathetic nerve activity in response to vestibular stimulation

    NASA Technical Reports Server (NTRS)

    Kerman, I. A.; McAllen, R. M.; Yates, B. J.

    2000-01-01

    Growing evidence suggests a role for the vestibular system in regulation of autonomic outflow during postural adjustments. In the present paper we review evidence for the patterning of sympathetic nerve activity elicited by vestibular stimulation. In response to electrical activation of vestibular afferents, firing of sympathetic nerves located throughout the body is altered. However, activity of the renal nerve is most sensitive to vestibular inputs. In contrast, high-intensity simultaneous activation of cutaneous and muscle inputs elicits equivalent changes in firing of the renal, superior mesenteric and lumbar colonic nerves. Responses of muscle vasoconstrictor (MVC) efferents to vestibular stimulation are either inhibitory (Type I) or are comprised of a combination of excitation and inhibition (Type II). Interestingly, single MVC units located in the hindlimb exhibited predominantly Type I responses while those located in the forelimb and face exhibited Type II responses. Furthermore, brachial and femoral arterial blood flows were dissociated in response to vestibular stimulation, such that brachial vascular resistance increased while femoral resistance decreased. These studies demonstrate that vestibulosympathetic reflexes are patterned according to both the anatomical location and innervation target of a particular sympathetic nerve, and can lead to distinct changes in local blood flow.

  10. Prefrontal Cortex Activity Related to Abstract Response Strategies

    PubMed Central

    Genovesio, Aldo; Brasted, Peter J.; Mitz, Andrew R.; Wise, Steven P.

    2005-01-01

    Overview In monkeys, foraging strategies depend not only on a context established by spatial or symbolic cues, but also on the relations among cues. Genovesio et al. recorded the activity of prefrontal cortex neurons while monkeys chose a strategy based on the relation between consecutive symbolic cues. For the same cues and actions, the monkeys also learned fixed responses to the same symbols. Many neurons had activity selective for a given strategy, others for whether the monkeys’ response choice depended on a symbol or the relation between symbols. These findings indicate that the primate prefrontal cortex contributes to implementing abstract strategies. PMID:16039571

  11. Somatostatin inhibits exocytosis in rat pancreatic α-cells by Gi2-dependent activation of calcineurin and depriming of secretory granules

    PubMed Central

    Gromada, Jesper; Høy, Marianne; Buschard, Karsten; Salehi, Albert; Rorsman, Patrik

    2001-01-01

    Measurements of cell capacitance were used to investigate the molecular mechanisms by which somatostatin inhibits Ca2+-induced exocytosis in single rat glucagon-secreting pancreatic α-cells. Somatostatin decreased the exocytotic responses elicited by voltage-clamp depolarisations by 80 % in the presence of cyclic AMP-elevating agents such as isoprenaline and forskolin. Inhibition was time dependent and half-maximal within 22 s. The inhibitory action of somatostatin was concentration dependent with an IC50 of 68 nm and prevented by pretreatment of the cells with pertussis toxin. The latter effect was mimicked by intracellular dialysis with specific antibodies to Gi1/2 and by antisense oligonucleotides against G proteins of the subtype Gi2. Somatostatin lacked inhibitory action when applied in the absence of forskolin or in the presence of the L-type Ca2+ channel blocker nifedipine. The size of the ω-conotoxin-sensitive and forskolin-independent component of exocytosis was limited to 60 fF. By contrast, somatostatin abolished L-type Ca2+ channel-dependent exocytosis in α-cells exposed to forskolin. The magnitude of the latter pool amounted to 230 fF. The inhibitory effect of somatostatin on exocytosis was mediated by activation of the serine/threonine protein phosphatase calcineurin and was prevented by pretreatment with cyclosporin A and deltamethrin or intracellularly applied calcineurin autoinhibitory peptide. Experiments using the stable ATP analogue AMP-PCP indicate that somatostatin acts by depriming of granules. We propose that somatostatin receptors associate with L-type Ca2+ channels and couple to Gi2 proteins leading to a localised activation of calcineurin and depriming of secretory granules situated close to the L-type Ca2+ channels. PMID:11533141

  12. Tumor necrosis factor-α increases brain-derived neurotrophic factor expression in trigeminal ganglion neurons in an activity-dependent manner.

    PubMed

    Bałkowiec-Iskra, E; Vermehren-Schmaedick, A; Balkowiec, A

    2011-04-28

    Many chronic trigeminal pain conditions, such as migraine or temporo-mandibular disorders, are associated with inflammation within peripheral endings of trigeminal ganglion (TG) sensory neurons. A critical role in mechanisms of neuroinflammation is attributed to proinflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α (TNFα) that also contribute to mechanisms of persistent neuropathic pain resulting from nerve injury. However, the mechanisms of cytokine-mediated synaptic plasticity and nociceptor sensitization are not completely understood. In the present study, we examined the effects of TNFα on neuronal expression of brain-derived neurotrophic factor (BDNF), whose role in synaptic plasticity and sensitization of nociceptive pathways is well documented. We show that 4- and 24-h treatment with TNFα increases BDNF mRNA and protein, respectively, in neuron-enriched dissociated cultures of rat TG. TNFα increases the phosphorylated form of the cyclic AMP-responsive element binding protein (CREB), a transcription factor involved in regulation of BDNF expression in neurons, and activates transcription of BDNF exon IV (former exon III) and, to a lesser extent, exon VI (former exon IV), but not exon I. TNFα-mediated increase in BDNF expression is accompanied by increase in calcitonin gene-related peptide (CGRP), which is consistent with previously published studies, and indicates that both peptides are similarly regulated in TG neurons by inflammatory mediators. The effect of TNFα on BDNF expression is dependent on sodium influx through TTX-sensitive channels and on p38-mitogen-activated protein kinase. Moreover, electrical stimulation and forskolin, known to increase intracellular cAMP, potentiate the TNFα-mediated upregulation of BDNF expression. This study provides new evidence for a direct action of proinflammatory cytokines on TG primary sensory neurons, and reveals a mechanism through which TNFα stimulates de novo synthesis of BDNF in

  13. Educating for Political Activity: A Younger Generational Response

    ERIC Educational Resources Information Center

    Mac an Ghaill, Mairtin

    2010-01-01

    This paper is a response to Professor Chitty's "Educational Review" Guest Lecture article, "Educating for political activity". I address the three sections of his paper: a global and national-based politics of war, corporate manipulation and parliamentary scandals. This provides a basis to draw upon empirical material from a recent critical…

  14. Dynamics of lung macrophage activation in response to helminth infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Most of our understanding of the development and phenotype of alternatively activated macrophages (AAM) has been obtained from studies investigating the response of bone marrow- and peritoneal-derived cells to IL-4 or IL-13 stimulation. Comparatively little is known about the development of the AAM...

