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Sample records for 8-bromo cyclic amp

  1. Induction of differentiation in v-Ha-ras-transformed MDCK cells by prostaglandin E2 and 8-bromo-cyclic AMP is associated with a decrease in steady-state level of inositol 1,4,5-trisphosphate.

    PubMed Central

    Wu, Y Y; Lin, M C

    1990-01-01

    We used Ha-ras-transformed Madin-Darby canine kidney (MDCK) cells as a model to study possible signal transduction mechanisms underlying the induction of glucagon responsiveness by the differentiation inducers prostaglandin E2 (PGE2) and 8-bromo-cyclic (8-Br-cAMP) AMP and the inhibition of induction by phorbol ester or a serum factor. The steady-state level of inositol 1,4,5-trisphosphate (IP3) was higher in Ha-ras-transformed MDCK cells than in parental MDCK cells. In contrast, the steady-state level of intracellular cAMP of transformed cells was similar to that of normal cells. PGE2 and 8-Br-cAMP increased cAMP content but decreased IP3 levels in a concentration-dependent fashion after 5 days of treatment. We examined the time course for effects of PGE2 and 8-Br-cAMP and found that there was a lag period of 8 to 16 h between elevation of cAMP after the addition of 8-Br-cAMP or PGE2 and the decrease of IP3 levels. Another lag period of 2 days existed before the induction of differentiation. Both the reduction of IP3 levels and the induction of glucagon responsiveness were blocked by phorbol-12-myristate-13-acetate or serum, suggesting that a decrease in the IP3 level might be causally involved in induction of differentiation in transformed MDCK cells. However, induction of differentiation was not due to changes in the expression or guanine nucleotide-binding properties of p21 protein. It is likely that cAMP has a direct regulatory effect on the phospholipid signaling pathway. We conclude that perturbation of the inositol phosphate signaling pathway may be responsible for the induction of differentiation by PGE2 and 8-Br-cAMP in transformed MDCK cells. Images PMID:2152966

  2. 8-Bromo-cyclic inosine diphosphoribose: towards a selective cyclic ADP-ribose agonist

    PubMed Central

    Kirchberger, Tanja; Moreau, Christelle; Wagner, Gerd K.; Fliegert, Ralf; Siebrands, Cornelia C.; Nebel, Merle; Schmid, Frederike; Harneit, Angelika; Odoardi, Francesca; Flügel, Alexander; Potter, Barry V. L.; Guse, Andreas H.

    2009-01-01

    cADPR (cyclic ADP-ribose) is a universal Ca2+ mobilizing second messenger. In T-cells cADPR is involved in sustained Ca2+ release and also in Ca2+ entry. Potential mechanisms for the latter include either capacitative Ca2+ entry, secondary to store depletion by cADPR, or direct activation of the non-selective cation channel TRPM2 (transient receptor potential cation channel, subfamily melastatin, member 2). Here we characterize the molecular target of the newly-described membrane-permeant cADPR agonist 8-Br-N1-cIDPR (8-bromo-cyclic IDP-ribose). 8-Br-N1-cIDPR evoked Ca2+ signalling in the human T-lymphoma cell line Jurkat and in primary rat T-lymphocytes. Ca2+ signalling induced by 8-Br-N1-cIDPR consisted of Ca2+ release and Ca2+ entry. Whereas Ca2+ release was sensitive to both the RyR (ryanodine receptor) blocker RuRed (Ruthenium Red) and the cADPR antagonist 8-Br-cADPR (8-bromo-cyclic ADP-ribose), Ca2+ entry was inhibited by the Ca2+ entry blockers Gd3+ (gadolinium ion) and SKF-96365, as well as by 8-Br-cADPR. To unravel a potential role for TRPM2 in sustained Ca2+ entry evoked by 8-Br-N1-cIDPR, TRPM2 was overexpressed in HEK (human embryonic kidney)-293 cells. However, though activation by H2O2 was enhanced dramatically in those cells, Ca2+ signalling induced by 8-Br-N1-cIDPR was almost unaffected. Similarly, direct analysis of TRPM2 currents did not reveal activation or co-activation of TRPM2 by 8-Br-N1-cIDPR. In summary, the sensitivity to the Ca2+ entry blockers Gd3+ and SKF-96365 is in favour of the concept of capacitative Ca2+ entry, secondary to store depletion by 8-Br-N1-cIDPR. Taken together, 8-Br-N1-cIDPR appears to be the first cADPR agonist affecting Ca2+ release and secondary Ca2+ entry, but without effect on TRPM2. PMID:19492987

  3. Effects of 8-bromo cyclic GMP and verapamil on depolarization-evoked Ca2+ signal and contraction in rat aorta.

    PubMed Central

    Salomone, S; Morel, N; Godfraind, T

    1995-01-01

    1. The pharmacological action of NO donors is usually attributed to a cellular rise in guanosine 3':5'-cyclic monophosphate (cyclic GMP), but this hypothesis is based only on indirect evidence. Therefore, we have studied the effects of cyclic GMP on Ca2+ movements and contraction in rat isolated endothelium-denuded aorta stimulated by KCl depolarizing solution using the permeant analogue 8-bromo cyclic GMP (BrcGMP). Isometric contraction and fura-2 Ca2+ signals were measured simultaneously in preparations treated with BrcGMP and with verapamil. The activation of calcium channels was estimated by measuring the quenching rate of the intracellular fura-2 signal by Mn2+ and by the depolarization-dependent influx of 45Ca2+. 2. Stimulation with 67 mM KCl-solution evoked an increase in cytosolic Ca2+ concentration ([Ca2+]cyt) and a contractile response which were inhibited by pretreatment with verapamil (0.1 microM) or BrcGMP (0.1-1 mM). However, the inhibition of the fura-2 Ca2+ signal was significantly higher with verapamil than with BrcGMP, whereas the contraction was inhibited to a similar extent. 3. When preparations were exposed to K(+)-depolarizing solution in which the calcium concentration was cumulatively increased, the related increase in fura-2 Ca2+ signal was barely affected by BrcGMP, whereas the contractile tension was strongly and significantly inhibited. 4. Cellular Ca2+ changes were also estimated with 45Ca2+. 45Ca2+ influx in resting preparations was significantly reduced by BrcGMP (0.1 mM) but not by verapamil (0.1 microM); 45Ca2+ influx in KCl-depolarized preparations was reduced by verapamil but was unaffected by BrcGMP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7599942

  4. 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

  5. Modulation of relaxation to levcromakalim by S-nitroso-N-acetylpenicillamine (SNAP) and 8-bromo cyclic GMP in the rat isolated mesenteric artery

    PubMed Central

    White, Richard; Hiley, C Robin

    1998-01-01

    Levcromakalim caused concentration-dependent relaxations of methoxamine-induced tone in both endothelium-denuded and intact vessels. Its potency was reduced by the nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP; 0.1 μM or 1 μM) in both denuded and intact vessels. The maximal relaxation (Rmax) was reduced only in denuded vessels. SNAP was more potent in endothelium-denuded than intact vessels but there were no differences in Rmax. Glibenclamide (10 μM) did not affect relaxation to SNAP in endothelium-denuded or intact vessels. The soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μM) increased the potency and Rmax of levcromakalim in endothelium-intact vessels. ODQ had no effect in denuded vessels. ODQ (10 μM) reduced the vasorelaxant potency of SNAP in both intact and endothelium-denuded vessels by 190-fold and 620-fold, respectively. 8-bromo cyclic GMP (10 or 30 μM) reduced both the potency and Rmax of levcromakalim in de-endothelialized vessels, but had no effect in intact vessels although it reduced both the potency and Rmax of levcromakalim in intact vessels incubated with ODQ (10 μM). In the presence of ODQ (10 μM), SNAP (0.1 μM or 1 μM) reduced the potency of levcromakalim in intact vessels, without altering Rmax, but had no effect in denuded vessels. SNAP (50 μM) reduced both the potency and Rmax of levcromakalim in intact and endothelium-denuded vessels. Therefore, although SNAP causes relaxation principally through generation of cyclic GMP, it can modulate the actions of levcromakalim through mechanisms both dependent on, and independent of, cyclic GMP; the former predominate in endothelium-denuded vessels and the latter in intact vessels. PMID:9720794

  6. Mutants of PC12 cells with altered cyclic AMP responses

    SciTech Connect

    Block, T.; Kon, C.; Breckenridge, B.M.

    1984-10-01

    PCl2 cells, derived from a rat pheochromocytoma, were mutagenized and selected in media containing agents known to elevate intracellular concentrations of cyclic AMP (cAMP). More than 40 clones were isolated by selection with cholera toxin or 2-chloroadenosine or both. The variants that were deficient in accumulating cAMP were obtained by using a protocol in which 1 ..mu..m 8-bromo-cAMP was included in addition to the agonist. Certain of these variants were partially characterized with respect to the site of altered cAMP metabolism. The profiles of adenylate cyclase activity responsiveness of certain variants to guanosine-5'-(BETA,..gamma..-imido) triphosphate and to forskolin resembled those of UNC and cyc phenotypes of S49 lymphoma cells, which are functionally deficient in the GTP-sensitive coupling protein, N/sub s/. Other variants were characterized by increased cyclic nucleotide phosphodiesterase activity at low substrate concentration. Diverse morphological traits were observed among the variants, but it was not possible to assign them to a particular cAMP phenotype. Two revertants of a PCl2 mutant were isolated and observed to have regained a cellular cAMP response to 2-chloroadenosine and to forskolin. It is hoped that these PCl2 mutants will have utility for defining cAMP-mediated functions, including any links to the action of nerve growth factor, in cells derived from the neural crest.

  7. Cyclic AMP modulates electrical signaling in a weakly electric fish.

    PubMed

    McAnelly, L; Silva, A; Zakon, H H

    2003-04-01

    Many species of electric fish show diurnal or socially elicited variation in electric organ discharge amplitude. In Sternopygus macrurus, activation of protein kinase A by 8-bromo-cAMP increases electrocyte sodium current magnitude. To determine whether the behavioral plasticity in electric organ discharge amplitude is controlled by electrocyte biophysical properties, we examined whether the effects of phosphorylation on ion currents in the electric organ translate directly into electric organ discharge changes. We injected the electric organ of restrained fish with 8-bromo-cAMP and monitored the electric organ discharge. The effect of protein kinase A activation on electrocyte action potentials was examined in isolated electric organ using two-electrode current clamp. Electric organ discharge and action potential amplitude and pulse duration increased in response to 8-bromo-cAMP. Pulse and action potential duration both increased by about 25%. However, the increase in electric organ discharge amplitude (approximately 400%) was several-fold greater than the action potential amplitude increase (approximately 40%). Resting membrane resistance decreased in electrocytes exposed to 8-bromo-cAMP. We propose that in the Thevenin equivalent circuit of the electric organ a moderate increase in action potential amplitude combined with a decrease in internal resistance produces a greater voltage drop across the external resistance (the water around the fish), accounting for the large increase in the externally recorded electric organ discharge.

  8. 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

  9. Effects of cyclic AMP on growth and differentiation of rat retinoblastoma-like tumor cells in vitro.

    PubMed

    Nishida, T; Mukai, N; Solish, S P; Pomeroy, M

    1982-02-01

    We examined the effects of cyclic AMP (cAMP) on the growth and differentiation of RAO 188 cells, a cultured cell line derived from a retinoblastoma-like tumor induced in an inbred rat by intravitreous inoculation with human adenovirus serotype 12. After adding cAMP analogs (dibutyryl cAMP and 8-bromo cAMP) and phosphodiesterase inhibitors (theophylline, amino-phylline, and 1-methyl-3-isobutyl xanthine) to the RAO 188 cell culture medium, we measured changes in cell incorporation of the DNA and RNA precursors 14C-thymidine and 3H-uridine, and we observed the morphologic alterations of RAO 188 by phase-contrast and transmission and scanning electron microscopy. Incorporation of the labeled precursors decreased with increased concentrations of cAMP analogs and phosphodiesterase inhibitors. Incorporation of the labeled precursors was inhibited shortly after the addition of dibutyryl cAMP to the culture medium. The effect was maximal at 8 hr and was sustained for up to 48 hr. Reversibility of cAMP effects on incorporation gradually decreased for 10 days; at 10 days these effects were essentially irreversible. Neuritelike processes developed shortly after cAMP analog treatment and formed a network after 24 hr. Transmission electron microscopy disclosed changes in the cell membrane and cytoplasm of cells treated with 8-bromo cAMP and theophylline: perturbation of the cell membrane and the appearance of intercellular junctional devices and microfilaments. The activity of glutamate decarboxylase, which is involved in the biosynthesis of gamma-aminobutyric acid, was increased in treated cells. These results show that cAMP decreases DNA and RNA synthesis and cell proliferation and facilitates morphologic and biochemical differentiation of RAO 188 cells.

  10. Pharmacological elevation of cyclic AMP and transmitter release at the mouse neuromuscular junction.

    PubMed

    Dryden, W F; Singh, Y N; Gordon, T; Lazarenko, G

    1988-03-01

    Intracellular recordings of spontaneous and evoked end-plate potentials have been made at the neuromuscular junction of mouse hemidiaphragms to determine a possible role of cyclic AMP (cAMP) in the release of acetylcholine from presynaptic terminals. Spontaneous release, as determined from the frequency of miniature end-plate potentials, was increased by drugs that inhibit phosphodiesterase: isobutylmethylxanthine (IBMX), SQ 20,009, theophylline, and caffeine; drugs that stimulate adenylate cyclase: forskolin, fluoride, and cholera toxin, and the stable analogue of cAMP: 8-bromo-cAMP but not dibutyryl cAMP. Release increased with time during maintained exposure to the drugs and generally followed a simple exponential time course with time constants ranging from 8 to 17 min at 20 degrees C, except for SQ 20,009 and cholera toxin which required longer exposure times for effect. The order of potency of the phosphodiesterase inhibitors was IBMX = SQ 20,009 greater than theophylline = caffeine. This is consistent with an effect mediated by an increase in cAMP concentrations within the nerve terminal. Evoked release, determined from the quantal content of the end-plate potential, was increased to a lesser extent than spontaneous release. The results are discussed with reference to the possible involvement of second messengers in the release of vesicles from nerve terminals in vertebrate synapses.

  11. Cyclic AMP in cervical mucus.

    PubMed

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

    1981-01-01

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

  12. 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 cAMP

  13. Changes in nephrogenous cyclic AMP excretion and plasma cyclic AMP following treatment of hyperthyroidism.

    PubMed

    Naafs, M A; van der Velden, P C; Fischer, H R; Koorevaar, G; van Duin, S; Hackeng, W H; Schopman, W; Silberbusch, J

    1984-08-01

    Plasma cyclic AMP (PcAMP) concentration and the excretion of cyclic AMP/dl GF were estimated in 11 thyrotoxic patients before and after medical treatment. PcAMP concentrations were significantly higher during hyperthyroidism (2.30 +/- 0.69 vs 1.88 +/- 0.71 nmol/dl; P less than 0.05), and total urinary cyclic AMP (TcAMP) excretion showed no significant changes (3.24 +/- 0.64 vs 3.44 +/- 1.77 nmol/dl GF). Nephrogenous (NcAMP) excretion rose significantly (1.00 +/- 0.82 vs 1.68 +/- 1.31 mmol/dl GF; P less than 0.025). The increase in NcAMP excretion correlated significantly with the decrease in serum T3 levels (r = -0.46; P less than 0.05). Serum iPTH levels showed no significant change. Both the serum Ca, corrected for serum total protein and TmPO4/GFR declined after treatment (respectively 2.44 +/- 0.13 vs 2.33 +/- 0.08 mmol/l; P less than 0.05 and 1.18 +/- 0.29 vs 1.05 +/- 0.22 mmol/l; P less than 0.05). It is concluded that the rise in NcAMP excretion corroborates the concept of increasing parathyroid activity following the treatment of hyperthyroidism. PMID:6206676

  14. 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...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1230 Cyclic AMP test system. (a) Identification. A cyclic AMP test system is a device intended...

  15. Cell cycle regulation of cyclin A gene expression by the cyclic AMP-responsive transcription factors CREB and CREM.

    PubMed Central

    Desdouets, C; Matesic, G; Molina, C A; Foulkes, N S; Sassone-Corsi, P; Brechot, C; Sobczak-Thepot, J

    1995-01-01

    Cyclin A is a pivotal regulatory protein which, in mammalian cells, is involved in the S phase of the cell cycle. Transcription of the human cyclin A gene is cell cycle regulated. We have investigated the role of the cyclic AMP (cAMP)-dependent signalling pathway in this cell cycle-dependent control. In human diploid fibroblasts (Hs 27), induction of cyclin A gene expression at G1/S is stimulated by 8-bromo-cAMP and suppressed by the protein kinase A inhibitor H89, which was found to delay S phase entry. Transfection experiments showed that the cyclin A promoter is inducible by activation of the adenylyl cyclase signalling pathway. Stimulation is mediated predominantly via a cAMP response element (CRE) located at positions -80 to -73 with respect to the transcription initiation site and is able to bind CRE-binding proteins and CRE modulators. Moreover, activation by phosphorylation of the activators CRE-binding proteins and CRE modulator tau and levels of the inducible cAMP early repressor are cell cycle regulated, which is consistent with the pattern of cyclin A inducibility by cAMP during the cell cycle. These results suggest that the CRE is, at least partly, implicated in stimulation of cyclin A transcription at G1/S. PMID:7760825

  16. 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.