  15. Sensorimotor-Independent Prefrontal Activity During Response Inhibition

    PubMed Central

    Cai, Weidong; Cannistraci, Christopher J.; Gore, John C.; Leung, Hoi-Chung

    2015-01-01

    A network of brain regions involving the ventral inferior frontal gyrus/anterior insula (vIFG/AI), presupplementary motor area (pre-SMA) and basal ganglia has been implicated in stopping impulsive, unwanted responses. However, whether this network plays an equal role in response inhibition under different sensorimotor contexts has not been tested systematically. Here, we conducted an fMRI experiment using the stop signal task, a sensorimotor task requiring occasional withholding of the planned response upon the presentation of a stop signal. We manipulated both the sensory modality of the stop signal (visual versus auditory) and the motor response modality (hand versus eye). Results showed that the vIFG/AI and the preSMA along with the right middle frontal gyrus were commonly activated in response inhibition across the various sensorimotor conditions. Our findings provide direct evidence for a common role of these frontal areas, but not striatal areas in response inhibition independent of the sensorimotor contexts. Nevertheless, these three frontal regions exhibited different activation patterns during successful and unsuccessful stopping. Together with the existing evidence, we suggest that the vIFG/AI is involved in the early stages of stopping such as triggering the stop process while the preSMA may play a role in regulating other cortical and subcortical regions involved in stopping. PMID:23798325

  16. Sensorimotor-independent prefrontal activity during response inhibition.

    PubMed

    Cai, Weidong; Cannistraci, Christopher J; Gore, John C; Leung, Hoi-Chung

    2014-05-01

    A network of brain regions involving the ventral inferior frontal gyrus/anterior insula (vIFG/AI), presupplementary motor area (pre-SMA) and basal ganglia has been implicated in stopping impulsive, unwanted responses. However, whether this network plays an equal role in response inhibition under different sensorimotor contexts has not been tested systematically. Here, we conducted an fMRI experiment using the stop signal task, a sensorimotor task requiring occasional withholding of the planned response upon the presentation of a stop signal. We manipulated both the sensory modality of the stop signal (visual versus auditory) and the motor response modality (hand versus eye). Results showed that the vIFG/AI and the preSMA along with the right middle frontal gyrus were commonly activated in response inhibition across the various sensorimotor conditions. Our findings provide direct evidence for a common role of these frontal areas, but not striatal areas in response inhibition independent of the sensorimotor contexts. Nevertheless, these three frontal regions exhibited different activation patterns during successful and unsuccessful stopping. Together with the existing evidence, we suggest that the vIFG/AI is involved in the early stages of stopping such as triggering the stop process while the preSMA may play a role in regulating other cortical and subcortical regions involved in stopping. PMID:23798325

  17. Phase response curves in the characterization of epileptiform activity

    NASA Astrophysics Data System (ADS)

    Perez Velazquez, J. L.; Galán, R. F.; Dominguez, L. Garcia; Leshchenko, Y.; Lo, S.; Belkas, J.; Erra, R. Guevara

    2007-12-01

    Coordinated cellular activity is a major characteristic of nervous system function. Coupled oscillator theory offers unique avenues to address cellular coordination phenomena. In this study, we focus on the characterization of the dynamics of epileptiform activity, based on some seizures that manifest themselves with very periodic rhythmic activity, termed absence seizures. Our approach consists in obtaining experimentally the phase response curves (PRCs) in the neocortex and thalamus, and incorporating these PRCs into a model of coupled oscillators. Phase preferences of the stationary states and their stability are determined, and these results from the model are compared with the experimental recordings, and interpreted in physiological terms.

  18. Bacterial lifestyle shapes the regulation of stringent response activation

    PubMed Central

    Boutte, Cara C.; Crosson, Sean

    2014-01-01

    Bacteria inhabit enormously diverse niches and have a correspondingly large array of regulatory mechanisms to adapt to often inhospitable and variable environments. The stringent response allows bacteria to quickly reprogram transcription in response to changes in nutrient availability. Although the proteins controlling this response are conserved in almost all bacterial species, recent work has illuminated considerable diversity in the starvation cues and regulatory mechanisms that activate stringent signaling proteins in bacteria from different environments. In this review we describe the signals and genetic circuitries that control the stringent signaling systems of a copiotroph, a bacteriovore, an oligotroph and a mammalian pathogen – Escherichia coli, Myxococcus xanthus, Caulobacter crescentus and Mycobacterium tuberculosis, respectively – and discuss how control of the stringent response in these species is adapted to their particular lifestyles. PMID:23419217

  19. Anticipating Human Activities Using Object Affordances for Reactive Robotic Response.

    PubMed

    Koppula, Hema S; Saxena, Ashutosh

    2016-01-01

    An important aspect of human perception is anticipation, which we use extensively in our day-to-day activities when interacting with other humans as well as with our surroundings. Anticipating which activities will a human do next (and how) can enable an assistive robot to plan ahead for reactive responses. Furthermore, anticipation can even improve the detection accuracy of past activities. The challenge, however, is two-fold: We need to capture the rich context for modeling the activities and object affordances, and we need to anticipate the distribution over a large space of future human activities. In this work, we represent each possible future using an anticipatory temporal conditional random field (ATCRF) that models the rich spatial-temporal relations through object affordances. We then consider each ATCRF as a particle and represent the distribution over the potential futures using a set of particles. In extensive evaluation on CAD-120 human activity RGB-D dataset, we first show that anticipation improves the state-of-the-art detection results. We then show that for new subjects (not seen in the training set), we obtain an activity anticipation accuracy (defined as whether one of top three predictions actually happened) of 84.1, 74.4 and 62.2 percent for an anticipation time of 1, 3 and 10 seconds respectively. Finally, we also show a robot using our algorithm for performing a few reactive responses. PMID:26656575

  20. [Bone marrow stromal damage mediated by immune response activity].

    PubMed

    Vojinović, J; Kamenov, B; Najman, S; Branković, Lj; Dimitrijević, H

    1994-01-01

    The aim of this work was to estimate influence of activated immune response on hematopoiesis in vitro, using the experimental model of BCG immunized BALB/c mice and in patients with chronic immunoactivation: long-lasting infections, autoimmunity or malignancy. We correlated changes in long term bone marrow cultures (Dexter) and NBT reduction with appearance of anemia in patients and experimental model of immunization by BCG. Increased spontaneous NBT reduction pointed out role of macrophage activation in bone marrow stroma damage. Long-term bone marrow cultures showed reduced number of hematopoietic cells, with predomination of fibroblasts and loss of fat cells. This results correlated with anemia and leucocytosis with stimulated myelopoiesis in peripheral blood. Activation of immune response, or acting of any agent that directly changes extracellular matrix and cellularity of bone marrow, may result in microenviroment bone marrow damage that modify hematopoiesis. PMID:18173180

  1. Humpback Dolphin (Genus Sousa) Behavioural Responses to Human Activities.

    PubMed

    Piwetz, Sarah; Lundquist, David; Würsig, Bernd

    2015-01-01

    Humpback dolphins (genus Sousa) use shallow, near-shore waters throughout their range. This coastal distribution makes them vulnerable to recreational and commercial disturbances, especially near heavily populated and industrialized areas. Most research focusing on Sousa and human activities has emphasized direct impacts and threats, involving injury and death, with relatively little focus on indirect effects on dolphins, such as changes in behaviour that may lead to deleterious effects. Understanding behaviour is important in resolving human-wildlife conflict and is an important component of conservation. This chapter gives an overview of animal behavioural responses to human activity with examples from diverse taxa; reviews the scientific literature on behavioural responses of humpback dolphins to human activity throughout their range, including marine vessel traffic, dolphin tourism, cetacean-fishery interactions, noise pollution, and habitat alteration; and highlights information and data gaps for future humpback dolphin research to better inform behaviour-based management decisions that contribute to conservation efforts. PMID:26555621

  2. CD83 Modulates B Cell Activation and Germinal Center Responses.

    PubMed

    Krzyzak, Lena; Seitz, Christine; Urbat, Anne; Hutzler, Stefan; Ostalecki, Christian; Gläsner, Joachim; Hiergeist, Andreas; Gessner, André; Winkler, Thomas H; Steinkasserer, Alexander; Nitschke, Lars

    2016-05-01

    CD83 is a maturation marker for dendritic cells. In the B cell lineage, CD83 is expressed especially on activated B cells and on light zone B cells during the germinal center (GC) reaction. The function of CD83 during GC responses is unclear. CD83(-/-) mice have a strong reduction of CD4(+) T cells, which makes it difficult to analyze a functional role of CD83 on B cells during GC responses. Therefore, in the present study we generated a B cell-specific CD83 conditional knockout (CD83 B-cKO) model. CD83 B-cKO B cells show defective upregulation of MHC class II and CD86 expression and impaired proliferation after different stimuli. Analyses of GC responses after immunization with various Ags revealed a characteristic shift in dark zone and light zone B cell numbers, with an increase of B cells in the dark zone of CD83 B-cKO mice. This effect was not accompanied by alterations in the level of IgG immune responses or by major differences in affinity maturation. However, an enhanced IgE response was observed in CD83 B-cKO mice. Additionally, we observed a strong competitive disadvantage of CD83-cKO B cells in GC responses in mixed bone marrow chimeras. Furthermore, infection of mice with Borrelia burgdorferi revealed a defect in bacterial clearance of CD83 B-cKO mice with a shift toward a Th2 response, indicated by a strong increase in IgE titers. Taken together, our results show that CD83 is important for B cell activation and modulates GC composition and IgE Ab responses in vivo. PMID:26983787

  3. Delphinid behavioral responses to incidental mid-frequency active sonar.

    PubMed

    Henderson, E Elizabeth; Smith, Michael H; Gassmann, Martin; Wiggins, Sean M; Douglas, Annie B; Hildebrand, John A

    2014-10-01

    Opportunistic observations of behavioral responses by delphinids to incidental mid-frequency active (MFA) sonar were recorded in the Southern California Bight from 2004 through 2008 using visual focal follows, static hydrophones, and autonomous recorders. Sound pressure levels were calculated between 2 and 8 kHz. Surface behavioral responses were observed in 26 groups from at least three species of 46 groups out of five species encountered during MFA sonar incidents. Responses included changes in behavioral state or direction of travel, changes in vocalization rates and call intensity, or a lack of vocalizations while MFA sonar occurred. However, 46% of focal groups not exposed to sonar also changed their behavior, and 43% of focal groups exposed to sonar did not change their behavior. Mean peak sound pressure levels when a behavioral response occurred were around 122 dB re: 1 μPa. Acoustic localizations of dolphin groups exhibiting a response gave insight into nighttime movement patterns and provided evidence that impacts of sonar may be mediated by behavioral state. The lack of response in some cases may indicate a tolerance of or habituation to MFA sonar by local populations; however, the responses that occur at lower received levels may point to some sensitization as well. PMID:25324099

  4. Characterizing wind turbine system response to lightning activity

    SciTech Connect

    McNiff, B.; LaWhite, N.; Muljadi, E.

    1998-07-01

    A lightning protection research program was instituted by National Renewable Energy Laboratory to minimize lightning damage to wind turbines and to further the understanding of effective damage mitigation techniques. To that end, a test program is under way to observe lightning activity, protection system response, and damage at a wind power plant in the Department of Energy (DOE) and Electric Power Research Institute (EPRI) Turbine Verification Program. The authors installed Lightning activated surveillance cameras along with a special storm tracking device to observe the activity in the wind plant area. They instrumented the turbines with lightning and ground current detection devices to log direct and indirect strike activity at each unit. They installed a surge monitor on the utility interface to track incoming activity from the transmission lines. Maintenance logs are used to verify damage and determine downtime and repair costs. Actual strikes to turbines were recorded on video and ancillary devices. The test setup and some results are discussed in this paper.

  5. Prostaglandin E2 and the Suppression of Phagocyte Innate Immune Responses in Different Organs

    PubMed Central

    Medeiros, Alexandra; Peres-Buzalaf, Camila; Fortino Verdan, Felipe; Serezani, C. Henrique

    2012-01-01

    The local and systemic production of prostaglandin E2 (PGE2) and its actions in phagocytes lead to immunosuppressive conditions. PGE2 is produced at high levels during inflammation, and its suppressive effects are caused by the ligation of the E prostanoid receptors EP2 and EP4, which results in the production of cyclic AMP. However, PGE2 also exhibits immunostimulatory properties due to binding to EP3, which results in decreased cAMP levels. The various guanine nucleotide-binding proteins (G proteins) that are coupled to the different EP receptors account for the pleiotropic roles of PGE2 in different disease states. Here, we discuss the production of PGE2 and the actions of this prostanoid in phagocytes from different tissues, the relative contribution of PGE2 to the modulation of innate immune responses, and the novel therapeutic opportunities that can be used to control inflammatory responses. PMID:23024463

  6. Regulation of β1- and β3-adrenergic agonist-stimulated lipolytic response in hyperthyroid and hypothyroid rat white adipocytes

    PubMed Central

    Germack, Renée; Starzec, Anna; Perret, Gérard Y

    2000-01-01

    This study examined the effects of thyroid status on the lipolytic responses of rat white adipocytes to β-adrenoceptor (β-AR) stimulation. The β1- and β3-AR mRNAs and proteins were measured by Northern and saturation analyses, respectively. Glycerol production and adenyl cyclase (AC) activity induced by various non-selective and selective β1/β3-AR agonists and drugs which act distal to the receptor in the signalling cascade were measured in cells from untreated, tri-iodothyronine (T3)-treated and thyroidectomized rats. The β3-AR density was enhanced (72%) by T3-treatment and reduced (50%) by introduction of a hypothyroid state while β1-AR number remained unaffected. The β1- and β3-AR density was correlated with the specific mRNA level in all thyroid status. The lipolytic responses to isoprenaline, noradrenaline (β1/β3/β3-AR agonists) and BRL 37344 (β3-AR agonist) were potentiated by 48, 58 and 48%, respectively in hyperthyroidism and reduced by about 80% in hypothyroidism. T3-treatment increased the maximal lipolytic response to the partial β3-AR (CGP 12177) and β1-AR (xamoterol) agonists by 234 and 260%, respectively, increasing their efficacy (intrinsic activity: 0.95 versus 0.43 and 1.02 versus 0.42). The maximal AC response to these agonists was increased by 84 and 58%, respectively, without changing their efficacy. In the hypothyroid state, the maximal lipolytic and AC responses were decreased with CGP (0.17±0.03 versus 0.41±0.08 μmol glycerol/106 adipocytes; 0.048±0.005 versus 0.114±0.006 pmol cyclic AMP min−1 mg−1) but not changed with xamoterol. The changes in lipolytic responses to postreceptor-acting agents (forskolin, enprofylline and dibutenyl cyclic AMP, (Bu)2cAMP) suggest the modifications on receptor coupling and phosphodiesterase levels in both thyroid states. Thyroid status affects lipolysis by modifying β3-AR density and postreceptor events without changes in the β1-AR functionality. PMID:10711342