  17. 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

  18. 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

  19. Effect of atrial natriuretic factor and 8-bromo cyclic guanosine 3':5'-monophosphate on ( sup 3 H)acetylcholine outflow from myenteric-plexus longitudinal muscle of the guinea pig

    SciTech Connect

    Matusak, O.; Kuchel, O.; Hamet, P. )

    1991-04-01

    We report that atrial natriuretic factor (ANF) inhibits electrically induced cholinergic twitches of longitudinal muscle in whole intestinal segments and myenteric-plexus longitudinal muscle (MPLM) strips from the guinea pig ileum. To elucidate the possible presynaptic mechanism of ANF's action, we studied spontaneous and stimulation-evoked radiolabeled acetylcholine (ACh) outflow from MPLM after incubation with ({sup 3}H)choline. We developed a method of mounting and treating MPLM preparations, which allowed us, at the same time, to record isometric contractions and to determine ({sup 3}H)ACh outflow upon electrical stimulation by a train of three pulses. ANF (5 x 10{sup {minus} 8}M), norepinephrine (2 x 10{sup {minus} 7}) M and 8-bromoguanosine 3':5'-cyclic monophosphate (10{sup {minus} 3} M) in nearly equieffective concentrations caused a similar inhibition of cholinergic twitches. However, ANF had no effect on ({sup 3}H)ACh outflow, whereas norepinephrine was found to suppress ({sup 3}H)ACh outflow and 8-bromoguanosine 3':5'-cGMP to enhanced ({sup 3}H)ACh outflow. ANF (5 x 10{sup {minus} 8} M) caused a 7.0-fold increase of cGMP over control values, predominantly in muscle layers, whereas Escherichia coli heat-stable toxin (12.5 U/ml) elicited a 35-fold increment of cGMP in the extramuscular layer. Thus, ANF is able to elevate cGMP in intestinal smooth muscle and to inhibit cholinergic contractions of MPLM. This inhibition is mimicked by exogenous cGMP and by endogenously generated cyclic nucleotides. We suggest that the depressive action of ANF on cholinergic contractions of MPLM is mediated via its postsynaptic impact implicating elevation of cGMP in smooth muscle.

  20. Detection of cyclic di-AMP using a competitive ELISA with a unique pneumococcal cyclic di-AMP binding protein

    PubMed Central

    Underwood, Adam J.; Zhang, Yang; Metzger, Dennis W.; Bai, Guangchun

    2014-01-01

    Cyclic di-AMP (c-di-AMP) is a signaling molecule that has been shown to play important roles in bacterial physiology and infections. Currently, c-di-AMP detection and quantification relies mostly on the use of high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS). In this study, a competitive enzyme-linked immunosorbent assay (ELISA) for the quantification of c-di-AMP was developed, which utilizes a novel pneumococcal c-di-AMP binding protein (CabP) and a newly commercialized c-di-AMP derivative. With this new method, c-di-AMP concentrations in biological samples can be quickly and accurately quantified. Furthermore, this assay is much more efficient than current methods as it requires less overall cost and training while processing many samples at once. Therefore, this assay can be extensively used in research into c-di-AMP signaling. PMID:25239824

  1. Cyclic AMP, a nonessential regulator of the cell cycle.

    PubMed Central

    Coffino, P; Gray, J W; Tomkins, G M

    1975-01-01

    Flow-microfluorimetric analysis has been carried out on populations of exponentially growing S49 mouse lymphoma cells treated with dibutyryl cyclic AMP. The drug produces a specific concentration-dependent block in the G-1 phase of the cell cycle while other phases of the cycle are not perceptibly altered. The cell cycle of a line of mutant cells lacking the cyclic AMP-dependent protein kinase is not affected by the drug. Since these mutant cells have been shown to maintain a normal cell cycle, even in the presence of high levels of cyclic AMP, periodic fluctuations in the levels of the cyclic nucleotide cannot be required for or determine progression through the cell cycle. PMID:165491

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

    PubMed Central

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

    2016-01-01

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

  3. 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

  4. 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.

  5. Smooth muscle cell expression of type I cyclic GMP-dependent protein kinase is suppressed by continuous exposure to nitrovasodilators, theophylline, cyclic GMP, and cyclic AMP.

    PubMed Central

    Soff, G A; Cornwell, T L; Cundiff, D L; Gately, S; Lincoln, T M

    1997-01-01

    A key component of the nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in smooth muscle cells (SMC) is the type I GMP-dependent protein kinase (PK-G I). Activation of PK-G I mediates the reduction of cytoplasmic calcium concentrations and vasorelaxation. In this manuscript, we demonstrate that continuous exposure of SMC in culture to the nitrovasodilators S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) results in approximately 75% suppression of PK-G I mRNA by 48 h. PK-G I mRNA and protein were also suppressed by continuous exposure to cGMP analogues 8-bromo- and 8-(4-chlorophenylthio) guanosine-3,5-monophosphate or the cAMP analogue dibutyryl cAMP. These results suggest that activation of one or both of the cyclic nucleotide-dependent protein kinases mediates PK-G I mRNA suppression. Using isoform-specific cDNA probes, only the PK-G I alpha was detected in SMC, either at baseline or after suppression, while PK-G I beta was not detected, indicating that isoform switch was not contributing to the gene regulation. Using the transcription inhibitor actinomycin D, the PK-G I mRNA half-life in bovine SMC was observed to be 5 h. The half-life was not affected by the addition of SNAP to actinomycin D, indicating no effect on PK-G I mRNA stability. Nuclear runoff studies indicated a suppression of PK-G I gene transcription by SNAP. PK-G I suppression was also observed in vivo in rats given isosorbide dinitrate in the drinking water, with a dose-dependent suppression of PK-G I protein in the aorta. PK-G I antigen in whole rat lung extract was also suppressed by administration of isosorbide or theophylline in the drinking water. These data may contribute to our understanding of nitrovasodilator resistance, a phenomenon resulting from continuous exposure to nitroglycerin or other nitrovasodilators. PMID:9366573

  6. Cyclic AMP inhibits secretion from electroporated human neutrophils.

    PubMed

    Smolen, J E; Stoehr, S J; Kuczynski, B

    1991-02-01

    It has long been known that intracellular cAMP inhibits and cGMP enhances intact neutrophil function. However, these effects are modest and require relatively high concentrations of the cyclic nucleotides. We decided to re-examine the effects of cyclic nucleotides on Ca2(+)-induced secretion by electroporated cells. This system allowed us to bypass normal cell surface receptor-ligand interactions as well as to directly expose the intracellular space to native cyclic nucleotides. We found that concentrations of cAMP as low as 3 microM inhibited Ca2(+)-induced secretion; 30-300 microM cAMP was maximally inhibitory. cAMP was actually slightly more potent than dibutyryl cAMP, a membrane-permeant derivative. In contrast, cGMP was only slightly stimulatory at 3 microM and modestly inhibitory at 300 microM; dibutyryl cGMP was ineffective. A more detailed investigation of the effects of cAMP showed that inhibition was only obtained in the presence of Mg2+. Half-maximal inhibition by cAMP occurred at 10-30 microM. Inhibition by cAMP was achieved by shifting the Ca2+ dose-response curve for secretion to the right; this was observed for the release of both specific granules (vitamin B12 binding protein) and azurophil granules (B-glucuronidase). We previously showed that ATP could enhance Ca2(+)-induced secretion in the presence of Mg2+, apparently by interacting with a cell surface purine receptor. However, increasing concentrations of ATP could not overcome inhibition by cAMP; this suggested that cAMP acted at some site other than the purine receptor. Inhibition by cAMP was also less apparent in the presence of the protein kinase C agonist phorbol myristate acetate (PMA), suggesting that the cyclic nucleotide did not produce systemic desensitization of the neutrophils. In summary, these results demonstrate that low, physiologically relevant concentrations of cAMP can modulate neutrophil responsiveness. PMID:1846904

  7. Direct activation of cardiac pacemaker channels by intracellular cyclic AMP.

    PubMed

    DiFrancesco, D; Tortora, P

    1991-05-01

    Cyclic AMP acts as a second messenger in the modulation of several ion channels that are typically controlled by a phosphorylation process. In cardiac pacemaker cells, adrenaline and acetylcholine regulate the hyperpolarization-activated current (if), but in opposite ways; this current is involved in the generation and modulation of pacemaker activity. These actions are mediated by cAMP and underlie control of spontaneous rate by neurotransmitters. Whether the cAMP modulation of if is mediated by channel phosphorylation is, however, still unknown. Here we investigate the action of cAMP on if in excised patches of cardiac pacemaker cells and find that cAMP activates if by a mechanism independent of phosphorylation, involving a direct interaction with the channels at their cytoplasmic side. Cyclic AMP activates if by shifting its activation curve to more positive voltages, in agreement with whole-cell results. This is the first evidence of an ion channel whose gating is dually regulated by voltage and direct cAMP binding.

  8. Cyclic nucleotides of cone-dominant retinas. Reduction of cyclic AMP levels by light and by cone degeneration.

    PubMed

    Farber, D B; Souza, D W; Chase, D G; Lolley, R N

    1981-01-01

    Dark-adapted retinas or whole eyes of 13-line ground squirrels (Citellus tridecemlineatus) and western fence lizards (Sceloporus occidentalis) contain higher levels of cyclic AMP than of cyclic GMP. In these cone-dominant retinas, light reduces cyclic AMP content selectively. Freezing of dark- or light-adapted retinas or eyes also reduces cyclic AMP content, with only minimal changes in cyclic GMP levels. In addition, exposure of frozen retinas of dark-adapted ground squirrel to light results in a significant decrease in cyclic AMP content. The destruction of cone visual cells of ground squirrel retina by iodoacetic acid injection decreases the cyclic nucleotide content of the dark-adapted retina. Considering the relative loss of cyclic nucleotides from cone degeneration, we estimate that the content of cyclic AMP in visual cells of ground squirrel retina is about four times greater than that of cyclic GMP. PMID:6256308

  9. 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 §...

  10. 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 §...

  11. 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 §...

  12. 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 §...

  13. Cyclic AMP negatively regulates prodigiosin production by Serratia marcescens.

    PubMed

    Kalivoda, Eric J; Stella, Nicholas A; Aston, Marissa A; Fender, James E; Thompson, Paul P; Kowalski, Regis P; Shanks, Robert M Q

    2010-03-01

    Many Serratia marcescens strains produce the red pigment prodigiosin, which has antimicrobial and anti-tumor properties. Previous reports suggest that cyclic AMP (cAMP) is a positive regulator of prodigiosin production. Supporting this model, the addition of glucose to growth medium inhibited pigment production in rich and minimal media. Unexpectedly, we observed highly elevated levels of prodigiosin production in isogenic strains with mutations in genes involved in cAMP production (cyaA and crr) and in cAMP-dependent transcriptional signaling (crp). Multicopy expression of the Escherichia coli cAMP-phosphodiesterase gene, cpdA, also conferred a striking increase in prodigiosin production. Exogenous cAMP decreased both pigment production and pigA-lacZ transcription in the wild-type (WT) strain, and pigA-lacZ transcription was significantly increased in a crp mutant relative to WT. Suppressor and epistasis analysis indicate that the hyperpigment phenotype was dependent upon pigment biosynthetic genes (pigA, pigB, pigC, pigD and pigM). These experiments establish cAMP as a negative regulator of prodigiosin production in S. marcescens.

  14. Cyclic AMP regulation of early gene expression in Dictyostelium discoideum: mediation via the cell surface cyclic AMP receptor.

    PubMed Central

    Mann, S K; Firtel, R A

    1987-01-01

    We examined two sets of genes expressed early in the developmental cycle of Dictyostelium discoideum that appear to be regulated by cyclic AMP (cAMP). The transcripts of both sets of genes were not detectable in vegetative cells. During normal development on filter pads, RNA complementary to these genes could be detected at about 2 h, peaked around 6 to 8 h, and decreased gradually thereafter. Expression of these genes upon starvation in shaking culture was stimulated by pulsing the cells with nanomolar levels of cAMP, a condition that mimics the in vivo pulsing during normal aggregation. Expression was inhibited by caffeine or by continuous levels of cAMP, a condition found later in development when in vivo expression of these genes decreased. The inhibition of caffeine could be overcome by pulsing cells with cAMP. These results suggest that the expression is mediated via the cell surface cAMP receptor, but does not require a rise in intracellular cAMP. mRNA from a gene of the second class was induced upon starvation, peaked by 2.5 h of development, and then declined. In contrast to the other genes, its expression was maintained by continuous levels of cAMP and repressed by cAMP pulses. These and other results on a number of classes of developmentally regulated genes indicates that changing levels of cAMP, acting via the cell surface cAMP receptor, are involved in controlling these groups of genes. We also examined the structure and partial sequence of the cAMP pulse-induced genes. The two tandemly duplicated M3 genes were almost continuously homologous over the sequenced portion of the protein-coding region except for a region near the N-terminal end. The two M3 genes had regions of homology in the 5' flanking sequence and showed slight homology to the same regions in gene D2, another cAMP pulse-induced gene. D2 showed extremely significant homology over its entire sequenced length to an acetylcholinesterase. The results presented here and by others suggest that

  15. Bacterial Cyclic AMP-Phosphodiesterase Activity Coordinates Biofilm Formation

    PubMed Central

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

    2013-01-01

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

  16. Cyclic Di-AMP Homeostasis in Bacillus subtilis

    PubMed Central

    Mehne, Felix M. P.; Gunka, Katrin; Eilers, Hinnerk; Herzberg, Christina; Kaever, Volkhard; Stülke, Jörg

    2013-01-01

    The genome of the Gram-positive soil bacterium Bacillus subtilis encodes three potential diadenylate cyclases that may synthesize the signaling nucleotide cyclic di-AMP (c-di-AMP). These enzymes are expressed under different conditions in different cell compartments, and they localize to distinct positions in the cell. Here we demonstrate the diadenylate cyclase activity of the so far uncharacterized enzymes CdaA (previously known as YbbP) and CdaS (YojJ). Our work confirms that c-di-AMP is essential for the growth of B. subtilis and shows that an excess of the molecule is also harmful for the bacteria. Several lines of evidence suggest that the diadenylate cyclase CdaA is part of the conserved essential cda-glm module involved in cell wall metabolism. In contrast, the CdaS enzyme seems to provide c-di-AMP for spores. Accumulation of large amounts of c-di-AMP impairs the growth of B. subtilis and results in the formation of aberrant curly cells. This phenotype can be partially suppressed by elevated concentrations of magnesium. These observations suggest that c-di-AMP interferes with the peptidoglycan synthesis machinery. The activity of the diadenylate cyclases is controlled by distinct molecular mechanisms. CdaA is stimulated by a regulatory interaction with the CdaR (YbbR) protein. In contrast, the activity of CdaS seems to be intrinsically restricted, and a single amino acid substitution is sufficient to drastically increase the activity of the enzyme. Taken together, our results support the idea of an important role for c-di-AMP in B. subtilis and suggest that the levels of the nucleotide have to be tightly controlled. PMID:23192352

  17. Intraoperative urinary cyclic AMP monitoring in primary hyperparathyroidism.

    PubMed Central

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

    1993-01-01

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

  18. Epidermal chalone and cyclic AMP: an in vivo study.

    PubMed

    Elgjo, K

    1975-01-01

    Water extracts of skin contain two factors that inhibit epidermal cell proliferation: one substance inhibits epidermal cells in the G2 phase (the epidermal G2 inhibitor), and another inhibits the transit of cells from the G1 phase into the S phase (the epidermal G1 inhibitor). Pretreatment of mice with a beta-receptor antagonist (propranolol) abolished the activity of the G2 inhibitor but not that of the G1 inhibitor. After pretreatment with both propranolol and a phosphodiesterase inhibitor (caffine)the G2 inhibitor had full effect. Cafine alone had a moderately inhibitory effect on epidermal G2 cells and enhanced the depressing effect of the G1 inhibitor on epidermal DNA synthesis. AMP level in epidermis to be active. Cyclic AMP is probably also involved in the regulation of the rate of transit of epidermal G1 cells into the S phase but the epidermal cyclic AMP level seems not to be so critical for the efficacy of the epidermal G2 inhibitor in epidermal cell differentiation. PMID:162919

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

    PubMed Central

    Škrnjug, Ivana

    2014-01-01

    The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP) after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice. A characteristic of the cGAMP-induced immune response is the slightly reduced induction of interleukin-17 as a hallmark of Th17 activity – a distinct feature that is not observed with other cyclic di-nucleotide adjuvants. We further characterize the innate immune stimulation activity in vitro on murine bone marrow-derived dendritic cells and human dendritic cells. The observed results suggest the consideration of cGAMP as a candidate mucosal adjuvant for human vaccines. PMID:25295996

  20. 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.