  7. The prolactin responses to active and passive heating in man.

    PubMed

    Low, David; Purvis, Alison; Reilly, Thomas; Cable, N Tim

    2005-11-01

    The aim of this study was to compare the prolactin and blood pressure responses at identical core temperatures during active and passive heat stresses, using prolactin as an indirect marker of central fatigue. Twelve male subjects cycled to exhaustion at 60% maximal oxygen uptake (VO2peak) in a room maintained at 33 degrees C (active). In a second trial they were passively heated (passive) in a water bath (41.56 +/- 1.65 degrees C) until core temperature was equal to the core temperature observed at exhaustion during the active trial. Blood samples were taken from an indwelling venous cannula for the determination of serum prolactin during active heating and at corresponding core temperatures during passive heating. Core temperature was not significantly different between the two methods of heating and averaged 38.81 +/- 0.53 and 38.82 +/- 0.70 degrees C (data expressed as means +/- s.d.) at exhaustion during active heating and at the end of passive heating, respectively (P > 0.05). Mean arterial blood pressure was significantly lower throughout passive heating (active, 73 +/- 9 mmHg; passive, 62 +/- 12 mmHg; P < 0.01). Despite the significantly reduced blood pressure responses during passive heating, during both forms of heating the prolactin response was the same (active, 14.9 +/- 12.6 ng ml(-1); passive, 13.3 +/- 9.6 ng ml(-1); n.s.). These results suggest that thermoregulatory, i.e. core temperature, and not cardiovascular afferents provide the key stimulus for the release of prolactin, an indirect marker of central fatigue, during exercise in the heat. PMID:16157657

  8. Changes in thyroid peroxidase activity in response to various chemicals.

    PubMed

    Song, Mee; Kim, Youn-Jung; Park, Yong-Keun; Ryu, Jae-Chun

    2012-08-01

    Thyroperoxidase (TPO) is a large heme-containing glycoprotein that catalyzes the transfer of iodine to thyroglobulin during thyroid hormone (TH) synthesis. Previously, we established an in vitro assay for TPO activity based on human recombinant TPO (hrTPO) stably transfected into human follicular thyroid carcinoma (FTC-238) cells. It is important to determine whether environmental chemicals can disrupt TPO activity because it is an important factor in the TH axis. In this study, we used our assay to examine the changes in TPO activity in response to various chemicals, including benzophenones (BPs), polycyclic aromatic hydrocarbons (PAHs), and persistent organic pollutants (POPs). Overall, BPs, PAHs, and POPs slightly altered TPO activity at low doses, as compared with the positive controls methimazole (MMI), genistein, and 2,2',4,4'-tetrahydroxy BP. Benzophenone, benzhydrol, 3-methylchloranthracene, pyrene, benzo(k)fluoranthene, benzo(e)pyrene, perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), and heptachlor decreased TPO activity, while 2,4-dihydroxy BP, 2,2'-dihydroxy-4-methoxy BP, and dibenzo(a,h)anthracene increased TPO activity. From these data, we can predict the disruption of TPO activity by various chemicals as a sensitive TH end point. TPO activity should be considered when enacting measures to regulate environmental exposure to thyroid-disrupting chemicals. PMID:22699773

  9. Response Activation in Overlapping Tasks and the Response-Selection Bottleneck

    ERIC Educational Resources Information Center

    Schubert, Torsten; Fischer, Rico; Stelzel, Christine

    2008-01-01

    The authors investigated the impact of response activation on dual-task performance by presenting a subliminal prime before the stimulus in Task 2 (S2) of a psychological refractory period (PRP) task. Congruence between prime and S2 modulated the reaction times in Task 2 at short stimulus onset asynchrony despite a PRP effect. This Task 2…

  10. Reduced brain activation in violent adolescents during response inhibition

    PubMed Central

    Qiao, Yi; Mei, Yi; Du, XiaoXia; Xie, Bin; Shao, Yang

    2016-01-01

    Deficits in inhibitory control have been linked to aggression and violent behaviour. This study aimed to observe whether violent adolescents show different brain activation patterns during response inhibition and to ascertain the roles these brain regions play. A self-report method and modified overt aggression scale (MOAS) were used to evaluate violent behaviour. Functional magnetic resonance imaging was performed in 22 violent adolescents and 17 matched healthy subjects aged 12 to 18 years. While scanning, a go/no-go task was performed. Between-group comparisons revealed that activation in the bilateral middle and superior temporal gyrus, hippocampus, and right orbitofrontal area (BA11) regions were significantly reduced in the violent group compared with the control group. Meanwhile, the violent group had more widespread activation in the prefrontal cortex than that observed in the control group. Activation of the prefrontal cortex in the violent group was widespread but lacking in focus, failing to produce intensive activation in some functionally related regions during response inhibition. PMID:26888566

  11. Organization and regulation of the D-xylose operons in Escherichia coli K-12: XylR acts as a transcriptional activator.

    PubMed Central

    Song, S; Park, C

    1997-01-01

    The metabolism of D-xylose in Escherichia coli K-12 is known to be mediated by the xylAB gene. However, the nearby xylFGHR genes were found by genome sequencing and predicted to be responsible for transport and regulation for xylose based on their sequence similarities to other functionally related genes. Here, we investigated transcriptional organization and functions of the xyl genes. An analysis with random transposon insertions revealed that the xyl genes are organized into two major transcriptional units, xylAB and xylFGHR, governed by the promoters PA and PF, respectively. However, there is an additional weak promoter, PR, which is specific for xylR. Sites of transcription initiation were determined by primer extension analysis. When studied with operon fusions to lacZ, the PA and PF promoters were activated by D-xylose and repressed by glucose. In contrast, the PR promoter was not regulated by these sugars. A mutation in xylR completely abolished expression from the PA and PF promoters, causing a defect in both growth and transport. Binding of XylR to the xyl promoter was enhanced by the presence of D-xylose, suggesting that transcription was positively regulated by XylR. In vivo footprinting analysis revealed that XylR binds to at least two DNA regions, IA and IF, each with a direct repeat. It is very likely that XylR interacts with IA and IF as a dimer. The presumed binding sites are located just upstream of the promoter consensus sequences (-35), while IA is additionally flanked by a cyclic AMP receptor protein-binding site on the other side. The proposed structure of xyl promoters is consistent with the regulation of xyl gene expression and with phenotypes of transposon insertions obtained in the promoter regions. PMID:9371449

  12. Effects of three activities on annoyance responses to recorded flyovers

    NASA Technical Reports Server (NTRS)

    Gunn, W. J.; Shepherd, W. T.; Fletcher, J. L.