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

    PubMed Central

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

    2015-01-01

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

  2. Cyclic AMP signalling pathways in the regulation of uterine relaxation

    PubMed Central

    Yuan, Wei; López Bernal, Andrés

    2007-01-01

    Studying the mechanism(s) of uterine relaxation is important and will be helpful in the prevention of obstetric difficulties such as preterm labour, which remains a major cause of perinatal mortality and morbidity. Multiple signalling pathways regulate the balance between maintaining relative uterine quiescence during gestation, and the transition to the contractile state at the onset of parturition. Elevation of intracellular cyclic AMP promotes myometrial relaxation, and thus quiescence, via effects on multiple intracellular targets including calcium channels, potassium channels and myosin light chain kinase. A complete understanding of cAMP regulatory pathways (synthesis and hydrolysis) would assist in the development of better tocolytics to delay or inhibit preterm labour. Here we review the enzymes involved in cAMP homoeostasis (adenylyl cyclases and phosphodiesterases) and possible myometrial substrates for the cAMP dependent protein kinase. We must emphasise the need to identify novel pharmacological targets in human pregnant myometrium to achieve safe and selective uterine relaxation when this is indicated in preterm labour or other obstetric complications. PMID:17570154

  3. Fluorescence study of Escherichia coli cyclic AMP receptor protein.

    PubMed

    Wasylewski, M; Małecki, J; Wasylewski, Z

    1995-07-01

    Time-resolved, steady-state fluorescence and fluorescence-detected circular dichroism (FDCD) have been used to resolve the fluorescence contributions of the two tryptophan residues, Trp-13 and Trp-85, in the cyclic AMP receptor protein (CRP). The iodide and acrylamide quenching data show that in CRP one tryptophan residue, Trp-85, is buried within the protein matrix and the other, Trp-13, is moderately exposed on the surface of the protein. Fluorescence-quenching-resolved spectra show that Trp-13 has emission at about 350 nm and contributes 76-83% to the total fluorescence emission. The Trp-85, unquenchable by iodide and acrylamide, has the fluorescence emission at about 337 nm. The time-resolved fluorescence measurements show that Trp-13 has a longer fluorescence decay time. The Trp-85 exhibits a shorter fluorescence decay time. In the CRP-cAMP complex the Trp-85, previously buried in the apoprotein becomes totally exposed to the iodide and acrylamide quenchers. The FDCD spectra indicate that in the CRP-cAMP complex Trp-85 remains in the same environment as in the protein alone. It has been proposed that the binding of cAMP to CRP is accompanied by a hinge reorientation of two protein domains. This allows for penetration of the quencher molecules into the Trp-85 residue previously buried in the protein matrix. PMID:8590598

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. Prostaglandins and muscarinic agonists induce cyclic AMP attenuation by two distinct mechanisms in the pregnant-rat myometrium. Interaction between cyclic AMP and Ca2+ signals.

    PubMed Central

    Goureau, O; Tanfin, Z; Harbon, S

    1990-01-01

    In pregnant-rat myometrium (day 21 of gestation), isoprenaline-induced cyclic AMP accumulation, resulting from receptor-mediated activation of adenylate cyclase, was negatively regulated by prostaglandins [PGE2, PGF2 alpha; EC50 (concn. giving 50% of maximal response) = 2 nM] and by the muscarinic agonist carbachol (EC50 = 2 microM). PG-induced inhibition was prevented by pertussis-toxin treatment, supporting the idea that it was mediated by the inhibitory G-protein Gi through the inhibitory pathway of the adenylate cyclase. Both isoprenaline-induced stimulation and PG-evoked inhibition of cyclic AMP were insensitive to Ca2+ depletion. By contrast, carbachol-evoked attenuation of cyclic AMP accumulation was dependent on Ca2+ and was insensitive to pertussis toxin. The inhibitory effect of carbachol was mimicked by ionomycin. Indirect evidence was thus provided for the enhancement of cyclic AMP degradation by a Ca2(+)-dependent phosphodiesterase activity in the muscarinic-mediated effect. The attenuation of cyclic AMP elicited by carbachol coincided with carbachol-stimulated inositol phosphate (InsP3, InsP2 and InsP) generation, which displayed an almost identical EC50 (3 microM) and was similarly unaffected by pertussis toxin. Both carbachol effects were reproduced by oxotremorine, whereas pilocarpine (a partial muscarinic agonist) failed to induce any decrease in cyclic AMP accumulation and concurrently was unable to stimulate the generation of inositol phosphates. These data support our proposal for a carbachol-mediated enhancement of a Ca2(+)-dependent phosphodiesterase activity, compatible with the rises in Ca2+ associated with muscarinic-induced increased generation of inositol phosphates. They further illustrate that a cross-talk between the two major transmembrane signalling systems contributed to an ultimate decrease in cyclic AMP in the pregnant-rat myometrium near term. PMID:1700899

  10. 3':5'-cyclic AMP and hormonal control of puparium formation in the fleshfly Sarcophaga bullata.

    PubMed

    Fraenkel, G; Blechl, A; Blechl, J; Herman, P; Seligman, M I

    1977-05-01

    Injection of 3':5'-cyclic AMP (cAMP) into larvae of the fly Sarcophaga bullata 3-4 hr before the beginning of puparium formation (red-spiracle stage) greatly accelerates the onset of tanning without affecting initiation of puparium formation (anterior retraction). Accelerated tanning resembles real tanning in two important respects: the solubility of cuticular proteins becomes reduced and [U-14C]tyrosine is incorporated into the cuticle. Of a number of cAMP analogues tested, 3':5'- cyclic GMP, 2':3'-cyclic AMP, and 5'-AMP were inactive, dibutyryl-3':5'-cAMP had only slight activity, and cyclic IMP and deoxy-3':5'-cAMP showed some activity. Theophylline enhanced the effect of small doses of cAMP or of blood, diluted 1:8, active in the puparium tanning factor. Injection of dopa, dopamine, acetyldopamine, or epinephrine, but not of tyrosine, had an accelerating effect similar to that of cAMP. The tanning-inhibiting effect of DL-alpha-methyl-alpha-hydrazino-beta-(3,4-dihydroxyphenyl)propionic acid monohydrate is reversed by dopamine or epinephrine, but not by tyrosine, dopa, or cAMP. Evidence is presented to indicate that the responses to cAMP are not artifacts but reflect actual biochemical events during tanning.

  11. Resonance Raman characterization of different forms of ground-state 8-bromo-7-hydroxyquinoline caged acetate in aqueous solutions.

    PubMed

    An, Hui-Ying; Ma, Chensheng; Nganga, Jameil L; Zhu, Yue; Dore, Timothy M; Phillips, David Lee

    2009-03-26

    The 8-bromo-7-hydroxyquinolinyl group (BHQ) is a derivative of 7-hydroxyquinoline (7-HQ) and BHQ molecules coexisting as different forms in aqueous solution. Absorption and resonance Raman spectroscopic methods were used to examine 8-bromo-7-hydroxyquinoline protected acetate (BHQ-OAc) in acetonitrile (MeCN), H(2)O/MeCN (60:40, v/v, pH 6 approximately 7), and NaOH-H(2)O/MeCN (60:40, v/v, pH 11 approximately 12) to obtain a better characterization of the forms of the ground-state species of BHQ-OAc in aqueous solutions and to examine their properties. The absorption spectra of BHQ-OAc in water show no absorption bands of the tautomeric species unlike the strong band at about 400 nm observed for the tautomeric form in 7-HQ aqueous solution. The resonance Raman spectra in conjunction with Raman spectra predicted from density functional theory (DFT) calculations reveal the observation of a double Raman band system characteristic of the neutral form (the nominal C=C ring stretching, C-N stretching, and O-H bending modes at 1564 and 1607 cm(-1)) and a single Raman band diagnostic of the enol-deprotonated anionic form (the nominal C=C ring, C-N, and C-O(-) stretching modes in the 1593 cm(-1) region). These results suggest that the neutral form of BHQ-OAc is the major species in neutral aqueous solution. There is a modest increase in the amount of the anionic form and a big decrease in the amount of the tautomeric form of the molecules for BHQ-OAc compared to 7-HQ in neutral aqueous solution. The presence of the 8-bromo group and/or competitive hydrogen bonding that hinder the formation and transfer process of a BHQ-OAc-water cyclic complex may be responsible for this large substituent effect. PMID:19296708

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

    PubMed Central

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

    1979-01-01

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

  13. Glucose-induced hyperaccumulation of cyclic AMP and defective glucose repression in yeast strains with reduced activity of cyclic AMP-dependent protein kinase.

    PubMed

    Mbonyi, K; van Aelst, L; Argüelles, J C; Jans, A W; Thevelein, J M

    1990-09-01

    Addition of glucose or related fermentable sugars to derepressed cells of the yeast Saccharomyces cerevisiae triggers a RAS-mediated cyclic AMP (cAMP) signal that induces a protein phosphorylation cascade. In yeast mutants (tpk1w1, tpk2w1, and tpk3w1) containing reduced activity of cAMP-dependent protein kinase, fermentable sugars, as opposed to nonfermentable carbon sources, induced a permanent hyperaccumulation of cAMP. This finding confirms previous conclusions that fermentable sugars are specific stimulators of cAMP synthesis in yeast cells. Despite the huge cAMP levels present in these mutants, deletion of the gene (BCY1) coding for the regulatory subunit of cAMP-dependent protein kinase severely reduced hyperaccumulation of cAMP. Glucose-induced hyperaccumulation of cAMP was also observed in exponential-phase glucose-grown cells of the tpklw1 and tpk2w1 strains but not the tpk3w1 strain even though addition of glucose to glucose-repressed wild-type cells did not induce a cAMP signal. Investigation of mitochondrial respiration by in vivo 31P nuclear magnetic resonance spectroscopy showed the tpk1w1 and tpk2w1 strains, to be defective in glucose repression. These results are consistent with the idea that the signal transmission pathway from glucose to adenyl cyclase contains a glucose-repressible protein. They also show that a certain level of cAMP-dependent protein phosphorylation is required for glucose repression. Investigation of the glucose-induced cAMP signal and glucose-induced activation of trehalase in derepressed cells of strains containing only one of the wild-type TPK genes indicates that the transient nature of the cAMP signal is due to feedback inhibition by cAMP-dependent protein kinase.

  14. The plasma cyclic AMP response to catecholamines as potentiated by phentolamine in rats.

    PubMed

    Kunitada, S; Ui, M

    1978-05-15

    Norepinephrine failed to increase plasma cyclic AMP when injected alone into fasted rats, in contrast with sharp increases elicited by isoproterenol, epinephrine or tyramine. In rats pretreated with 6-hydroxydopamine or cocain, however, there was significant increase in plasma cyclic AMP after norepinephrine injection, suggesting that the rapid neuronal catecholamine uptake was at least partly responsible for the lack of norepinephrine action. Phentolamine was very effective in enhancing the epinephrine-, norepinephrine- or tyramine-induced increase in plasma cyclic AMP but without effect on the isoproterenol-induced increase. Blockade of postsynaptic alpha-adrenoceptors, rather than of presynaptic receptors, is likely to be involved in the phentolamine potentiation, since it was even observed in rats treated with 6-hydroxydopamine or cocaine. A discussion is presented regarding the mechanism by which cyclic AMP generation is influenced by the alpha- and beta-adrenoceptor interaction on effector cell membranes.

  15. 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.

  16. Trehalase activity and cyclic AMP content during early development of Mucor rouxii spores.

    PubMed Central

    Dewerchin, M A; Van Laere, A J

    1984-01-01

    Incubation of Mucor rouxii sporangiospores in complex medium under aerobic conditions resulted in a transient 20-fold increase in trehalase activity. Maximum activity was reached after 15 min. Simultaneously, the cyclic AMP (cAMP) content increased approximately eightfold, reaching a maximum within 10 min. Increases in trehalase activity and cAMP content were also observed under anaerobic conditions (CO2). The extent of trehalase activation and the changes in cAMP content, during both aerobic and anaerobic incubation, varied with the medium used. Trehalase was activated in vitro by a cAMP- and ATP-dependent process. An even faster activation was obtained when cAMP was replaced by the catalytic subunit of beef heart protein kinase. The coincidence of, and the correlation between, increased cAMP contents and trehalase activities support the involvement of a cAMP-dependent phosphorylation in the in vivo regulation of trehalase activity. PMID:6327611

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

    PubMed Central

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

    1994-01-01

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

  18. 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

  19. Cyclic Amp-Dependent Resuscitation of Dormant Mycobacteria by Exogenous Free Fatty Acids

    PubMed Central

    Shleeva, Margarita; Goncharenko, Anna; Kudykina, Yuliya; Young, Danielle; Young, Michael; Kaprelyants, Arseny

    2013-01-01

    One third of the world population carries a latent tuberculosis (TB) infection, which may reactivate leading to active disease. Although TB latency has been known for many years it remains poorly understood. In particular, substances of host origin, which may induce the resuscitation of dormant mycobacteria, have not yet been described. In vitro models of dormant (“non-culturable”) cells of Mycobacterium smegmatis (mc2155) and Mycobacterium tuberculosis H37Rv were used. We found that the resuscitation of dormant M. smegmatis and M. tuberculosis cells in liquid medium was stimulated by adding free unsaturated fatty acids (FA), including arachidonic acid, at concentrations of 1.6–10 µM. FA addition enhanced cAMP levels in reactivating M. smegmatis cells and exogenously added cAMP (3–10 mM) or dibutyryl-cAMP (0.5–1 mM) substituted for FA, causing resuscitation of M. smegmatis and M. tuberculosis dormant cells. A M. smegmatis null-mutant lacking MSMEG_4279, which encodes a FA-activated adenylyl cyclase (AC), could not be resuscitated by FA but it was resuscitated by cAMP. M. smegmatis and M. tuberculosis cells hyper-expressing AC were unable to form non-culturable cells and a specific inhibitor of AC (8-bromo-cAMP) prevented FA-dependent resuscitation. RT-PCR analysis revealed that rpfA (coding for resuscitation promoting factor A) is up-regulated in M. smegmatis in the beginning of exponential growth following the cAMP increase in lag phase caused by FA-induced cell activation. A specific Rpf inhibitor (4-benzoyl-2-nitrophenylthiocyanate) suppressed FA-induced resuscitation. We propose a novel pathway for the resuscitation of dormant mycobacteria involving the activation of adenylyl cyclase MSMEG_4279 by FAs resulted in activation of cellular metabolism followed later by increase of RpfA activity which stimulates cell multiplication in exponential phase. The study reveals a probable role for lipids of host origin in the resuscitation of dormant mycobacteria

  20. Interaction of alpha-melanocyte-stimulating hormone, melatonin, cyclic AMP and cyclic GMP in the control of melanogenesis in hair follicle melanocytes in vitro.

    PubMed

    Weatherhead, B; Logan, A

    1981-07-01

    In short-term (48 h) cultures of hair follicles alpha-melanocyte-stimulating hormone (alpha-MSH) and cyclic AMP stimulated melanogenesis through an increase in tyrosinase activity. In contrast cyclic GMP mimicked the effects of melatonin by inhibiting melanin production without causing a concomitant decrease in tyrosinase activity. Both cyclic GMP and melatonin blocked the stimulatory effects of cyclic AMP and alpha-MSH on melanin production but they left the increased levels of tyrosinase activity unaffected. Phosphodiesterase inhibitors (3-isobutyl-1--methylxanthine and papaverine) simultaneously stimulated tyrosinase activity and inhibited melanin production, presumably by allowing endogenous cyclic AMP and cyclic GMP to accumulate intracellularly. It is suggested that whereas MSH stimulates melanogenesis through a cyclic AMP-dependent mechanism there must also be an inhibitory cyclic GMP-dependent mechanism, perhaps activated by melatonin, which operates at some post-tyrosinase step in the melanin biosynthetic pathway. PMID:6267154

  1. 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.

  2. Cyclic-AMP inhibition of fimbriae and prodigiosin production by Serratia marcescens is strain-dependent.

    PubMed

    Stella, Nicholas A; Shanks, Robert M Q

    2014-05-01

    The cyclic-nucleotide 3',5'-cyclic AMP (cAMP) is an ancient and widespread regulatory molecule. Previous studies have shown that fimbria production and secondary metabolite production are inhibited by cAMP in the prokaryote Serratia marcescens. This study used genetic manipulations to test the strain specificity of cAMP-cyclic-AMP receptor protein regulation of fimbria production and of the red pigment, prodigiosin. A surprising amount of variation was observed, as multicopy expression of the cAMP-phosphodiesterase gene, cpdS, conferred either an increase or decrease in fimbriae-dependent yeast agglutination and prodigiosin production depending upon the strain background. Mutation of crp, the gene coding for the cAMP-receptor protein, similarly conferred strain-dependent phenotypes. This study shows that three distinct biological properties, modulated by a conserved genetic regulatory molecule, can vary significantly among strains. Such variation can complicate the functional analysis of bacterial phenotypic properties which are dependent upon global genetic regulators such as cAMP.

  3. Increase in Endogenous and Exogenous Cyclic AMP Levels Inhibits Sclerotial Development in Sclerotinia sclerotiorum

    PubMed Central

    Rollins, Jeffrey A.; Dickman, Martin B.

    1998-01-01

    Growth and development of a wild-type Sclerotinia sclerotiorum isolate were examined in the presence of various pharmacological compounds to investigate signal transduction pathways that influence the development of sclerotia. Compounds known to increase endogenous cyclic AMP (cAMP) levels in other organisms by inhibiting phosphodiesterase activity (caffeine and 3-isobutyl-1-methyl xanthine) or by activating adenylate cyclase (NaF) reduced or eliminated sclerotial development in S. sclerotiorum. Growth in the presence of 5 mM caffeine correlated with increased levels of endogenous cAMP in mycelia. In addition, incorporation of cAMP into the growth medium decreased or eliminated the production of sclerotia in a concentration-dependent manner and increased the accumulation of oxalic acid. Inhibition of sclerotial development was cAMP specific, as exogenous cyclic GMP, AMP, and ATP did not influence sclerotial development. Transfer of developing cultures to cAMP-containing medium at successive time points demonstrated that cAMP inhibits development prior to or during sclerotial initiation. Together, these results indicate that cAMP plays a role in the early transition between mycelial growth and sclerotial development. PMID:9647827

  4. Cyclic AMP Can Decrease Expression of Genes Subject to Catabolite Repression in Saccharomyces cerevisiae

    PubMed Central

    Zaragoza, Oscar; Lindley, Chris; Gancedo, Juana M.