    1975-01-01

    Subjects participated in an experiment in which they were engaged in TV viewing, telephone listening, or reverie (no activity) for a 1/2-hour session. During the session, they were exposed to a series of recorded aircraft sounds at the rate of one flight every 2 minutes. Within each session, four levels of flyover noise, separated by 5dB increments, were presented several times in a Latin Square balanced sequence. The peak level of the noisiest flyover in any session was fixed at 95, 90, 85, 75, or 70 dBA. At the end of the test session, subjects recorded their responses to the aircraft sounds, using a bipolar scale which covered the range from 'very pleasant' to 'extremely annoying'. Responses to aircraft noises were found to be significantly affected by the particular activity in which the subjects were engaged. Furthermore, not all subjects found the aircraft sounds to be annoying.

  13. Activation and Regulation of DNA-Driven Immune Responses

    PubMed Central

    2015-01-01

    SUMMARY The innate immune system provides early defense against infections and also plays a key role in monitoring alterations of homeostasis in the body. DNA is highly immunostimulatory, and recent advances in this field have led to the identification of the innate immune sensors responsible for the recognition of DNA as well as the downstream pathways that are activated. Moreover, information on how cells regulate DNA-driven immune responses to avoid excessive inflammation is now emerging. Finally, several reports have demonstrated how defects in DNA sensing, signaling, and regulation are associated with susceptibility to infections or inflammatory diseases in humans and model organisms. In this review, the current literature on DNA-stimulated innate immune activation is discussed, and important new questions facing this field are proposed. PMID:25926682

  14. Hemodynamic responses to functional activation accessed by optical imaging

    NASA Astrophysics Data System (ADS)

    Ni, Songlin; Li, Pengcheng; Yang, Yuanyuan; Lv, Xiaohua; Luo, Qingming

    2006-01-01

    A multi-wavelength light-emitting diode (LED) and laser diode (LD) based optical imaging system was developed to visualize the changes in cerebral blood flow, oxygenation following functional activation simultaneously in rodent cortex. The 2-D blood flow image was accessed by laser speckle contrast imaging, and the spectroscopic imaging of intrinsic signal was used for the calculation of oxyhemoglobin (HbO), deoxyhemoglobin (Hb) and total hemoglobin (HbT) concentration. The combination of spectroscopic imaging and laser speckle contrast imaging provides the capability to simultaneously investigate the spatial and temporal blood flow and hemoglobin concentration changes with high resolution, which may lead to a better understanding of the coupling between neuronal activation and vascular responses. The optical imaging system been built is compact and convenient to investigators. And it is reliable to acquire raw data. In present study, the hemodynamic responses to cortical spreading depression (CSD) in parietal cortex of ~-chloralose/urethan anesthetized rats were demonstrated.

  15. Photodynamic therapy for cancer and activation of immune response

    NASA Astrophysics Data System (ADS)

    Mroz, Pawel; Huang, Ying-Ying; Hamblin, Michael R.

    2010-02-01

    Anti-tumor immunity is stimulated after PDT for cancer due to the acute inflammatory response, exposure and presentation of tumor-specific antigens, and induction of heat-shock proteins and other danger signals. Nevertheless effective, powerful tumor-specific immune response in both animal models and also in patients treated with PDT for cancer, is the exception rather than the rule. Research in our laboratory and also in others is geared towards identifying reasons for this sub-optimal immune response and discovering ways of maximizing it. Reasons why the immune response after PDT is less than optimal include the fact that tumor-antigens are considered to be self-like and poorly immunogenic, the tumor-mediated induction of CD4+CD25+foxP3+ regulatory T-cells (T-regs), that are able to inhibit both the priming and the effector phases of the cytotoxic CD8 T-cell anti-tumor response and the defects in dendritic cell maturation, activation and antigen-presentation that may also occur. Alternatively-activated macrophages (M2) have also been implicated. Strategies to overcome these immune escape mechanisms employed by different tumors include combination regimens using PDT and immunostimulating treatments such as products obtained from pathogenic microorganisms against which mammals have evolved recognition systems such as PAMPs and toll-like receptors (TLR). This paper will cover the use of CpG oligonucleotides (a TLR9 agonist found in bacterial DNA) to reverse dendritic cell dysfunction and methods to remove the immune suppressor effects of T-regs that are under active study.

  16. Autophagic activity dictates the cellular response to oncogenic RAS

    PubMed Central

    Wang, Yihua; Wang, Xiao Dan; Lapi, Eleonora; Sullivan, Alexandra; Jia, Wei; He, You-Wen; Ratnayaka, Indrika; Zhong, Shan; Goldin, Robert D.; Goemans, Christoph G.; Tolkovsky, Aviva M.; Lu, Xin

    2012-01-01

    RAS is frequently mutated in human cancers and has opposing effects on autophagy and tumorigenesis. Identifying determinants of the cellular responses to RAS is therefore vital in cancer research. Here, we show that autophagic activity dictates the cellular response to oncogenic RAS. N-terminal Apoptosis-stimulating of p53 protein 2 (ASPP2) mediates RAS-induced senescence and inhibits autophagy. Oncogenic RAS-expressing ASPP2(Δ3/Δ3) mouse embryonic fibroblasts that escape senescence express a high level of ATG5/ATG12. Consistent with the notion that autophagy levels control the cellular response to oncogenic RAS, overexpressing ATG5, but not autophagy-deficient ATG5 mutant K130R, bypasses RAS-induced senescence, whereas ATG5 or ATG3 deficiency predisposes to it. Mechanistically, ASPP2 inhibits RAS-induced autophagy by competing with ATG16 to bind ATG5/ATG12 and preventing ATG16/ATG5/ATG12 formation. Hence, ASPP2 modulates oncogenic RAS-induced autophagic activity to dictate the cellular response to RAS: to proliferate or senesce. PMID:22847423

  17. Dynamical theory of active cellular response to external stress.

    PubMed

    De, Rumi; Safran, Samuel A

    2008-09-01

    We present a comprehensive, theoretical treatment of the orientational response to external stress of active, contractile cells embedded in a gel-like elastic medium. The theory includes both the forces that arise from the deformation of the matrix as well as forces due to the internal regulation of the stress fibers and focal adhesions of the cell. We calculate the time-dependent response of both the magnitude and the direction of the elastic dipole that characterizes the active forces exerted by the cell, for various situations. For static or quasistatic external stress, cells orient parallel to the stress while for high frequency dynamic external stress, cells orient nearly perpendicular. Both numerical and analytical calculations of these effects are presented. In addition we predict the relaxation time for the cellular response for both slowly and rapidly varying external stresses; several characteristic scaling regimes for the relaxation time as a function of applied frequency are predicted. We also treat the case of cells for which the regulation of the stress fibers and focal adhesions is controlled by strain (instead of stress) and show that the predicted dependence of the cellular orientation on the Poisson ratio of the matrix can differentiate strain vs stress regulation of cellular response. PMID:18851081

  18. Dynamical theory of active cellular response to external stress

    NASA Astrophysics Data System (ADS)

    de, Rumi; Safran, Samuel A.