    1999-01-01

    External cyclic AMP (cAMP) hindered the derepression of gluconeogenic enzymes in a pde2 mutant of Saccharomyces cerevisiae, but it did not prevent invertase derepression. cAMP reduced nearly 20-fold the transcription driven by upstream activation sequence (UAS1FBP1) from FBP1, encoding fructose-1,6-bisphosphatase; it decreased 2-fold the activation of transcription by UAS2FBP1. Nuclear extracts from cells derepressed in the presence of cAMP were impaired in the formation of specific UASFBP1-protein complexes in band shift experiments. cAMP does not appear to act through the repressing protein Mig1. Control of FBP1 transcription through cAMP is redundant with other regulatory mechanisms. PMID:10198033

  5. Protein Kinase A-Dependent and -Independent Signaling Pathways Contribute to Cyclic AMP-Stimulated Proliferation

    PubMed Central

    Cass, Lisa A.; Summers, Scott A.; Prendergast, Gregory V.; Backer, Jonathan M.; Birnbaum, Morris J.; Meinkoth, Judy L.

    1999-01-01

    The effects of cyclic AMP (cAMP) on cell proliferation are cell type specific. Although the growth-inhibitory effects of cAMP have been well studied, much less is known regarding how cAMP stimulates proliferation. We report that cAMP stimulates proliferation through both protein kinase A (PKA)-dependent and PKA-independent signaling pathways and that phosphatidylinositol 3-kinase (PI3K) is required for cAMP-stimulated mitogenesis. In cells where cAMP is a mitogen, cAMP-elevating agents stimulate membrane ruffling, Akt phosphorylation, and p70 ribosomal S6 protein kinase (p70s6k) activity. cAMP effects on ruffle formation and Akt were PKA independent but sensitive to wortmannin. In contrast, cAMP-stimulated p70s6k activity was repressed by PKA inhibitors but not by wortmannin or microinjection of the N-terminal SH2 domain of the p85 regulatory subunit of PI3K, indicating that p70s6k and Akt can be regulated independently. Microinjection of highly specific inhibitors of PI3K or Rac1, or treatment with the p70s6k inhibitor rapamycin, impaired cAMP-stimulated DNA synthesis, demonstrating that PKA-dependent and -independent pathways contribute to cAMP-mediated mitogenesis. Direct elevation of PI3K activity through microinjection of an antibody that stimulates PI3K activity or stable expression of membrane-localized p110 was sufficient to confer hormone-independent DNA synthesis when accompanied by elevations in p70s6k activity. These findings indicate that multiple pathways contribute to cAMP-stimulated mitogenesis, only some of which are PKA dependent. Furthermore, they demonstrate that the ability of cAMP to stimulate both p70s6k- and PI3K-dependent pathways is an important facet of cAMP-regulated cell cycle progression. PMID:10454535

  6. The interplay between cyclic AMP, MAPK, and NF-κB pathways in response to proinflammatory signals in microglia.

    PubMed

    Ghosh, Mousumi; Aguirre, Vladimir; Wai, Khine; Felfly, Hady; Dietrich, W Dalton; Pearse, Damien D

    2015-01-01

    Cyclic AMP is an important intracellular regulator of microglial cell homeostasis and its negative perturbation through proinflammatory signaling results in microglial cell activation. Though cytokines, TNF-α and IL-1β, decrease intracellular cyclic AMP, the mechanism by which this occurs is poorly understood. The current study examined which signaling pathways are responsible for decreasing cyclic AMP in microglia following TNF-α stimulation and sought to identify the role cyclic AMP plays in regulating these pathways. In EOC2 microglia, TNF-α produced a dramatic reduction in cyclic AMP and increased cyclic AMP-dependent PDE activity that could be antagonized by Rolipram, myristoylated-PKI, PD98059, or JSH-23, implicating a role for PDE4, PKA, MEK, and NF-κB in this regulation. Following TNF-α there were significant increases in iNOS and COX-2 immunoreactivity, phosphorylated ERK1/2 and NF-κB-p65, IκB degradation, and NF-κB p65 nuclear translocation, which were reduced in the presence of high levels of cyclic AMP, indicating that reductions in cyclic AMP during cytokine stimulation are important for removing its inhibitory action on NF-κB activation and subsequent proinflammatory gene expression. Further elucidation of the signaling crosstalk involved in decreasing cyclic AMP in response to inflammatory signals may provide novel therapeutic targets for modulating microglial cell activation during neurological injury and disease. PMID:25722974

  7. Both cyclic-AMP and cyclic-GMP can act as regulators of the phenylpropanoid pathway in Arabidopsis thaliana seedlings.

    PubMed

    Pietrowska-Borek, Małgorzata; Nuc, Katarzyna

    2013-09-01

    Cyclic nucleotides (cAMP and cGMP) are important signaling molecules that control a range of cellular functions and modulate different reactions. It is known that under abiotic or biotic stress plant cells synthesize these nucleotides and that they also enhance the activity of the phenylpropanoid pathway. Wondering what is the relation between these two facts, we investigated how the exogenously applied membrane-permeable derivatives, 8-Br-cAMP or 8-Br-cGMP, which are believed to act as the original cyclic nucleotides, affect the expression of the genes for and the specific activity of three enzymes of the phenylpropanoid pathway in Arabidopsis thaliana seedlings. We found that the expression of the genes of phenylalanine ammonia-lyase (PAL2), 4-coumarate:coenzyme A ligase (4CL1) and chalcone synthase (CHS), and the specific activities of PAL (EC 4.3.1.5), 4CL (EC 6.2.1.12) and CHS (EC 2.3.1.74) were induced in the same way by either of these cyclic nucleotides used at 5 μM concentration. None of the possible cAMP and cGMP degradation products (AMP, GMP, adenosine or guanosine) evoked such effects. Expression of PAL1, 4CL2 and 4CL3 were practically not affected. Although the investigated nucleotides induced rapid expression of the aforementioned enzymes, they did not affect the level of anthocyanins within the same period. We discuss the effects exerted by the exogenously administered cyclic nucleotides, their relation with stress and the role which the phenylpropanoid pathways the cyclic nucleotides may play in plants.

  8. Cyclic AMP-dependent protein phosphorylation in isolated neuronal growth cones from developing rat forebrain.

    PubMed

    Lockerbie, R O; Eddé, B; Prochiantz, A

    1989-03-01

    We have shown recently that neuronal growth cones isolated from developing rat forebrain possess an appreciable activity of adenylate cyclase, which produces cyclic AMP and can be stimulated by various neurotransmitter receptor agonists and by forskolin. To investigate cyclic AMP-mediated biochemical mechanisms in isolated growth cones, we have centered the present study on cyclic AMP-dependent protein phosphorylation. One-dimensional gel electrophoretic analysis showed that cyclic AMP analogs increased incorporation of 32P into several phosphoproteins in molecular mass ranges of 50-58 and 76-82 kilodaltons, including those of 82, 76, and 51 kilodaltons. Two-dimensional electrophoresis, using isoelectric focusing in the first dimension, resolved phosphorylated alpha- and beta-tubulin species, actin, a very acidic protein (isoelectric point 4.0) with a molecular mass of 93 kilodaltons, and two proteins (x and x') closely neighboring beta-tubulin. Two other phosphoproteins seen in the gels had molecular masses of 56 and 51 kilodaltons (respective isoelectric points, 4.5 and 4.4) and, along with the 93-kilodalton phosphoprotein, were highly enriched in the isolated growth cones. Only the tubulin and actin species were major proteins in the isolated growth cones. Cyclic AMP analogs enhanced incorporation of 32P into phosphoproteins x and x', and, as assessed by immunoprecipitation, into beta-tubulin. Peptide digest experiments suggested that phosphoproteins x and x' are unrelated to beta-tubulin. Nonequilibrium two-dimensional electrophoresis resolved many phosphoproteins, of which a 79- and 75-kilodalton doublet, a 74-kilodalton species, and a 58-kilodalton doublet showed enhanced incorporation of 32P in the presence of cyclic AMP.

  9. 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.

  10. 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

  11. An Essential Poison: Synthesis and Degradation of Cyclic Di-AMP in Bacillus subtilis

    PubMed Central

    Gundlach, Jan; Mehne, Felix M. P.; Herzberg, Christina; Kampf, Jan; Valerius, Oliver; Kaever, Volkhard

    2015-01-01

    ABSTRACT Gram-positive bacteria synthesize the second messenger cyclic di-AMP (c-di-AMP) to control cell wall and potassium homeostasis and to secure the integrity of their DNA. In the firmicutes, c-di-AMP is essential for growth. The model organism Bacillus subtilis encodes three diadenylate cyclases and two potential phosphodiesterases to produce and degrade c-di-AMP, respectively. Among the three cyclases, CdaA is conserved in nearly all firmicutes, and this enzyme seems to be responsible for the c-di-AMP that is required for cell wall homeostasis. Here, we demonstrate that CdaA localizes to the membrane and forms a complex with the regulatory protein CdaR and the glucosamine-6-phosphate mutase GlmM. Interestingly, cdaA, cdaR, and glmM form a gene cluster that is conserved throughout the firmicutes. This conserved arrangement and the observed interaction between the three proteins suggest a functional relationship. Our data suggest that GlmM and GlmS are involved in the control of c-di-AMP synthesis. These enzymes convert glutamine and fructose-6-phosphate to glutamate and glucosamine-1-phosphate. c-di-AMP synthesis is enhanced if the cells are grown in the presence of glutamate compared to that in glutamine-grown cells. Thus, the quality of the nitrogen source is an important signal for c-di-AMP production. In the analysis of c-di-AMP-degrading phosphodiesterases, we observed that both phosphodiesterases, GdpP and PgpH (previously known as YqfF), contribute to the degradation of the second messenger. Accumulation of c-di-AMP in a gdpP pgpH double mutant is toxic for the cells, and the cells respond to this accumulation by inactivation of the diadenylate cyclase CdaA. IMPORTANCE Bacteria use second messengers for signal transduction. Cyclic di-AMP (c-di-AMP) is the only second messenger known so far that is essential for a large group of bacteria. We have studied the regulation of c-di-AMP synthesis and the role of the phosphodiesterases that degrade this second

  12. Cyclic-AMP inhibition of fimbriae and prodigiosin production by Serratia marcescens is strain-dependent

    PubMed Central

    Stella, Nicholas A.; Shanks, Robert M. Q.

    2014-01-01

    The cyclic-nucleotide 3’,5’-cyclic AMP (cAMP) is an ancient and wide spread regulatory molecule. Previous studies have shown that fimbria production and secondary metabolite production are inhibited by cAMP in the prokaryote Serratia marcescens. This study used genetic manipulations to test the strain specificity of cAMP-CRP regulation of fimbria production and of the red pigment, prodigiosin. A surprising amount of variation was observed, as multicopy expression of the cAMP-phosphodiesterase gene, cpdS, conferred either an increase or decrease in fimbriae-dependent yeast agglutination and prodigiosin production depending upon the strain background. Mutation of crp, the gene coding for the cAMP-receptor protein similarly conferred strain-dependent phenotypes. This study shows that three distinct biological properties, modulated by a conserved genetic regulatory molecule, can vary significantly among strains. Such variation can complicate the functional analysis of bacterial phenotypic properties which are dependent upon global genetic regulators such as cAMP. PMID:24619531

  13. Heparin modulates intracellular cyclic AMP in human trabecular bone cells and adherent rheumatoid synovial cells.

    PubMed Central

    Crisp, A J; Roelke, M S; Goldring, S R; Krane, S M

    1984-01-01

    Cells were cultured from explants of human trabecular bone excised from eight patients and incubated usually for 20 minutes with bovine parathyroid hormone, salmon calcitonin, prostaglandin E2, or heparin. The intracellular content of cyclic AMP was measured by radioimmunoassay and was significantly increased by parathyroid hormone in four, by calcitonin in two, by prostaglandin E2 in eight, and by heparin in seven out of eight cultures. In the two cultures containing calcitonin-responsive cells heparin inhibited the cyclic AMP response induced by calcitonin. Heparin did not affect the cyclic AMP response to parathyroid hormone or prostaglandin E2. Heparin also increased the cyclic AMP content of cultured adherent rheumatoid synovial cells. It is proposed that, in certain situations of focal pathological bone resorption, although concentrations of circulating hormones may be normal, the local release of products such as heparin may modify the effect of hormones which regulate connective tissue homoeostasis. local changes in hormone responses could contribute to the enhanced bone resorption associated with inflammatory processes such as rheumatoid arthritis. Images PMID:6089675

  14. Modulation of norepinephrine-stimulated cyclic AMP accumulation in rat pinealocytes by n-3 fatty acids.

    PubMed

    Delton-Vandenbroucke, I; Sarda, N; Molière, P; Lagarde, M; Gharib, A

    1996-10-01

    This work showed that docosahexaenoic (22:6n-3) and eicosapentaenoic (20:5n-3) acid supplementation for 48 h have opposite effects on the norepinephrine-stimulated cyclic AMP accumulation in rat pinealocytes. We found that 22:6n-3 supplementation of pineal cells, done by increasing specifically 22:6n-3 in phospholipid and triacylglycerol pools, led to inhibition of norepinephrine-stimulated cyclic AMP production whereas 20:5n-3 supplementation, by increasing 20:5n-3, and 22:5n-3 and 22:6n-3 in the same pools, stimulated it. In contrast, direct treatment of pinealocytes with each fatty acid (50 microM) did not affect cyclic AMP production in the presence of (0.1-10 microM) norepinephrine. The results indicate that, using pharmacological agents such as forskolin or prazosin: (a) neither basal nor forskolin-stimulated cyclic AMP levels were modified in fatty acid-supplemented cells compared to control cells; (b) in the presence of 1 microM prazosin, the activation by 20:5n-3 was still effective whereas no additional inhibition of norepinephrine stimulation was observed in 22:6n-3-supplemented cells. Taken together our results suggest that 22:6n-3 or 20:5n-3 supplementation modulates specifically the alpha 1- or beta-adrenoceptors in the rat pineal gland.

  15. Role of cyclic AMP in pulmonary xenobiotic metabolism with special emphasis on benzo(a)pyrene

    SciTech Connect

    Schaeffer, V.H.

    1986-01-01

    This thesis was intended to investigate the role of the intracellular regulator, cAMP, on pulmonary xenobiotic metabolism using the well-studied carcinogen, benzo(a)pyrene (BP) as a representative xenobiotic. Lung slices from rats administered N/sup 6/, O/sup 2/', dibutyryl cAMP (DcAMP), theophylline or forskolin, all of which elevated biologically reactive cAMP levels in the lung, showed an increased ability to metabolize (/sup 3/H)-BP. This effect occurred beyond 6 hr following treatment and reached a maximum at 12 hr, at a time when cAMP content had already peaked and returned to basal levels. The perfusion of BP through the isolated lungs of animals administered DcAMP in vivo indicated that the BP metabolites primarily responsible for the cyclic nucleotide-induced increase in metabolism were the 3-hydroxy BP, 9-hydroxy BP, BP 9, 10 diol, BP-glucuronides and BP-glutathione conjugates. Kinetic analysis indicated that the Km component of these reactions was altered without a corresponding change in Vmax, suggesting that elevated pulmonary cAMP content may be affecting the detoxication enzymes, UDP-glucuronyltransferase and sulfotransferase. Studies with pulmonary microsomes from DcAMP-treated animals indicated that the cyclic nucleotide not only enhanced the hydroxylation of BP but also the cytochrome P450-dependent hydroxylation of coumarin. This is supported by the fact that DcAMP administration in vivo also enhanced phosphorylation of two classes of nuclear proteins, histones and nuclear acidic proteins, believed to play a role in the transcription of RNA and DNA.

  16. Cell behavior in Dictyostelium discoideum: preaggregation response to localized cyclic AMP pulses

    PubMed Central

    1982-01-01

    The motion of cells in the aggregation phase of Dictyostelium discoideum development is complex. To probe its mechanisms we applied precisely timed (+/- 1 s) and positioned (+/-2 micrometers) pulses of cyclic AMP to fields of cells of moderate density using a micropipette. We recorded cell behavior by time lapse microcinematography and extracted cell motion data from the film with our Galatea computer system. Analysis of these data reveals: (a) Chemotaxis lasts only about as long as the cyclic AMP signal; in particular, brief pulses (approximately 5 s) do not induce chemotaxis. (b) Chemotactic competence increases gradually from within an hour after the initiation of development (starvation) to full competence at approximately 15 h when aggregation begins under our conditions. (c) Cell motion reverses rapidly (within 20 s) when the external gradient is reversed. There is no refractory period for motion. We present a new description of the process of aggregation consistent with our result and other recent findings. (d) The behavioral response to cyclic AMP includes a phenomenon we call "cringing." In a prototypical cringe the cell speed drops within 3 s after a brief cyclic AMP stimulus, and the cell stops and rounds and then resumes motion after 25 s. (e) The development of the speed response in cringing as the cells age closely parallels the development of the cyclic AMP-induced light-scattering response of cells in suspension. (f) Cringing occurs in natural populations during weak oriented movement. The computerized analysis of cell behavior proves to be a powerful technique which can reveal significant phenomena that are not apparent to the eye even after repeated examination of the film. PMID:6282894

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

    PubMed

    Minder, C; Vanden Driessche, T

    1978-05-26

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

  18. 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

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

    PubMed Central

    Klein, Matthias; Bopp, Tobias

    2016-01-01

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

  20. 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.

  1. 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.