    2008-09-01

    We present a comprehensive, theoretical treatment of the orientational response to external stress of active, contractile cells embedded in a gel-like elastic medium. The theory includes both the forces that arise from the deformation of the matrix as well as forces due to the internal regulation of the stress fibers and focal adhesions of the cell. We calculate the time-dependent response of both the magnitude and the direction of the elastic dipole that characterizes the active forces exerted by the cell, for various situations. For static or quasistatic external stress, cells orient parallel to the stress while for high frequency dynamic external stress, cells orient nearly perpendicular. Both numerical and analytical calculations of these effects are presented. In addition we predict the relaxation time for the cellular response for both slowly and rapidly varying external stresses; several characteristic scaling regimes for the relaxation time as a function of applied frequency are predicted. We also treat the case of cells for which the regulation of the stress fibers and focal adhesions is controlled by strain (instead of stress) and show that the predicted dependence of the cellular orientation on the Poisson ratio of the matrix can differentiate strain vs stress regulation of cellular response.

  19. Activation of the DNA Damage Response by RNA Viruses.

    PubMed

    Ryan, Ellis L; Hollingworth, Robert; Grand, Roger J

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  20. Activation of the DNA Damage Response by RNA Viruses

    PubMed Central

    Ryan, Ellis L.; Hollingworth, Robert; Grand, Roger J.

    2016-01-01

    RNA viruses are a genetically diverse group of pathogens that are responsible for some of the most prevalent and lethal human diseases. Numerous viruses introduce DNA damage and genetic instability in host cells during their lifecycles and some species also manipulate components of the DNA damage response (DDR), a complex and sophisticated series of cellular pathways that have evolved to detect and repair DNA lesions. Activation and manipulation of the DDR by DNA viruses has been extensively studied. It is apparent, however, that many RNA viruses can also induce significant DNA damage, even in cases where viral replication takes place exclusively in the cytoplasm. DNA damage can contribute to the pathogenesis of RNA viruses through the triggering of apoptosis, stimulation of inflammatory immune responses and the introduction of deleterious mutations that can increase the risk of tumorigenesis. In addition, activation of DDR pathways can contribute positively to replication of viral RNA genomes. Elucidation of the interactions between RNA viruses and the DDR has provided important insights into modulation of host cell functions by these pathogens. This review summarises the current literature regarding activation and manipulation of the DDR by several medically important RNA viruses. PMID:26751489

  1. Cytoplasmic Forms of Human T-Cell Leukemia Virus Type 1 Tax Induce NF-κB Activation

    PubMed Central

    Nicot, Christophe; Tie, Feng; Giam, Chou-Zen

    1998-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) Tax targets I-κBα and I-κBβ for phosphorylation, ubiquitination, and proteasome-mediated degradation, causing the nuclear translocation of NF-κB/Rel proteins and transcription induction of many cellular genes. The mechanism by which a nuclear protein such as Tax stimulates I-κB phosphorylation and degradation remains unclear. Here, we describe two cytoplasmic mutants of Tax, designated TaxΔN81 and TaxΔN109, from which the domains important for cyclic AMP response element binding factor (CREB) and serum response factor (SRF) binding and nuclear transport have been removed. These mutants were unable to trans activate from the HTLV-1 21-bp repeats or the serum response element in the c-fos promoter. In contrast, they activated NF-κB reporters, suggesting that activation of NF-κB by Tax occurs in the cytoplasm. Incorporation of the nuclear localization signal (NLS) of the simian virus 40 large T antigen into TaxΔN81 and TaxΔN109 redirected both proteins predominantly to the nucleus yet did not restore trans activation via CREB or SRF. The NLS fusion had little effect on TaxΔN81 but reduced NF-κB trans activation by TaxΔN109, possibly because of its proximity to the NF-κB-activating domain of Tax. In contrast to wild-type Tax, the cytoplasmic TaxΔN mutants are not cytotoxic. Stable expression of TaxΔN109 in HeLa cells resulted in a significant reduction in the intracellular level of I-κBα, with the constitutive presence of NF-κB in the nucleus and concomitant activation of the NF-κB enhancer. These results are suggestive of a potential application of the TaxΔN109-like mutants in targeting I-κB degradation and NF-κB activation. Interestingly, a Tax species with a molecular mass similar to that of TaxΔN109 was identified in many HTLV-1-transformed T cells, suggesting that TaxΔN109-like species might play a role in HTLV-1-induced leukemogenesis. PMID:9658126

  2. Substance P primes lipoteichoic acid- and Pam3CysSerLys4-mediated activation of human mast cells by up-regulating Toll-like receptor 2.

    PubMed

    Tancowny, Brian P; Karpov, Victor; Schleimer, Robert P; Kulka, Marianna

    2010-10-01

    Substance P (SP) is a neuropeptide with neuroimmunoregulatory activity that may play a role in susceptibility to infection. Human mast cells, which are important in innate immune responses, were analysed for their responses to pathogen-associated molecules via Toll-like receptors (TLRs) in the presence of SP. Human cultured mast cells (LAD2) were activated by SP and TLR ligands including lipopolysaccharide (LPS), Pam3CysSerLys4 (Pam3CSK4) and lipoteichoic acid (LTA), and mast cell leukotriene and chemokine production was assessed by enzyme-linked immunosorbent assay (ELISA) and gene expression by quantitative PCR (qPCR). Mast cell degranulation was determined using a β-hexosaminidase (β-hex) assay. SP treatment of LAD2 up-regulated mRNA for TLR2, TLR4, TLR8 and TLR9 while anti-immunoglobulin E (IgE) stimulation up-regulated expression of TLR4 only. Flow cytometry and western blot confirmed up-regulation of TLR2 and TLR8. Pretreatment of LAD2 with SP followed by stimulation with Pam3CSK4 or LTA increased production of leukotriene C4 (LTC(4) ) and interleukin (IL)-8 compared with treatment with Pam3CSK4 or LTA alone (>2-fold; P<0·01). SP alone activated 5-lipoxygenase (5-LO) nuclear translocation but also augmented Pam3CSK4 and LTA-mediated 5-LO translocation. Pam3CSK4, LPS and LTA did not induce LAD2 degranulation. SP primed LTA and Pam3CSK4-mediated activation of JNK, p38 and extracellular-signal-regulated kinase (ERK) and activated the nuclear translocation of c-Jun, nuclear factor (NF)-κB, activating transcription factor 2 (ATF-2) and cyclic-AMP-responsive element binding protein (CREB) transcription factors. Pretreatment with SP followed by LTA stimulation synergistically induced production of chemokine (C-X-C motif) ligand 8 (CXCL8)/IL-8, chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein 1 (MCP-1), tumour necrosis factor (TNF) and IL-6 protein. SP primes TLR2-mediated activation of human mast cells by up-regulating TLR expression and