  2. 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

  3. From drought sensing to developmental control: evolution of cyclic AMP signaling in social amoebas.

    PubMed

    Ritchie, Allyson V; van Es, Saskia; Fouquet, Celine; Schaap, Pauline

    2008-10-01

    Amoebas and other protists commonly encyst when faced with environmental stress. Although little is known of the signaling pathways that mediate encystation, the analogous process of spore formation in dictyostelid social amoebas is better understood. In Dictyostelium discoideum, secreted cyclic AMP (cAMP) mediates the aggregation of starving amoebas and induces the differentiation of prespore cells. Intracellular cAMP acting on cAMP-dependent protein kinase (PKA) triggers the maturation of spores and prevents their germination under the prevalent conditions of high osmolality in the spore head. The osmolyte-activated adenylate cyclase, ACG, produces cAMP for prespore differentiation and inhibition of spore germination. To retrace the origin of ACG function, we investigated ACG gene conservation and function in species that span the dictyostelid phylogeny. ACG genes, osmolyte-activated ACG activity, and osmoregulation of spore germination were detected in species that represent the 4 major groups of Dictyostelia. Unlike the derived species D. discoideum, many basal Dictyostelia have retained the ancestral mechanism of encystation from solitary amoebas. In these species and in solitary amoebas, encystation is independently triggered by starvation or by high osmolality. Osmolyte-induced encystation was accompanied by an increase in cAMP and prevented by inhibition of PKA, indicating that ACG and PKA activation mediate this response. We propose that high osmolality signals drought in soil amoebas and that developmental cAMP signaling in the Dictyostelia has evolved from this stress response.

  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. Effects of dibutyryl cyclic AMP and papaverine on intrahepatocytic bile acid transport. Role of vesicle transport.

    PubMed

    Hoshino, M; Ohiwa, T; Hayakawa, T; Kamiya, Y; Tanaka, A; Hirano, A; Kumai, T; Katagiri, K; Miyaji, M; Takeuchi, T

    1993-09-01

    The secondary messenger cyclic AMP plays an important role in regulating biliary excretory function by stimulating the transcytotic vesicle transport system, whereas papaverine exerts an inhibitory effect on this system. We therefore investigated their effects on bile acid-induced cytotoxicity and intrahepatocytic content of bile acid in primary cultured rat hepatocytes. Simultaneous addition of 1 mM dibutyryl cyclic AMP (DBcAMP), an analogue of cAMP, with 1 mM taurochenodeoxycholic acid (TCDCA) significantly decreased the release of lactate dehydrogenase (LDH) as compared with the case with 1 mM TCDCA alone (7.1 +/- 0.13% of total versus 10.7 +/- 0.3%). In contrast, 0.1 mM papaverine approximately doubled the amount of LDH (22.0 +/- 0.6% of total versus 10.7 +/- 0.3%; P < 0.01). The intracellular content of TCDCA 180 min after the administration of 1 mM TCDCA alone was 20.8 +/- 0.7 nmol/mg protein, that after simultaneous administration of 1 mM DBcAMP, 16.2 +/- 1.0 nmol/mg protein, and that after the simultaneous administration of 0.1 mM papaverine, 38.5 +/- 1.9 nmol/mg protein. A clear correlation between the release of LDH from hepatocytes and the intracellular content of TCDCA was thus observed. When given together with 1 mM taurocholic acid (TCA) or 1 mM tauroursodeoxycholic acid (TUDCA), papaverine exerted little effect on cytotoxicity or intrahepatocytic bile acid content. When cells were bathed in a medium free of bile acid after pretreatment with 1 mM TCDCA and 1 mM DBcAMP, additional exposure to DBcAMP for 30 min significantly stimulated reduction of intracellular TCDCA content (30.2 +/- 0.4% of total versus 44.0 +/- 1.4%).(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Cyclic AMP levels during induction and repression of cellulase biosynthesis in Thermomonospora curvata

    SciTech Connect

    Wood, W.E.; Neubauer, D.G.; Stutzenberger, F.J.

    1984-12-01

    Specific cellulase production rates (SCPR) were compared with intracellular cyclic AMP (cAMP) levels in the thermophilic actinomycete, Thermomonospora curvata, during growth on several carbon sources in a chemically defined medium. SCPR and cAMP levels were 0.03 U (endoglucanase (EG) units) and 2 pmol per mg of dry cells, respectively, during exponential growth on glucose. These values increased to about 6 and 25, respectively, during growth on cellulose. Detectable EG production ceased when cAMP levels dropped below 10. Cellobiose (usually considered to be a cellulase inducer) caused a sharp decrease in cAMP levels and repressed EG production when added to cellulose-grown cultures. 2-deoxy-D-glucose, although nometabolizable in T. curvata, depressed cAMP to levels observed with glucose, but unlike glucose, the 2DG effect persisted until cells were washed and transferred to fresh medium. SCPR values and cAMP levels in cells grown in continuous culture under conditions of cellobiose limitation were markedly influenced by dilution rate (D). The maxima for both occurred at D = 0.085 (culture generation time of 11.8 h). When D was held constant and cellobiose concentration was increased over a 14-fold range to support higher steady state population levels, SCPR values decreased about fivefold, indicating that extracellular catabolite accumulation may be a factor in EG repression. The role of cAMP in the mechanism of this repression appears to be neither simple nor direct, since large changes (up to 200-fold) in SCPR accompany relatively small changes (10-fold) in cellular cAMP levels.

  7. Association of the cyclic AMP chemotaxis receptor with the detergent- insoluble cytoskeleton of Dictyostelium discoideum

    PubMed Central

    1984-01-01

    Treatment of 6-h differentiated Dictyostelium discoideum cells with the nonionic detergent Triton X-100 dissolves away membranes and soluble components, as judged by marker enzyme distributions, leaving intact a cytoskeletal residue that contains approximately 10% of the cell protein and 50% of the actin. Nitrobenzooxadiazo-phallacidin staining for F-actin and electron microscopy of detergent-extracted whole-mounts indicate that the cytoskeletons retain the size and shape of intact cells and contain F-actin in cortical meshworks. The cytoskeletons contain little if any remaining membrane material by morphological criteria, and the plasma membrane enzymes cyclic nucleotide phosphodiesterase and alkaline phosphatase are absent from the insoluble residue, which retains only 15% of the membrane concanavalin A-binding glycoproteins. This detergent-insoluble residue retains a specific [3H]cAMP-binding site with the nucleotide specificity, rapid kinetics and approximate affinity of the cAMP receptor on intact cells. Upon detergent extraction of cells, the number of cAMP-binding sites increases 20-70%. The binding site is attached to the insoluble residue whether or not the cAMP receptor is occupied at the time of detergent addition. The pH dependence for recovery of the insoluble cAMP-binding site is much sharper than that on intact cells or membranes with an optimum at pH 6.1. Conditions of pH and ionic composition that lead to disruption of the cytoskeleton upon detergent treatment also result in the loss of cAMP binding. During differentiation, the detergent- insoluble cAMP binding increases in parallel with cell surface cAMP receptors and chemotaxis to cAMP. PMID:6693497

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

    PubMed

    Aykaç Fas, Burcu; Tutar, Yusuf; Haliloğlu, Türkan

    2013-01-01

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

  9. Cloning and sequencing of a calcium-binding protein regulated by cyclic AMP in the thyroid.

    PubMed Central

    Lefort, A; Lecocq, R; Libert, F; Lamy, F; Swillens, S; Vassart, G; Dumont, J E

    1989-01-01

    p24 is a thyroid protein (Mr 24,000) identified by two-dimensional gel electrophoresis on the basis that its synthesis and phosphorylation are up-regulated by thyrotropin and cyclic AMP agonists. p24 cDNA was cloned from a lambda gt11 cDNA library using a polyclonal antibody raised against the protein recovered from a Western blot spot. The encoded polypeptide (189 residues) displays a putative target-site for phosphorylation by cyclic AMP-dependent protein kinase and belongs to the superfamily of proteins binding Ca2+ through 'EF hand' domains. It presents four such domains of which two agree closely with the consensus. The ability of p24 to bind Ca2+ has been directly confirmed on Western blots. p24 was detected in many tissues including the salivary glands, the lung and the brain. The ubiquitous nature of p24, together with its regulatory and sequence characteristics suggest that it constitutes an important target common to the cyclic AMP and Ca2+-phosphatidylinositol cascades. Images PMID:2540953

  10. 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.

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

    SciTech Connect

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

    1985-09-25

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

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

    PubMed Central

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

    2016-01-01

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

  13. alpha-Tocopherol decreases the somatostatin receptor-effector system and increases the cyclic AMP/cyclic AMP response element binding protein pathway in the rat dentate gyrus.

    PubMed

    Hernández-Pinto, A M; Puebla-Jiménez, L; Arilla-Ferreiro, E

    2009-08-01

    Neuronal survival has been shown to be enhanced by alpha-tocopherol and modulated by cyclic AMP (cAMP). Somatostatin (SST) receptors couple negatively to adenylyl cyclase (AC), thus leading to decreased cAMP levels. Whether alpha-tocopherol can stimulate neuronal survival via regulation of the somatostatinergic system, however, is unknown. The aim of this study was to investigate the effects of alpha-tocopherol on the SST signaling pathway in the rat dentate gyrus. To that end, 15-week-old male Sprague-Dawley rats were treated daily for 1 week with (+)-alpha-tocopherol or vehicle and sacrificed on the day following the last administration. No changes in either SST-like immunoreactivity (SST-LI) content or SST mRNA levels were detected in the dentate gyrus as a result of alpha-tocopherol treatment. A significant decrease in the density of the SST binding sites and an increase in the dissociation constant, however, were detected. The lower SST receptor density in the alpha-tocopherol-treated rats correlated with a significant decrease in the protein levels of the SST receptor subtypes SSTR1-SSTR4, whereas the corresponding mRNA levels were unaltered. G-protein-coupled-receptor kinase 2 expression was decreased by alpha-tocopherol treatment. This vitamin induced a significant increase in both basal and forskolin-stimulated AC activity, as well as a decrease in the inhibitory effect of SST on AC. Whereas the protein levels of AC type V/VI were not modified by alpha-tocopherol administration, ACVIII expression was significantly enhanced, suggesting it might account for the increase in AC activity. In addition, this treatment led to a reduction in Gialpha1-3 protein levels and in Gi functionality. alpha-Tocopherol did not affect the expression of the regulator of G-protein signaling 6/7 (RGS6/7). Finally, alpha-tocopherol induced an increase in the levels of phosphorylated cAMP response element binding protein (p-CREB) and total CREB in the dentate gyrus. Since CREB

  14. SCAP/SREBP pathway is required for the full steroidogenic response to cyclic AMP.

    PubMed

    Shimizu-Albergine, Masami; Van Yserloo, Brian; Golkowski, Martin G; Ong, Shao-En; Beavo, Joseph A; Bornfeldt, Karin E

    2016-09-20

    Luteinizing hormone (LH) stimulates steroidogenesis largely through a surge in cyclic AMP (cAMP). Steroidogenic rates are also critically dependent on the availability of cholesterol at mitochondrial sites of synthesis. This cholesterol is provided by cellular uptake of lipoproteins, mobilization of intracellular lipid, and de novo synthesis. Whether and how these pathways are coordinated by cAMP are poorly understood. Recent phosphoproteomic analyses of cAMP-dependent phosphorylation sites in MA10 Leydig cells suggested that cAMP regulates multiple steps in these processes, including activation of the SCAP/SREBP pathway. SCAP [sterol-regulatory element-binding protein (SREBP) cleavage-activating protein] acts as a cholesterol sensor responsible for regulating intracellular cholesterol balance. Its role in cAMP-mediated control of steroidogenesis has not been explored. We used two CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated protein 9) knockout approaches to test the role of SCAP in steroidogenesis. Our results demonstrate that SCAP is required for progesterone production induced by concurrent inhibition of the cAMP phosphodiesterases PDE4 and PDE8. These inhibitors increased SCAP phosphorylation, SREBP2 activation, and subsequent expression of cholesterol biosynthetic genes, whereas SCAP deficiency largely prevented these effects. Reexpression of SCAP in SCAP-deficient cells restored SREBP2 protein expression and partially restored steroidogenic responses, confirming the requirement of SCAP-SREBP2 in steroidogenesis. Inhibitors of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase and isoprenylation attenuated, whereas exogenously provided cholesterol augmented, PDE inhibitor-induced steroidogenesis, suggesting that the cholesterol substrate needed for steroidogenesis is provided by both de novo synthesis and isoprenylation-dependent mechanisms. Overall, these results demonstrate a novel role for LH/cAMP in SCAP

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

    PubMed

    Lin, Ching-Ting; Lin, Tien-Huang; Wu, Chien-Chen; Wan, Lei; Huang, Chun-Fa; Peng, Hwei-Ling

    2016-01-01

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

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

    PubMed

    Lin, Ching-Ting; Lin, Tien-Huang; Wu, Chien-Chen; Wan, Lei; Huang, Chun-Fa; Peng, Hwei-Ling

    2016-01-01

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

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

    PubMed Central

    Lin, Ching-Ting; Lin, Tien-Huang; Wu, Chien-Chen; Wan, Lei; Huang, Chun-Fa; Peng, Hwei-Ling

    2016-01-01

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

  18. Cyclic AMP in female mouse brain is altered by the adrenocorticotropic hormone(4-9) analogue organon 2766.

    PubMed

    Schneider, D R; Felt, B T; Murphy, S; Goldman, H

    1981-09-01

    Cyclic AMP content was determined in 12 brain regions of young adult female mice at 30 min and at 24 h following an intraperitoneal injection of the tri-substituted adrenocorticotropic hormone(4-9) [ACTH(4-9)] analogue Organon 2766 [ORG 2766]. Animals were killed by focused 3.5 kW microwave radiation applied for 350 ms. Unlike previously reported responses in male mice, at 30 min post-injection there were no detectable differences in cyclic AMP content between the placebo and ORG 2766-treated animals. By contrast, 24 h after injection, the content of cyclic AMP was changed significantly in 8 of the 12 brain regions examined: medulla-pons, septal area, thalamus, hypothalamus, hippocampus, olfactory bulb, and parietal and occipital cortices. In most of the regions examined, differences consisted of 50% or greater reductions of tissue cyclic AMP content. The changes were unrelated to the estrus cycle of these animals.

  19. 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

  20. Effect of Glucagon on Net Splanchnic Cyclic AMP Production in Normal and Diabetic Men

    PubMed Central

    Liljenquist, John E.; Bomboy, James D.; Lewis, Stephen B.; Sinclair-Smith, Bruce C.; Felts, Philip W.; Lacy, William W.; Crofford, Oscar B.; Liddle, Grant W.

    1974-01-01

    Glucagon activates hepatic adenylate cyclase, thereby increasing acutely the liver content of cyclic AMP (cAMP) as well as the release of cAMP into the hepatic vein. Insulin, on the other hand, antagonizes this glucagon-mediated cAMP production, thus providing a hypothetical mechanism through which insulin might correct some of the metabolic abnormalities of diabetes. To study this hormonal interaction in man, net splanchnic cAMP production (NScAMPP) was investigated in normal and insulin-dependent diabetic men under basal conditions and in response to intravenous glucagon, 50 ng/kg/min for 2 h. In normals (n=19), basal hepatic vein cAMP concentration was 23.6±1.1 nM and NScAMPP was 1.7±0.6 nmol/min. Glucagon stimulated NScAMPP in four normal subjects to a peak of 99.6±43 nmol/min at 25 min with a subsequent fall to 12.4±5.1 nmol/min by 90 min despite continuing glucagon infusion. Endogenous insulin secretion was stimulated as indicated by rising levels of immunoreactive insulin and C-peptide (connecting peptide) immunoreactivity, raising the possibility that endogenous insulin might be responsible for the fall in NScAMPP that followed the initial spike. In the diabetics (n=8), basal hepatic vein cAMP concentration was 24.7±1.2 nM and NScAMPP was undetectable. Glucagon stimulated NScAMPP in five diabetics to a peak of 169.9±42.6 with a subsequent fall to 17.4±3.9 nmol/min by 90 min even though endogenous insulin secretion was not stimulated (no rise in C-peptide immunoreactivity). Although the mean increase in NScAMPP was greater in the diabetics, the two groups did not differ significantly. Conclusions. In normal resting man the liver is a significant source of circulating cAMP. Diabetics do not release abnormally large amounts of hepatic cAMP under basal conditions. Glucagon markedly enhances hepatic cAMP release with a spike-decline pattern in both normal and diabetic men. The decline in hepatic cAMP release despite continuing glucagon stimulation is due

  1. 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.

  2. 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.

  3. Carbachol and bradykinin elevate cyclic AMP and rapidly deplete ATP in cultured rat sympathetic neurons.

    PubMed Central

    Suidan, H S; Murrell, R D; Tolkovsky, A M

    1991-01-01

    The agonists carbachol (CCh) and bradykinin (BK) and 54 mM KCl (high K+) were among the most potent stimulants of cyclic AMP (cAMP) production in cultured rat sympathetic neurons, measured with the use of a high-fidelity assay developed for small samples. The rise in cAMP evoked by CCh (through muscarinic receptors), BK, and high K+ was inhibited in Ca2(+)-depleted medium (1.3 mM Ca2+ and 2 mM BAPTA or EGTA), which also prevented the sustained rise in [Ca2+]i evoked by each of these stimuli, showing that elevation of cAMP requires extracellular Ca2+ and, possibly, Ca2+ influx. Preliminary results obtained with the novel calmodulin inhibitor CGS 9343B, which blocked the elevation of cAMP, and with the cyclogenase inhibitor indomethacin, which partially blocked the actions of the agonists but not those of high K+, suggest that calmodulin and arachidonate metabolites may be two components of the signaling pathway. In addition to their effects on cAMP metabolism, CCh, muscarine, and BK, but not nicotine, caused a 30-40% decrease in ATP levels. This effect was much greater than that evoked by high K+ and was largely inhibited by CGS 9343B but slightly enhanced in the Ca(+)-depleted medium, showing that agonists are still active in the absence of [Ca2+]o. Thus, agonists that activate phosphoinositide metabolism can also increase cAMP production and substantially deplete cells of ATP. These novel actions may have to be taken into account when the mechanisms by which such agonists regulate cell function are being considered. PMID:1848792

  4. Luteinizing hormone-releasing hormone (LHRH) attenuates morphine-induced inhibition of cyclic AMP (cAMP) in opioid-responsive SK-N-SH cells.