  3. Placebo-Activated Neural Systems are Linked to Antidepressant Responses

    PubMed Central

    Peciña, Marta; Bohnert, Amy S. B.; Sikora, Magdalena; Avery, Erich T.; Langenecker, Scott A.; Mickey, Brian J.; Zubieta, Jon-Kar

    2016-01-01

    Importance High placebo responses have been observed across a wide range of pathologies, severely impacting drug development. Objective Here we examined neurochemical mechanisms underlying the formation of placebo effects in patients with Major Depressive Disorder (MDD). Participants Thirty-five medication-free MDD patients. Design and Intervention We performed a single-blinded two-week cross-over randomized controlled trial of two identical oral placebos (described as having either “active” or “inactive” fast-acting antidepressant-like effects) followed by a 10-week open-label treatment with a selective serotonin reuptake inhibitor (SSRI) or in some cases, another agent as clinically indicated. The volunteers were studied with PET and the μ-opioid receptor (MOR)-selective radiotracer [11C]carfentanil after each 1-week “inactive” and “active” oral placebo treatment. In addition, 1 mL of isotonic saline was administered intravenously (i.v.) within sight of the volunteer during PET scanning every 4 min over 20 min only after the 1-week active placebo treatment, with instructions that the compound may be associated with the activation of brain systems involved in mood improvement. This challenge stimulus was utilized to test the individual capacity to acutely activate endogenous opioid neurotransmision under expectations of antidepressant effect. Setting A University Health System. Main Outcomes and Measures Changes in depressive symptoms in response to “active” placebo and antidepressant. Baseline and activation measures of MOR binding. Results Higher baseline MOR binding in the nucleus accumbens (NAc) was associated with better response to antidepressant treatment (r=0.48; p=0.02). Reductions in depressive symptoms after 1-week of “active” placebo treatment, compared to the “inactive”, were associated with increased placebo-induced μ-opioid neurotransmission in a network of regions implicated in emotion, stress regulation, and the

  4. Cinobufagin Modulates Human Innate Immune Responses and Triggers Antibacterial Activity

    PubMed Central

    Xie, Shanshan; Spelmink, Laura; Codemo, Mario; Subramanian, Karthik; Pütsep, Katrin

    2016-01-01

    The traditional Chinese medicine Chan-Su is widely used for treatment of cancer and cardiovascular diseases, but also as a remedy for infections such as furunculosis, tonsillitis and acute pharyngitis. The clinical use of Chan-Su suggests that it has anti-infective effects, however, the mechanism of action is incompletely understood. In particular, the effect on the human immune system is poorly defined. Here, we describe previously unrecognized immunomodulatory activities of cinobufagin (CBG), a major bioactive component of Chan-Su. Using human monocyte-derived dendritic cells (DCs), we show that LPS-induced maturation and production of a number of cytokines was potently inhibited by CBG, which also had a pro-apoptotic effect, associated with activation of caspase-3. Interestingly, CBG triggered caspase-1 activation and significantly enhanced IL-1β production in LPS-stimulated cells. Finally, we demonstrate that CBG upregulates gene expression of the antimicrobial peptides (AMPs) hBD-2 and hBD-3 in DCs, and induces secretion of HNP1-3 and hCAP-18/LL-37 from neutrophils, potentiating neutrophil antibacterial activity. Taken together, our data indicate that CBG modulates the inflammatory phenotype of DCs in response to LPS, and triggers an antibacterial innate immune response, thus proposing possible mechanisms for the clinical effects of Chan-Su in anti-infective therapy. PMID:27529866

  5. Cinobufagin Modulates Human Innate Immune Responses and Triggers Antibacterial Activity.

    PubMed

    Xie, Shanshan; Spelmink, Laura; Codemo, Mario; Subramanian, Karthik; Pütsep, Katrin; Henriques-Normark, Birgitta; Olliver, Marie

    2016-01-01

    The traditional Chinese medicine Chan-Su is widely used for treatment of cancer and cardiovascular diseases, but also as a remedy for infections such as furunculosis, tonsillitis and acute pharyngitis. The clinical use of Chan-Su suggests that it has anti-infective effects, however, the mechanism of action is incompletely understood. In particular, the effect on the human immune system is poorly defined. Here, we describe previously unrecognized immunomodulatory activities of cinobufagin (CBG), a major bioactive component of Chan-Su. Using human monocyte-derived dendritic cells (DCs), we show that LPS-induced maturation and production of a number of cytokines was potently inhibited by CBG, which also had a pro-apoptotic effect, associated with activation of caspase-3. Interestingly, CBG triggered caspase-1 activation and significantly enhanced IL-1β production in LPS-stimulated cells. Finally, we demonstrate that CBG upregulates gene expression of the antimicrobial peptides (AMPs) hBD-2 and hBD-3 in DCs, and induces secretion of HNP1-3 and hCAP-18/LL-37 from neutrophils, potentiating neutrophil antibacterial activity. Taken together, our data indicate that CBG modulates the inflammatory phenotype of DCs in response to LPS, and triggers an antibacterial innate immune response, thus proposing possible mechanisms for the clinical effects of Chan-Su in anti-infective therapy. PMID:27529866

  6. Platelet activation and apoptosis modulate monocyte inflammatory responses in dengue

    PubMed Central

    Hottz, Eugenio D.; Medeiros-de-Moraes, Isabel M.; Vieira-de-Abreu, Adriana; de Assis, Edson F.; Vals-de-Souza, Rogério; Castro-Faria-Neto, Hugo C.; Weyrich, Andrew S.; Zimmerman, Guy A.; Bozza, Fernando A.; Bozza, Patrícia T.

    2014-01-01

    Background Dengue is the most prevalent human arbovirus disease in the world. Dengue infection has a large spectrum of clinical manifestations from self-limited febrile illness to severe syndromes accompanied by bleeding and shock. Thrombocytopenia and vascular leak with altered cytokine profiles in plasma are features of severe dengue. Although monocytes have been recognized as important sources of cytokines in dengue, the contributions of platelet-monocyte interactions to inflammatory responses in dengue have not been addressed. Patients/Methods Patients with dengue were investigated for platelet-monocyte aggregate formation and markers of monocyte activation. Platelet-induced cytokine responses by monocytes and underlying mechanisms were also investigated in vitro. Results We observed increased levels of platelet-monocyte aggregates in blood samples from patients with dengue, especially patients with thrombocytopenia and increased vascular permeability. Moreover, the exposure of monocytes from healthy volunteers to platelets from patients with dengue induced the secretion of the cytokines IL-1β, IL-8, IL-10 and MCP-1, while the exposure to platelets from healthy volunteers only induced the secretion of MCP-1. In addition to the well-established modulation of monocyte cytokine responses by activated platelets through P-selectin binding, we found that interaction of monocytes with apoptotic platelets mediate IL-10 secretion through phosphatidylserine recognition in platelet-monocyte aggregates. Moreover, IL-10 secretion required platelet-monocyte contact but not phagocytosis. Conclusions Together, our results demonstrate that activated and apoptotic platelets aggregate with monocytes during dengue infection and signal specific cytokine responses that may contribute to the pathogenesis of dengue. PMID:25015827

  7. Lysyl oxidase activity regulates oncogenic stress response and tumorigenesis.

    PubMed

    Wiel, C; Augert, A; Vincent, D F; Gitenay, D; Vindrieux, D; Le Calvé, B; Arfi, V; Lallet-Daher, H; Reynaud, C; Treilleux, I; Bartholin, L; Lelievre, E; Bernard, D

    2013-01-01

    Cellular senescence, a stable proliferation arrest, is induced in response to various stresses. Oncogenic stress-induced senescence (OIS) results in blocked proliferation and constitutes a fail-safe program counteracting tumorigenesis. The events that enable a tumor in a benign senescent state to escape from OIS and become malignant are largely unknown. We show that lysyl oxidase activity contributes to the decision to maintain senescence. Indeed, in human epithelial cell the constitutive expression of the LOX or LOXL2 protein favored OIS escape, whereas inhibition of lysyl oxidase activity was found to stabilize OIS. The relevance of these in vitro observations is supported by in vivo findings: in a transgenic mouse model of aggressive pancreatic ductal adenocarcinoma (PDAC), increasing lysyl oxidase activity accelerates senescence escape, whereas inhibition of lysyl oxidase activity was found to stabilize senescence, delay tumorigenesis, and increase survival. Mechanistically, we show that lysyl oxidase activity favors the escape of senescence by regulating the focal-adhesion kinase. Altogether, our results demonstrate that lysyl oxidase activity participates in primary tumor growth by directly impacting the senescence stability. PMID:24113189

  8. The unfolded protein response selectively targets active smoothened mutants.

    PubMed

    Marada, Suresh; Stewart, Daniel P; Bodeen, William J; Han, Young-Goo; Ogden, Stacey K

    2013-06-01

    The Hedgehog signaling pathway, an essential regulator of developmental patterning, has been implicated in playing causative and survival roles in a range of human cancers. The signal-transducing component of the pathway, Smoothened, has revealed itself to be an efficacious therapeutic target in combating oncogenic signaling. However, therapeutic challenges remain in cases where tumors acquire resistance to Smoothened antagonists, and also in cases where signaling is driven by active Smoothened mutants that exhibit reduced sensitivity to these compounds. We previously demonstrated that active Smoothened mutants are subjected to prolonged endoplasmic reticulum (ER) retention, likely due to their mutations triggering conformation shifts that are detected by ER quality control. We attempted to exploit this biology and demonstrate that deregulated Hedgehog signaling driven by active Smoothened mutants is specifically attenuated by ER stressors that induce the unfolded protein response (UPR). Upon UPR induction, active Smoothened mutants are targeted by ER-associated degradation, resulting in attenuation of inappropriate pathway activity. Accordingly, we found that the UPR agonist thapsigargin attenuated mutant Smoothened-induced phenotypes in vivo in Drosophila melanogaster. Wild-type Smoothened and physiological Hedgehog patterning were not affected, suggesting that UPR modulation may provide a novel therapeutic window to be evaluated for targeting active Smoothened mutants in disease. PMID:23572559

  9. Identification of a novel interleukin-6 response element containing an Ets-binding site and a CRE-like site in the junB promoter.

    PubMed Central

    Nakajima, K; Kusafuka, T; Takeda, T; Fujitani, Y; Nakae, K; Hirano, T

    1993-01-01

    Interleukin-6 (IL-6) activation of the immediate-early gene junB has been shown to require both a tyrosine kinase and an unknown 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7)-sensitive pathway. Here we report the identification and characterization of an IL-6 immediate-early response element in the junB promoter (designated JRE-IL6) in HepG2 cells. The JRE-IL6 element, located at -149 to -124, contains two DNA motifs, an Ets-binding site (EBS) (CAGGAAGC) and a CRE-like site (TGACGCGA). Functional studies using variously mutated JRE-IL6 elements showed that both motifs were necessary and sufficient for IL-6 response of the promoter. The EBS of the JRE-IL6 element (JEBS) appears to bind a protein in the Ets family or a related protein which could also form a major complex with the EBSs of the murine sarcoma virus long terminal repeat or human T-cell leukemia virus type 1 long terminal repeat. The CRE-like site appears to weakly bind multiple CREB-ATF family proteins. Despite the similarity in the structure between the JRE-IL6 element and the polyomavirus enhancer PyPEA3, composed of an EBS and an AP1-binding site and known to be activated by a variety of oncogene signals, JRE-IL6 could not be activated by activated Ha-Ras, Raf-1, or 12-O-tetradecanoylphorbol-13-acetate. We show that IL-6 activates JRE-IL6 through an H7-sensitive pathway that does not involve protein kinase C, cyclic AMP-dependent kinase, Ca(2+)- or calmodulin-dependent kinases, Ras, Raf-1, or NF-IL6 (C/EBP beta). The combination of JEBS and the CRE-like site appears to form the basis for the selective and efficient response of JRE-IL6 to IL-6 signals, but not to signals generated by activated Ha-Ras, Raf-1, or protein kinase C. Images PMID:8386318

  10. Adrenalectomy mediated alterations in adrenergic activation of adenylate cyclase in rat liver

    SciTech Connect

    El-Refai, M.; Chan, T.

    1986-05-01

    Adrenalectomy caused a large increase in the number of ..beta..-adrenergic binding sites on liver plasma membranes as measured by /sup 125/I-iodocyanopindolol (22 and 102 fmol/mg protein for control and adrenalectomized (ADX) rats). Concomitantly an increase in the number of binding sites for /sup 3/H-yohimbine was also observed (104 and 175 fmol/mg protein for control and adx membranes). Epinephrine-stimulated increase in cyclic AMP accumulation in isolated hepatocytes were greater in cells from ADX rats. This increase in ..beta..-adrenergic mediated action was much less than what may be expected as a result of the increase in the ..beta..-adrenergic binding in ADX membranes. In addition phenoxybenzamine (10 ..mu..M) further augmented this action of epinephrine in both control and ADX cells. To test the hypothesis that the increase in the number of the inhibitory ..cap alpha../sub 2/-adrenergic receptors in adrenalectomy is responsible for the muted ..beta..-adrenergic response, the authors injected rats with pertussis toxin (PT). This treatment may cause the in vivo ribosylation of the inhibitory binding protein (Ni). Adenylate cyclase (AC) activity in liver plasma membranes prepared from treated and untreated animals was measured. In contrast with control rats, treatment of ADX rats with PT resulted in a significant increase in the basal activity of AC (5.5 and 7.7 pmol/mg protein/min for untreated and treated rats respectively). Isoproterenol (10 ..mu..M), caused AC activity to increase to 6.5 and 8.4 pmol/mg protein/min for membranes obtained from ADX untreated and ADX treated rats respectively. The ..cap alpha..-adrenergic antagonists had no significant effect on the ..beta..-adrenergic-mediated activation of AC in liver plasma membranes from PT treated control and ADX rats. The authors conclude that the ..beta..-adrenergic activation of AC is attenuated by Ni protein both directly and as a result of activation of ..cap alpha..-ad