    PubMed

    Ratka, A; Simpkins, J W

    1997-04-01

    SK-N-SH cells were used to assess the effects of luteinizing hormone-releasing hormone (LHRH) on opioid receptor-mediated changes in cyclic AMP (cAMP). Prostaglandin E1 (PGE1, 1 microM) caused a dramatic increase in cAMP levels. Treatment with 10 microM morphine (MOR) significantly inhibited the stimulatory effect of PGE1, LHRH (0.8 microM) caused an increase in the basal level of intracellular cAMP and potentiated the stimulatory effect of PGE1 on cAMP accumulation. In cells pretreated with LHRH the inhibitory effect of MOR on cAMP accumulation was significantly attenuated. An LHRH antagonist had no effect on cAMP. The involvement of pertussis toxin (PTX)-sensitive G proteins in the actions of LHRH was studied. PTX increased the stimulatory effect of PGE1 on cAMP and attenuated the inhibitory effect of MOR. However, PTX pretreatment prevented the effects of LHRH on the intracellular actions of PGE1 but exerted an additive effect with LHRH in blocking the MOR-induced decrease in cAMP levels. We conclude that LHRH attenuates the inhibitory, opioid receptor-mediated effect of MOR on intracellular cAMP accumulation in SK-N-SH cells, and that the G protein-independent mechanism may be involved in LHRH-induced attenuation of the inhibitory effect of MOR on neuronal cAMP.

  5. Role of nitric oxide/cyclic GMP and cyclic AMP in beta3 adrenoceptor-chronotropic response.

    PubMed

    Sterin-Borda, Leonor; Bernabeo, Gustavo; Ganzinelli, Sabrina; Joensen, Lilian; Borda, Enri

    2006-04-01

    In this study we determine different signaling pathways involved in beta(3) adrenoceptor (beta(3)-AR) dependent frequency stimulation in isolated rodent atria. Promiscuous coupling between different G-proteins and beta(3)-AR could explain the multiple functional effects of beta(3)-AR stimulation. We examine the mechanisms and functional consequences of dual adenylate cyclase and guanylate cyclase pathways coupling to beta(3)-AR in isolated rodent atria. The beta(3)-AR selective agonists ZD 7114 and ICI 215001 stimulated in a dose-dependent manner the contraction frequency that significantly correlated with cyclic AMP (cAMP) accumulation. Inhibition of adenylate cyclase shifted the chronotropic effect to the right. On the other hand, the ZD 7114 activity on frequency was enhanced by the inhibition of nitric oxide synthase (NOS) and soluble guanylate cyclase. This countervailing negative chronotropic nitric oxide-cyclic GMP (NO-cGMP) significantly correlated with the increase on NOS activity and cGMP accumulation. Current analysis showed a negative cross talk between cAMP chronotropic and NO-cGMP effects by inhibition of phospholipase C (PLC), calcium/calmodulin (CaM), protein kinase C (PKC), NOS isoforms and Gi-protein on the effects of beta(3)-AR stimulation. RT-PCR detected both eNOS and nNOS in isolated rat atria. NOS isoforms performed independently. Only nNOS participated in limiting the effect of beta(3)-AR stimulation. In eNOS-KO (eNOS-/-) mice the chronotropic effect of beta(3)-AR agonists did not differ from wild type (WT) mice atria, but it was increased by the inhibition of nNOS activity. Our results suggest that the increase in frequency by beta(3)-AR activation on isolated rodent atria is associated to a parallel increases in cAMP. The nNOS-cGMP pathway negatively modulates beta(3)-AR activation. Multiple signal transduction pathways between G-protein and beta(3)-AR may protect myocardium from catecholamine-induced cardiotoxic effects. PMID:16510153

  6. 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

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

    PubMed Central

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

    1990-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-02-01

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

  11. Cyclic AMP and the mechanism of leucocyte lysosomal enzyme release during an immediate hypersensitivity reaction in vivo.

    PubMed

    Deporter, D A

    1977-11-01

    The pleural cavity of rats was used to study the effect of altering leucocyte cyclic AMP content on the release of B-glucuronidase activity during an immediate hypersensitivity reaction. The effect on intravenous colchicine was also studied. Despite an increase of 135 to 235% in leucocyte cyclic AMP content no decrease in B-glucuronidase release was observed. Similarly, colchicine had no effect on enzyme release. It was concluded that the cyclic nucleotides and leucocyte microtubules may have no significant role to play in the release of lysosomal enzymes during acute inflammation in vivo. PMID:201738

  12. 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.

  13. Phosphodiesterase 8B and cyclic AMP signaling in the adrenal cortex.

    PubMed

    Leal, Leticia Ferro; Szarek, Eva; Faucz, Fabio; Stratakis, Constantine A

    2015-09-01

    Bilateral adrenocortical hyperplasia (BAH) in humans and mice has been recently linked to phosphodiesterase (PDE) 8B (PDE8B) and 11 (PDE11A) defects. These findings have followed the discovery that defects of primary genes of the cyclic monophosphatase (cAMP) signaling pathway, such as guanine nucleotide binding alpha subunit and PRKAR1A, are involved in the pathogenesis of BAH in humans; complete absence of Prkar1a in the adrenal cortex of mice also led to pathology that mimicked the human disease. Here, we review the most recent findings in human and mouse studies on PDE8B, a cAMP-specific PDE that appears to be highly expressed in the adrenal cortex and whose deficiency may underlie predisposition to BAH and possibly other human diseases. PMID:25971952

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

    PubMed

    Malamed, S; Carsia, R V

    1983-03-01

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

  15. ATP and noradrenaline activate CREB in astrocytes via noncanonical Ca(2+) and cyclic AMP independent pathways.

    PubMed

    Carriba, Paulina; Pardo, Luis; Parra-Damas, Arnaldo; Lichtenstein, Mathieu P; Saura, Carlos A; Pujol, Aurora; Masgrau, Roser; Galea, Elena

    2012-09-01

    In neurons, it is well established that CREB contributes to learning and memory by orchestrating the translation of experience into the activity-dependent (i.e., driven by neurotransmitters) transcription of plasticity-related genes. The activity-dependent CREB-triggered transcription requires the concerted action of cyclic AMP/protein kinase A and Ca(2+) /calcineurin via the CREB-regulated transcription co-activator (CRTC). It is not known, however, whether a comparable molecular sequence occurs in astrocytes, despite the unquestionable contribution of these cells to brain plasticity. Here we sought to determine whether and how ATP and noradrenaline cause CREB-dependent transcription in rat cortical astrocyte cultures. Both transmitters induced CREB phosphorylation (Western Blots), CREB-dependent transcription (CRE-luciferase reporter assays), and the transcription of Bdnf, a canonical regulator of synaptic plasticity (quantitative RT-PCR). We indentified a Ca(2+) and diacylglycerol-independent protein kinase C at the uppermost position of the cascade leading to CREB-dependent transcription. Notably, CREB-dependent transcription was partially dependent on ERK1/2 and CRTC, but independent of cyclic AMP/protein kinase A or Ca(2+) /calcineurin. We conclude that ATP and noradrenaline activate CREB-dependent transcription in cortical astrocytes via an atypical protein kinase C. It is of relevance that the signaling involved be starkly different to the one described in neurons since there is no convergence of Ca(2+) and cyclic AMP-dependent pathways on CRTC, which, moreover, exerts a modulatory rather than a central role. Our data thus point to the existence of an alternative, non-neuronal, glia-based role of CREB in plasticity.

  16. GABAB receptors modulate catecholamine secretion in chromaffin cells by a mechanism involving cyclic AMP formation.

    PubMed Central

    Oset-Gasque, M. J.; Parramón, M.; González, M. P.

    1993-01-01

    1. The function of gamma-aminobutyric acidB (GABAB) receptors in modulation of catecholamine secretion by chromaffin cells and the possible mechanism involved in this action have been examined. 2. The GABAB agonists (-)-baclofen and 3-aminopropylphosphinic acid (3-APPA) were found to induce a dose-dependent increase of basal catecholamine secretion. The EC50s were 151 +/- 35 microM and 225 +/- 58 microM for baclofen and 3-APPA, respectively. This stimulatory effect was specific since it could be blocked by 0.5 mM of the specific GABAB antagonist CGP-35348. 3. In contrast, preincubation of chromaffin cells with the GABAB agonists was found to inhibit, in a dose-dependent manner, the catecholamine secretion evoked by 10 microM nicotine and 200 microM muscimol. 4. The effects of GABAB agonists on both basal and evoked catecholamine secretion were found to be accompanied by parallel changes in intracellular calcium concentration ([Ca2+]i). GABAB agonists produced a dose-dependent increase in [Ca2+]i which was partially blocked by CGP 35348, but they produced a strong inhibition of the [Ca2+]i increase induced by nicotine and muscimol. 5. The GABAB agonists also produced a dose-dependent increase in intracellular cyclic AMP levels, there being a direct correlation between both increase in catecholamine secretion and in intracellular cyclic AMP levels. 6. The pretreatment of chromaffin cells with pertussis toxin doubled the catecholamine secretion and increased by four times the intracellular cyclic AMP levels evoked by GABAB agonists. 7. The possible involvement of adenylate cyclase in the mechanism of GABAA receptor modulation of catecholamine secretion is discussed. PMID:8306105

  17. 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

  18. 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.

  19. Human dermal microvascular endothelial cells in vitro: effect of cyclic AMP on cellular morphology and proliferation rate.

    PubMed

    Davison, P M; Karasek, M A

    1981-02-01

    Macrovascular endothelial cells isolated from the human umbilical vein and microvessel endothelium from the newborn foreskin dermis differ in their requirements for optimal growth in vitro. In the presence of 5 X 10(-4) M dibutyryl cyclic AMP (Bt2cAMP), human dermal microvessel endothelial cell proliferation rate increased to give a cell number of 203% of controls values by day 10 in culture. The cells retained their characteristic endothelial cell morphology, reached confluence, and could be serially passaged. Cells grown in the absence of Bt2cAMP did not proliferate readily and grew in a disorganized pattern. The effect of Bt2cAMP on microvascular endothelial cell proliferation rate and morphology could be duplicated by cholera toxin (CT) used together with isobutyl methylxanthine (IMX). These agents were found to elevate intracellular levels of cyclic AMP in microvascular endothelium over 40-fold. Human umbilical vein cells in culture failed to respond to either Bt2cAMP or CT together with IMX. The growth-promoting effect of dibutyryl cyclic AMP (Bt2cAMP) on human foreskin dermal microvascular endothelium in vitro is in marked contrast to the lack of response of human umbilical vein cells. These results provide further evidence of differences in the mechanisms that regulate macro and microvessel endothelial cell proliferation in vitro.

  20. The Role of Cyclic AMP in Normalizing the Function of Engineered Human Blood Microvessels in Microfluidic Collagen Gels

    PubMed Central

    Wong, Keith H. K.; Truslow, James G.; Tien, Joe

    2010-01-01

    Nearly all engineered tissues must eventually be vascularized to survive. To this end, we and others have recently developed methods to synthesize extracellular matrix-based scaffolds that contain open microfluidic networks. These scaffolds serve as templates for the formation of endothelial tubes that can be perfused; whether such microvascular structures are stable and/or functional is largely unknown. Here, we show that compounds that elevate intracellular concentrations of the second messenger cyclic AMP (cAMP) strongly normalize the phenotype of engineered human microvessels in microfluidic type I collagen gels. Cyclic AMP-elevating agents promoted vascular stability and barrier function, and reduced cellular turnover. Under conditions that induced the highest levels of cAMP, the physiology of engineered microvessels in vitro quantitatively mirrored that of native vessels in vivo. Computational analysis indicated that cAMP stabilized vessels partly via its enhancement of barrier function. PMID:20303168

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

    PubMed

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

    2016-02-16

    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.

  2. 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

  3. 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.

  4. 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

  5. Biguanides suppress hepatic glucagon signalling by decreasing production of cyclic AMP.

    PubMed

    Miller, Russell A; Chu, Qingwei; Xie, Jianxin; Foretz, Marc; Viollet, Benoit; Birnbaum, Morris J

    2013-02-14

    Glucose production by the liver is essential for providing a substrate for the brain during fasting. The inability of insulin to suppress hepatic glucose output is a major aetiological factor in the hyperglycaemia of type-2 diabetes mellitus and other diseases of insulin resistance. For fifty years, one of the few classes of therapeutics effective in reducing glucose production has been the biguanides, which include phenformin and metformin, the latter the most frequently prescribed drug for type-2 diabetes. Nonetheless, the mechanism of action of biguanides remains imperfectly understood. The suggestion a decade ago that metformin reduces glucose synthesis through activation of the enzyme AMP-activated protein kinase (AMPK) has recently been challenged by genetic loss-of-function experiments. Here we provide a novel mechanism by which metformin antagonizes the action of glucagon, thus reducing fasting glucose levels. In mouse hepatocytes, metformin leads to the accumulation of AMP and related nucleotides, which inhibit adenylate cyclase, reduce levels of cyclic AMP and protein kinase A (PKA) activity, abrogate phosphorylation of critical protein targets of PKA, and block glucagon-dependent glucose output from hepatocytes. These data support a mechanism of action for metformin involving antagonism of glucagon, and suggest an approach for the development of antidiabetic drugs.

  6. Beta-adrenergic regulation of cyclic AMP synthesis in cultured human syncytiotrophoblast.

    PubMed

    Grullon, K; Jacobs, M M; Li, S X; Illsley, N P

    1995-10-01

    Isolated elements of the beta-adrenergic/adenyl cyclase signal transduction system have been studied previously using purified membranes. We used cultured syncytiotrophoblast cells to identify components of this signalling system and the interactions which regulate syncytial adenyl cyclase. Generation of cyclic AMP (cAMP) was stimulated in these cells by both forskolin and isoproterenol but not by dopamine, adenosine, carbachol or prostaglandin E1. Synthesis was also stimulated by treatment with cholera toxin, indicating the involvement of the G-protein, Gs. Somatostatin inhibited isoproterenol- or forskolin-stimulated cAMP generation, an effect which could be blocked by pretreatment of the cells with pertussis toxin, demonstrating the mediation of somatostatin action by Gi. Furthermore, secretion of human chorionic gonadotrophin (hCG) was increased significantly by isoproterenol while somatostatin blocked the isoproterenol-stimulated release of hCG. These results clearly demonstrate that adenyl cyclase in syncytiotrophoblast is controlled by a stimulatory pathway operating through Gs and inhibitory pathway acting through Gi.

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

    PubMed

    Górna, M; Wieckowski, W

    1982-01-01

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

  8. 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

  9. Ethanol increases and vitamin D metabolites decrease the production of cyclic AMP by canine renal cortical membranes.

    PubMed

    Bergmann, P J; Nijs, N; Corvilain, J

    1988-12-01

    Ethanol 0.16% increased cyclic AMP production by canine renal cortical membranes in the basal state and when challenged with different parathyroid hormone or fluoride concentrations. 1,25-dihydroxycholecalciferol (1,25(OH)2D3) 40 pM completely inhibited this effect of ethanol and reversed cyclic AMP production to the level observed in buffer alone. The same inhibitory effect was observed with 25OHD3 and with 24,25-dihydroxycholecalciferol (24,25(OH)2D3). The inhibitory effect was related to the vitamin D metabolites' concentration and was maximal for 160 pM; it was independent of their biological activity. This suggests that the effect is mediated through an interaction with the membrane lipids. The effect of vitamin D metabolites on cyclic AMP production was also observed in the presence of serum proteins and should be taken into account if unextracted plasma is assayed in the renal cortical membrane system for PTH bioactivity.

  10. Histamine induced elevation of cyclic AMP phosphodiesterase activity in human monocytes.

    PubMed

    Holden, C A; Chan, S C; Norris, S; Hanifin, J M

    1987-10-01

    We have previously reported histamine desensitization of human blood mononuclear leukocytes resulting in reduced cAMP responses to beta-adrenergic agonists, histamine and prostaglandin E1. This heterologous desensitization occurred at low, micromolar histamine concentrations and was accompanied by elevation of cAMP-phosphodiesterase (PDE) activity in these cells. We have now investigated the activity of PDE in the lymphocyte and monocyte fractions of mononuclear leukocytes to determine the site of histamine effect. PDE activity per cell was higher in monocytes (0.075 +/- 0.070 units) than lymphocytes (0.026 +/- 0.08) units). Monocytes responded to 10(-6) M histamine stimulation with a much greater increase in PDE activity (0.354 +/- 0.1 units) than did lymphocytes (0.047 +/- 0.015 units). Histamine receptor studies, using thiazolylethylamine and chlorpheniramine as H1-agonist and antagonist respectively and dimaprit and cimetidine as H2-agonists and antagonists respectively, indicated that the histamine stimulation of PDE activity is mediated predominantly through H1 histamine receptor in the monocytes and the H1 receptor in the lymphocytes. Previously histamine had been thought to increase cyclic AMP by acting on H2 receptors to activate adenylate cyclase. Our studies show that stimulation of H1 or H2 receptors by low histamine concentration can cause the opposite effect i.e. increased catabolism and a net reduction in cAMP levels. The localization of this effect predominantly to monocytes indicates a potentially important mechanism for histamine action on immune regulation. PMID:2891264

  11. Effect of triiodothyronine on cyclic AMP and pulmonary function tests in bronchial asthma.

    PubMed

    abdel Khalek, K; el Kholy, M; Rafik, M; Fathalla, M; Heikal, E

    1991-01-01

    This study was undertaken to evaluate the effect of triiodothyronine, determined by pulmonary function tests and c-AMP plasma and sputum levels, in asthmatic children. Twenty-three children clinically euthyroid and complaining of chronic bronchial asthma were given a triiodothyronine (T3) supply for a period of 30 days. Pulmonary function tests, plasma and sputum cyclic AMP and plasma T3 levels were performed prior to and after T3 therapy. Patients were requested to continue on their usual antiasthma medicines and to try reduction of the doses of the drugs they needed as possible. All patients tolerated well the T3 regimen without any adverse effect. They all reported at the end of the 30 days an obvious subjective improvement of their asthmatic conditions with a decrease in the number of exacerbations. Seven patients stopped their usual antiasthmatic medicines, being maintained on T3 only and 3 have decreased the amount of bronchodilators needed. A significant improvement of pulmonary function tests was noted in all patients. Also, significantly increased levels of plasma and sputum c-AMP were observed after T3 administration in comparison to the control and pretest values. No statistical differences were found in plasma T3 between the control and the patients either before or after T3 therapy. The study revealed that T3 administration to clinically euthyroid chronic asthmatic children induced a beneficial effect. This might be through improvement of c-AMP synthesis. T3 in the doses used is devoid of side effects, proves to be a useful adjuvant to classic antiasthma therapy, and may reduce the amount of bronchodilators needed.

  12. 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.

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

    PubMed

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

    2016-02-16

    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

  14. Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness.

    PubMed

    Lolicato, Marco; Bucchi, Annalisa; Arrigoni, Cristina; Zucca, Stefano; Nardini, Marco; Schroeder, Indra; Simmons, Katie; Aquila, Marco; DiFrancesco, Dario; Bolognesi, Martino; Schwede, Frank; Kashin, Dmitry; Fishwick, Colin W G; Johnson, A Peter; Thiel, Gerhard; Moroni, Anna

    2014-06-01

    cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder β-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system. PMID:24776929

  15. Regulatory Action of Calcium Ion on Cyclic AMP-Enhanced Expression of Implantation-Related Factors in Human Endometrial Cells

    PubMed Central

    Kusama, Kazuya; Yoshie, Mikihiro; Tamura, Kazuhiro; Imakawa, Kazuhiko; Isaka, Keiichi; Tachikawa, Eiichi

    2015-01-01

    Decidualization of human endometrial stroma and gland development is mediated through cyclic AMP (cAMP), but the role of intracellular calcium ion (Ca2+) on cAMP mediated-signaling in human endometrial stroma and glandular epithelia has not been well-characterized. The present study was designed to investigate the role of intracellular Ca2+ on cAMP mediated-decidualization and gland maturation events, which can be identified by the up-regulation of prolactin and IGF-binding protein (IGFBP)1 in human endometrial stromal cells (ESCs), and cyclooxygenase 2 (COX2) and prostaglandin E2 (PGE2) and glandular epithelial EM-1 cells. Increases in decidual prolactin and IGFBP-1 transcript levels, induced by cAMP-elevating agents forskolin or dibutyryl cyclic AMP, were inhibited by Ca2+ influx into ESCs with Ca2+ ionophores (alamethicin, ionomycin) in a dose-dependent manner. Conversely, inhibitors of Ca2+ influx through L-type voltage-dependent Ca2+ channel (VDCC), nifedipine and verapamil, enhanced the decidual gene expression. Furthermore, dantrolene, an inhibitor of Ca2+ release from the intracellular Ca2+ store, up-regulated prolactin and IGFBP-1 expression. Ca2+ ionophores decreased intracellular cAMP concentrations, whereas nifedipine, verapamil or dantrolene increased cAMP concentrations in ESCs. In glandular epithelial cells, similar responses in COX2 expression and PGE2 production were found when intracellular cAMP levels were up-regulated by decreases in Ca2+ concentrations. Thus, a marked decrease in cytosolic Ca2+ levels caused the elevation of cAMP concentrations, resulting in enhanced expression of implantation-related factors including decidual markers. These findings suggest that fluctuation in cytosolic Ca2+ concentrations alters intracellular cAMP levels, which then regulate differentiation of endometrial stromal and glandular epithelial cells. PMID:26161798

  16. Regulatory Action of Calcium Ion on Cyclic AMP-Enhanced Expression of Implantation-Related Factors in Human Endometrial Cells.

    PubMed

    Kusama, Kazuya; Yoshie, Mikihiro; Tamura, Kazuhiro; Imakawa, Kazuhiko; Isaka, Keiichi; Tachikawa, Eiichi

    2015-01-01

    Decidualization of human endometrial stroma and gland development is mediated through cyclic AMP (cAMP), but the role of intracellular calcium ion (Ca2+) on cAMP mediated-signaling in human endometrial stroma and glandular epithelia has not been well-characterized. The present study was designed to investigate the role of intracellular Ca2+ on cAMP mediated-decidualization and gland maturation events, which can be identified by the up-regulation of prolactin and IGF-binding protein (IGFBP)1 in human endometrial stromal cells (ESCs), and cyclooxygenase 2 (COX2) and prostaglandin E2 (PGE2) and glandular epithelial EM-1 cells. Increases in decidual prolactin and IGFBP-1 transcript levels, induced by cAMP-elevating agents forskolin or dibutyryl cyclic AMP, were inhibited by Ca2+ influx into ESCs with Ca2+ ionophores (alamethicin, ionomycin) in a dose-dependent manner. Conversely, inhibitors of Ca2+ influx through L-type voltage-dependent Ca2+ channel (VDCC), nifedipine and verapamil, enhanced the decidual gene expression. Furthermore, dantrolene, an inhibitor of Ca2+ release from the intracellular Ca2+ store, up-regulated prolactin and IGFBP-1 expression. Ca2+ ionophores decreased intracellular cAMP concentrations, whereas nifedipine, verapamil or dantrolene increased cAMP concentrations in ESCs. In glandular epithelial cells, similar responses in COX2 expression and PGE2 production were found when intracellular cAMP levels were up-regulated by decreases in Ca2+ concentrations. Thus, a marked decrease in cytosolic Ca2+ levels caused the elevation of cAMP concentrations, resulting in enhanced expression of implantation-related factors including decidual markers. These findings suggest that fluctuation in cytosolic Ca2+ concentrations alters intracellular cAMP levels, which then regulate differentiation of endometrial stromal and glandular epithelial cells. PMID:26161798

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

    PubMed Central

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

    2014-01-01

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

  18. 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

  19. 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

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

    SciTech Connect

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

    1989-01-01

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

  1. DhhP, a Cyclic di-AMP Phosphodiesterase of Borrelia burgdorferi, Is Essential for Cell Growth and Virulence

    PubMed Central

    Ye, Meiping; Zhang, Jun-Jie; Fang, Xin; Lawlis, Gavin B.; Troxell, Bryan; Zhou, Yan; Gomelsky, Mark

    2014-01-01

    Cyclic di-AMP (c-di-AMP) is a recently discovered second messenger in bacteria. Most of work on c-di-AMP signaling has been done in Gram-positive bacteria, firmicutes, and actinobacteria, where c-di-AMP signaling pathways affect potassium transport, cell wall structure, and antibiotic resistance. Little is known about c-di-AMP signaling in other bacteria. Borrelia burgdorferi, the causative agent of Lyme disease, is a spirochete that has a Gram-negative dual membrane. In this study, we demonstrated that B. burgdorferi BB0619, a DHH-DHHA1 domain protein (herein designated DhhP), functions as c-di-AMP phosphodiesterase. Recombinant DhhP hydrolyzed c-di-AMP to pApA in a Mn2+- or Mg2+-dependent manner. In contrast to c-di-AMP phosphodiesterases reported thus far, DhhP appears to be essential for B. burgdorferi growth both in vitro and in the mammalian host. Inactivation of the chromosomal dhhP gene could be achieved only in the presence of a plasmid-encoded inducible dhhP gene. The conditional dhhP mutant had a dramatic increase in intracellular c-di-AMP level in comparison to the isogenic wild-type strain. Unlike what has been observed in Gram-positive bacteria, elevated cellular c-di-AMP in B. burgdorferi did not result in an increased resistance to β-lactamase antibiotics, suggesting that c-di-AMP's functions in spirochetes differ from those in Gram-positive bacteria. In addition, the dhhP mutant was defective in induction of the σS factor, RpoS, and the RpoS-dependent outer membrane virulence factor OspC, which uncovers an important role of c-di-AMP in B. burgdorferi virulence. PMID:24566626

  2. Cyclic AMP Control Measured in Two Compartments in HEK293 Cells: Phosphodiesterase KM Is More Important than Phosphodiesterase Localization

    PubMed Central

    Matthiesen, Karina; Nielsen, Jacob

    2011-01-01

    The intracellular second messenger cyclic AMP (cAMP) is degraded by phosphodiesterases (PDE). The knowledge of individual families and subtypes of PDEs is considerable, but how the different PDEs collaborate in the cell to control a cAMP signal is still not fully understood. In order to investigate compartmentalized cAMP signaling, we have generated a membrane-targeted variant of the cAMP Bioluminiscence Resonance Energy Transfer (BRET) sensor CAMYEL and have compared intracellular cAMP measurements with it to measurements with the cytosolic BRET sensor CAMYEL in HEK293 cells. With these sensors we observed a slightly higher cAMP response to adenylyl cyclase activation at the plasma membrane compared to the cytosol, which is in accordance with earlier results from Fluorescence Resonance Energy Transfer (FRET) sensors. We have analyzed PDE activity in fractionated lysates from HEK293 cells using selective PDE inhibitors and have identified PDE3 and PDE10A as the major membrane-bound PDEs and PDE4 as the major cytosolic PDE. Inhibition of membrane-bound or cytosolic PDEs can potentiate the cAMP response to adenylyl cyclase activation, but we see no significant difference between the potentiation of the cAMP response at the plasma membrane and in cytosol when membrane-bound and cytosolic PDEs are inhibited. When different levels of stimulation were tested, we found that PDEs 3 and 10 are mainly responsible for cAMP degradation at low intracellular cAMP concentrations, whereas PDE4 is more important for control of cAMP at higher concentrations. PMID:21931705

  3. EppA, a Putative Substrate of DdERK2, Regulates Cyclic AMP Relay and Chemotaxis in Dictyostelium discoideum

    PubMed Central

    Chen, Songyang; Segall, Jeffrey E.

    2006-01-01

    The mitogen-activated protein kinase DdERK2 is critical for cyclic AMP (cAMP) relay and chemotaxis to cAMP and folate, but the details downstream of DdERK2 are unclear. To search for targets of DdERK2 in Dictyostelium discoideum,32PO43−-labeled protein samples from wild-type and Dderk2− cells were resolved by 2-dimensional electrophoresis. Mass spectrometry was used to identify a novel 45-kDa protein, named EppA (ERK2-dependent phosphoprotein A), as a substrate of DdERK2 in Dictyostelium. Mutation of potential DdERK2 phosphorylation sites demonstrated that phosphorylation on serine 250 of EppA is DdERK2 dependent. Changing serine 250 to alanine delayed development of Dictyostelium and reduced Dictyostelium chemotaxis to cAMP. Although overexpression of EppA had no significant effect on the development or chemotaxis of Dictyostelium, disruption of the eppA gene led to delayed development and reduced chemotactic responses to both cAMP and folate. Both eppA gene disruption and overexpression of EppA carrying the serine 250-to-alanine mutation led to inhibition of intracellular cAMP accumulation in response to chemoattractant cAMP, a pivotal process in Dictyostelium chemotaxis and development. Our studies indicate that EppA regulates extracellular cAMP-induced signal relay and chemotaxis of Dictyostelium. PMID:16835457

  4. Cyclic AMP level of lymphocytes in patients with systemic lupus erythematosus and its relation to disease activity.

    PubMed

    Phi, N C; Takáts, A; Binh, V H; Vien, C V; González-Cabello, R; Gergely, P

    1989-11-01

    The basal and stimulated intracellular cyclic AMP (cAMP) levels of peripheral blood mononuclear cells (PBMC) of 16 control subjects and 14 patients with systemic lupus erythematosus (SLE), all fulfilling the ARA criteria, were studied. No significant difference in basal cAMP level was observed between SLE patients and controls. SLE lymphocytes (both active and inactive) elicited a diminished response to aminophylline and prostaglandin E2 (PGE2). No correlation was seen between disease activity and either baseline cAMP levels or response to these stimulators. We suggest an intrinsic (not disease activity-related) impairment of the adenylate cyclase-dependent regulatory mechanism in the PBMC of SLE patients, which may result in a defective IL-2 production and IL-2 dependent biological functions. PMID:2558072

  5. Cyclic di-AMP Is Critical for Listeria monocytogenes Growth, Cell Wall Homeostasis, and Establishment of Infection

    PubMed Central

    Witte, Chelsea E.; Whiteley, Aaron T.; Burke, Thomas P.; Sauer, John-Demian; Portnoy, Daniel A.; Woodward, Joshua J.

    2013-01-01

    ABSTRACT Listeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection. PMID:23716572

  6. 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

  7. 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.

  8. Distribution of cyclic AMP phosphodiesterase in microdissected periportal and perivenous rat liver tissue with different dietary states.

    PubMed

    Runge, D; Jungermann, K

    1991-01-01

    Cyclic AMP phosphodiesterase was measured in liver homogenates and microdissected periportal and perivenous liver tissue from rats in different dietary states under different conditions of substrate saturation and effector stimulation. A radiochemical microtest, more sensitive by 2-3 orders of magnitude than the usual assay, was established for the determination of the activity in liver samples corresponding to 200-800 ng dry weight. At saturating cyclic AMP concentrations (46 microM) phosphodiesterase was homogeneously distributed within the liver acinus of fed rats. Starvation for 48 h led to a decrease in the overall activity and to a heterogenous distribution with slightly higher activities in the perivenous zone. At physiological cyclic AMP concentrations (1.8 microM) phosphodiesterase showed a flat zonal gradient in livers of fed rats with higher levels in the periportal zone; after 48 h starvation it was homogeneously distributed. In the presence of cyclic GMP (2 microM) the basal activity at physiological substrate concentrations was stimulated to a greater extent in the perivenous zone. This led to a homogeneous activity distribution in the fed state and to a heterogenous pattern with a slight perivenous maximum in the fasted state. Thus there was no or only a small zonal heterogeneity of signal transmitting enzymes such as cyclic AMP phosphodiesterase and glucagon-stimulated adenylate cyclase (Zierz and Jungermann 1984). This similar signal transducing capacity in the periportal and the perivenous area will contribute to maintain the zonation of signal input due to the hormone concentration gradients across the liver acinus.

  9. A novel indirect sequence readout component in the E. coli cyclic AMP receptor protein operator.

    PubMed

    Lindemose, Søren; Nielsen, Peter Eigil; Valentin-Hansen, Poul; Møllegaard, Niels Erik

    2014-03-21

    The cyclic AMP receptor protein (CRP) from Escherichia coli has been extensively studied for several decades. In particular, a detailed characterization of CRP interaction with DNA has been obtained. The CRP dimer recognizes a consensus sequence AANTGTGANNNNNNTCACANTT through direct amino acid nucleobase interactions in the major groove of the two operator half-sites. Crystal structure analyses have revealed that the interaction results in two strong kinks at the TG/CA steps closest to the 6-base-pair spacer (N6). This spacer exhibits high sequence variability among the more than 100 natural binding sites in the E. coli genome, but the exact role of the N6 region in CRP interaction has not previously been systematic examined. Here we employ an in vitro selection system based on a randomized N6 spacer region to demonstrate that CRP binding to the lacP1 site may be enhanced up to 14-fold or abolished by varying the N6 spacer sequences. Furthermore, on the basis of sequence analysis and uranyl (UO2(2+)) probing data, we propose that the underlying mechanism relies on N6 deformability.

  10. RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messenger Cyclic di-AMP.

    PubMed

    Kellenberger, Colleen A; Chen, Chen; Whiteley, Aaron T; Portnoy, Daniel A; Hammond, Ming C

    2015-05-27

    Cyclic di-AMP (cdiA) is a second messenger predicted to be widespread in Gram-positive bacteria, some Gram-negative bacteria, and Archaea. In the human pathogen Listeria monocytogenes, cdiA is an essential molecule that regulates metabolic function and cell wall homeostasis, and decreased levels of cdiA result in increased antibiotic susceptibility. We have generated fluorescent biosensors for cdiA through fusion of the Spinach2 aptamer to ligand-binding domains of cdiA riboswitches. The biosensor was used to visualize intracellular cdiA levels in live L. monocytogenes strains and to determine the catalytic domain of the phosphodiesterase PdeA. Furthermore, a flow cytometry assay based on this biosensor was used to screen for diadenylate cyclase activity and confirmed the enzymatic activity of DisA-like proteins from Clostridium difficile and Methanocaldococcus jannaschii. Thus, we have expanded the development of RNA-based biosensors for in vivo metabolite imaging in Gram-positive bacteria and have validated the first dinucleotide cyclase from Archaea.

  11. 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

  12. Cyclic AMP inhibits and putrescine represses expression of the speA gene encoding biosynthetic arginine decarboxylase in Escherichia coli.

    PubMed Central

    Moore, R C; Boyle, S M

    1991-01-01

    The speA gene of Escherichia coli encodes biosynthetic arginine decarboxylase (ADC), the first of two enzymes in a putrescine biosynthetic pathway. The activity of ADC is negatively regulated by mechanisms requiring cyclic AMP (cAMP) and cAMP receptor protein (CRP) or putrescine. A 2.1-kb BamHI fragment containing the speA-metK intergenic region, speA promoter, and 1,389 bp of the 5' end of the speA coding sequence was used to construct transcriptional and translational speA-lacZ fusion plasmids. A single copy of either type of speA-lacZ fusion was transferred into the chromosomes of Escherichia coli KC14-1, CB806, and MC4100, using bacteriophage lambda. The speA gene in lysogenized strains remained intact and served as a control. Addition of 5 mM cAMP to lysogenic strains resulted in 10 to 37% inhibition of ADC activity, depending on the strain used. In contrast, the addition of 5 or 10 mM cAMP to these strains did not inhibit the activity of beta-galactosidase (i.e., ADC::beta-galactosidase). Addition of 10 mM putrescine to lysogenized strains resulted in 24 to 31% repression of ADC activity and 41 to 47% repression of beta-galactosidase activity. E. coli strains grown in 5 mM cAMP and 10 mM putrescine produced 46 to 61% less ADC activity and 41 to 52% less beta-galactosidase activity. cAMP (0.1 to 10 mM) did not inhibit ADC activity assayed in vitro. The effects of cAMP and putrescine on ADC activity were additive, indicating the use of independent regulatory mechanisms. These results show that cAMP acts indirectly to inhibit ADC activity and that putrescine causes repression of speA transcription. PMID:1646785

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

    PubMed

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

    2015-11-13

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

  14. Additional evidence for the cyclic GMP signaling pathway resulting in the photophobic behavior of Stentor coeruleus.

    PubMed

    Walerczyk, M; Fabczak, S

    2001-12-01

    We report that exo- and endogenous guanosine 3',5'-cyclic monophosphate (cGMP) specifically influenced the photophobic response. In behavioral experiments the slowly hydrolyzable and membrane-permeable analogs of cGMP (8-bromo-cGMP [Br-cGMP] and N6,2'-o-dibutyryl-cGMP) dramatically prolonged the time for ciliary stop response and decreased the duration of ciliary reversal in a dose-dependent manner. When analogs of adenosine 3',5'-cyclic monophosphate (cAMP) (8-bromo-cAMP or N6,2'-o-dibutyryl-cAMP) were used, no essential effects were detected on the kinetics of the photophobic response. Both nonspecific cyclic nucleotide phosphodiesterase (PDE) activity inhibitors (3-isobutyl-1-methylxanthine [IBMX] and 1,3-dimethylxanthine [theophylline]) and the highly specific cGMP-PDE activity inhibitor 1,4-dihydro-5-[2-propoxyphenyl]-7H-1,2,3-triazolo[4,5-d]pyrimidine-7-one (zaprinast) mimicked the effects of cGMP analogs. Treatment of cells with an inhibitor of guanylate cyclase activity (6-anilino-5,8-quinolinedione [LY 83583]) exerted an effect opposite to that of cGMP analogs and PDE activity inhibitors. The positive physiological effect of LY 83583 was significantly diminished in ciliates that were treated simultaneously with Br-cGMP. In an assay of cell cyclic nucleotide content, the exposure of dark-adapted Stentor to light evoked a transient decrease in the basal level of intracellular cGMP. Alterations in internal cGMP levels were more distinct when the intensity of applied illumination was increased. In the presence of IBMX or theophylline the basal content of cGMP was markedly enhanced, and the photoinduced changes in cGMP level were less pronounced. In this paper the possible whole molecular mechanism by which the ciliary orientation in Stentor is controlled by light is presented. PMID:11783940

  15. Electrical transients in the cell-volume response to cyclic AMP of the tsetse fly Malpighian tubule

    PubMed

    Isaacson; Nicolson

    1996-01-01

    1. Using cyclic AMP to stimulate perfused tsetse fly Malpighian tubules bathed in SO42- Ringer frequently causes an immediate but transient peak in transtubular potential (Vt), before stabilisation of Vt at an increased value. 2. These transients were investigated by monitoring the associated changes in cable properties and current­voltage (I/V) relationships. Tubules were perfused and bathed in either Cl- Ringer or SO42- Ringer (containing 8 mmol l-1 Cl-). 3. Tubules bathed in Cl- Ringer showed a transient swelling of the cells on exposure to cyclic AMP. Cable analysis confirmed the visually observed narrowing of the tubular lumen and revealed transient increases in core resistance (Rc) and transtubular resistance (Rt). As the cells returned to their initial volume, the lumen became distended, and Rc and Rt fell below their initial levels. These changes were accompanied by an increase, and a subsequent decrease, in the slope of the I/V plot. 4. None of the above changes was apparent in SO42- Ringer, other than a fall in Rt and in the slope of the I/V plot. 5. The results suggest that, in Cl- Ringer, cyclic AMP induces swelling of the tubular cells by promoting increased basolateral solute (and water) entry and that the subsequent rapid return to normal cell volume, with a concomitant progressive increase in the rate of tubular secretion, reflects the operation of a specific cell-volume regulatory mechanism of transepithelial transport. 6. The cyclic-AMP-induced peak that occurs in Vt in SO42- Ringer appears to be primarily due to a transient overshoot in the fall in series resistance (i.e. an increase in basolateral Na+ conductance), accompanied by a proportionately lesser increase in shunt resistance.

  16. Role of ecdysone, pupariation factors, and cyclic AMP in formation and tanning of the puparium of the fleshfly Sarcophaga bullata.

    PubMed

    Seligman, M; Blechl, A; Blechl, J; Herman, P; Fraenkel, G

    1977-10-01

    Two pupariation factors, anterior retraction factor (ARF) and puparium tanning factor (PTF), are absent from the hemolymph of larvae at the time of tanning accelerated by ARF/PTF, cyclic AMP, or dopamine. ARF and PTF are not involved in derepression of dopa decarboxylase (aromatic L-amino-acid decarboxylase, aromatic L-amino-acid carboxy-lyase, EC 4.1.1.28) synthesis initiated by ecdysone. Tanning is entirely inhibited by injection of two transcriptional inhibitors, actinomycin and BrdUrd, and two translational inhibitors, puromycin and cycloheximide. Retraction activity is more severely inhibited by the transcriptional than by the translational inhibitors. A tanning response is initiated by cyclic AMP in the presence of the transcriptional but not the translational inhibitors. Dihydric tanning substances (dopa, dopamine) initiate tanning in the presence of both types of inhibitors. Release of ARF and PTF from the central nervous system is inhibited by the four inhibitors. ARF totally reverses the inhibitory effects on retraction, whereas PTF does not reverse inhibition of tanning. These data are interpreted to mean that PTF is concerned with the regulation of two components of the tanning response: (i) acceleration of synthesis of a particular protein (associated with the tyrosine hydroxylation complex), and (ii) activation via cyclic AMP of a component of the tyrosine hydroxylating system.

  17. Group B Streptococcus Degrades Cyclic-di-AMP to Modulate STING-Dependent Type I Interferon Production.

    PubMed

    Andrade, Warrison A; Firon, Arnaud; Schmidt, Tobias; Hornung, Veit; Fitzgerald, Katherine A; Kurt-Jones, Evelyn A; Trieu-Cuot, Patrick; Golenbock, Douglas T; Kaminski, Pierre-Alexandre

    2016-07-13

    Induction of type I interferon (IFN) in response to microbial pathogens depends on a conserved cGAS-STING signaling pathway. The presence of DNA in the cytoplasm activates cGAS, while STING is activated by cyclic dinucleotides (cdNs) produced by cGAS or from bacterial origins. Here, we show that Group B Streptococcus (GBS) induces IFN-β production almost exclusively through cGAS-STING-dependent recognition of bacterial DNA. However, we find that GBS expresses an ectonucleotidase, CdnP, which hydrolyzes extracellular bacterial cyclic-di-AMP. Inactivation of CdnP leads to c-di-AMP accumulation outside the bacteria and increased IFN-β production. Higher IFN-β levels in vivo increase GBS killing by the host. The IFN-β overproduction observed in the absence of CdnP is due to the cumulative effect of DNA sensing by cGAS and STING-dependent sensing of c-di-AMP. These findings describe the importance of a bacterial c-di-AMP ectonucleotidase and suggest a direct bacterial mechanism that dampens activation of the cGAS-STING axis. PMID:27414497

  18. Adenylate cyclase and the cyclic AMP receptor protein modulate stress resistance and virulence capacity of uropathogenic Escherichia coli.

    PubMed

    Donovan, Grant T; Norton, J Paul; Bower, Jean M; Mulvey, Matthew A

    2013-01-01

    In many bacteria, the second messenger cyclic AMP (cAMP) interacts with the transcription factor cAMP receptor protein (CRP), forming active cAMP-CRP complexes that can control a multitude of cellular activities, including expanded carbon source utilization, stress response pathways, and virulence. Here, we assessed the role of cAMP-CRP as a regulator of stress resistance and virulence in uropathogenic Escherichia coli (UPEC), the principal cause of urinary tract infections worldwide. Deletion of genes encoding either CRP or CyaA, the enzyme responsible for cAMP synthesis, attenuates the ability of UPEC to colonize the bladder in a mouse infection model, dependent on intact innate host defenses. UPEC mutants lacking cAMP-CRP grow normally in the presence of glucose but are unable to utilize alternate carbon sources like amino acids, the primary nutrients available to UPEC within the urinary tract. Relative to the wild-type UPEC isolate, the cyaA and crp deletion mutants are sensitive to nitrosative stress and the superoxide generator methyl viologen but remarkably resistant to hydrogen peroxide (H(2)O(2)) and acid stress. In the mutant strains, H(2)O(2) resistance correlates with elevated catalase activity attributable in part to enhanced translation of the alternate sigma factor RpoS. Acid resistance was promoted by both RpoS-independent and RpoS-dependent mechanisms, including expression of the RpoS-regulated DNA-binding ferritin-like protein Dps. We conclude that balanced input from many cAMP-CRP-responsive elements, including RpoS, is critical to the ability of UPEC to handle the nutrient limitations and severe environmental stresses present within the mammalian urinary tract.

  19. 8-Chloro-cyclic AMP inhibits autocrine and angiogenic growth factor production in human colorectal and breast cancer.

    PubMed

    Bianco, C; Tortora, G; Baldassarre, G; Caputo, R; Fontanini, G; Chinè, S; Bianco, A R; Ciardiello, F

    1997-03-01

    8-Chloro-cyclic AMP (8-Cl-cAMP) is a cAMP analogue that specifically down-regulates type I protein kinase A, a signaling protein directly involved in cell proliferation and neoplastic transformation, and that causes growth inhibition in a variety of human cancer cell types. In this report, we have investigated the effects of 8-Cl-cAMP on the expression of several growth factors in human colon (GEO and LS174T) and breast (MDA-MB468) cancer cell lines. 8-Cl-cAMP treatment caused in the three cancer cell lines a significant dose- and time-dependent inhibition in the expression of various endogenous autocrine growth factors, such as transforming growth factor alpha, amphiregulin, and CRIPTO, and of two angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor, at both the mRNA and protein levels. Furthermore, 8-Cl-cAMP treatment markedly inhibited the ability of all three cell lines to invade a basement membrane matrix in a chemoinvasion assay. Finally, 8-Cl-cAMP-induced inhibition of GEO tumor growth in nude mice was accompanied by a significant suppression of transforming growth factor alpha, amphiregulin, CRIPTO, basic fibroblast growth factor, and vascular endothelial growth factor production by the tumor cells, and of neoangiogenesis, as detected by factor VIII staining of host blood cells. These results demonstrate that 8-Cl-cAMP is a novel anticancer drug that inhibits the production of various autocrine and paracrine tumor growth factors that are important in sustaining autonomous local growth and facilitate invasion and metastasis.

  20. Cyclic AMP and its functional relationship in Tetrahymena: a comparison between phagocytosis and glucose uptake.

    PubMed

    Csaba, G; Nagy, S U; Lantos, T

    1978-01-01

    In Tetrahymena, an increase in the level of cAMP is accompanied by an increased phagocytotic rate, whereas increased sugar uptake is parallelled by a decreased cAMP level. The increase in cAMP level seems to be decisive with respect to phagocytosis as a basic phenomenon of life. In the action of epinephrine, however, some mechanism other than cAMP mediation may be involved. Depending on concentration, one hormone may provoke either an increase or a decrease in cAMP level, and this in turn triggers the corresponding function.

  1. 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

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

    PubMed

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

    2015-10-20

    TREX1 is an exonuclease that digests DNA in the cytoplasm. Loss-of-function mutations of TREX1 are linked to Aicardi-Goutieres Syndrome (AGS) and systemic lupus erythematosus (SLE) in humans. Trex1(-/-) mice exhibit autoimmune and inflammatory phenotypes that are associated with elevated expression of interferon (IFN)-induced genes (ISGs). Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates the IFN pathway. Upon binding to DNA, cGAS is activated to catalyze the synthesis of cGAMP, which functions as a second messenger that binds and activates the adaptor protein STING to induce IFNs and other cytokines. Here we show that genetic ablation of cGas in Trex1(-/-) mice eliminated all detectable pathological and molecular phenotypes, including ISG induction, autoantibody production, aberrant T-cell activation, and lethality. Even deletion of just one allele of cGas largely rescued the phenotypes of Trex1(-/-) mice. Similarly, deletion of cGas in mice lacking DNaseII, a lysosomal enzyme that digests DNA, rescued the lethal autoimmune phenotypes of the DNaseII(-/-) mice. Through quantitative mass spectrometry, we found that cGAMP accumulated in mouse tissues deficient in Trex1 or DNaseII and that this accumulation was dependent on cGAS. These results demonstrate that cGAS activation causes the autoimmune diseases in Trex1(-/-) and DNaseII(-/-) mice and suggest that inhibition of cGAS may lead to prevention and treatment of some human autoimmune diseases caused by self-DNA. PMID:26371324

  3. Effects of flavonoids on cyclic AMP phosphodiesterase and lipid mobilization in rat adipocytes.

    PubMed

    Kuppusamy, U R; Das, N P

    1992-10-01

    Thirty-one flavonoids were tested for their effects on low Km phosphodiesterase with cyclic AMP as the substrate. Quercetin, luteolin, scutellarein, phloretin and genistein showed inhibitory potencies comparable to or greater than 3-isobutyl-2-methylxanthine (EC50 30-50 microM). Only four compounds namely, catechin, epicatechin, taxifolin and fustin stimulated the enzyme activity (stimulatory EC50 130-240 microM). The most potent phosphodiesterase (PDE) inhibitors were aglycones that had a C2.3 double bond, a keto group at C4 and hydroxyls at C3' and/or C4'. However, when the C-ring is opened then the requirement for the C2.3 double bond is eliminated. The same series of flavonoids were also tested for their lipolytic activity. The structural features required for effective synergistic lipolysis (with epinephrine) were generally similar to that required for potent PDE inhibition except that, for lipolytic activity, an intact C-ring was necessary. Fisetin and quercetin having the above-mentioned structure showed a dose- and time-dependent increase in lipolysis which was synergistic with epinephrine. Only butein and hesperetin showed inhibition of epinephrine-induced lipolysis, and their effect was dose-dependent. A time-course study indicated that hesperetin was able to delay the lipolytic action of epinephrine. It is most likely that the lipolytic effects of these compounds were not a result of PDE inhibition, as the orders of potency for the two activities had poor correlation. Apparently, the effective lipolytic flavonoids were also potent PDE inhibitors but not all the PDE inhibitors were able to induce lipolysis.

  4. 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

  5. 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.

  6. On the mechanism of action of lead in the testis: in vitro suppression of FSH receptors, cyclic AMP and steroidogenesis.

    PubMed

    Wiebe, J P; Salhanick, A I; Myers, K I

    1983-04-25

    Previous evidence has shown that prenatal and neonatal exposure to low levels of Pb result in decreased FSH binding and steroidogenesis in the testes at the onset of puberty. The purpose of the present study was to determine by in vitro methods, if Pb acts by interfering directly with hormone binding, cyclic AMP production and steroidogenic enzyme activity. Sertoli cells were isolated from testes of prepubertal rats and cultured in the presence of 2.64 x 10(-4)M of either NaAc (control) or PbAc for 1, 4, 24, 48, 96 or 144 hr. There was no reduction in FSH binding and in FSH-induced cyclic AMP after a 1-4 hr exposure to Pb. After a 24-hr exposure to Pb, the cells exhibited a 10-20% decrease in FSH binding and cyclic AMP production and after 96 hr there was a 75% decrease in these 2 parameters. The inhibition was greater in cells from 16 day old than from 20 day old rats, so that in the former, after a 144 hr exposure the FSH-induced cyclic AMP of the Pb exposed cells was only 3% of the amount produced by the NaAc exposed cells (i.e. a 97% inhibition). After in vitro exposure to Pb for 48 hr, the steroidogenic activity (progesterone conversion to steroid metabolites) of Sertoli cells was significantly reduced and their steroidogenesis was no longer stimulated by FSH. A crude testicular enzyme preparation containing 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) exhibited approximately 25% reduction in activity if the assay buffer contained PbCl2 instead of the equivalent in NaCl. Prolonged in vivo exposure to Pb resulted in approximately 50% reduction in 3 beta-HSD activity. This is the first indication that in the testis Pb may act directly (immediate effect) by suppressing enzyme activities, and indirectly (long term effect) by reducing gonadotropin-receptor binding and the resultant cyclic AMP production.

  7. Cyclic AMP Analog Blocks Kinase Activation by Stabilizing Inactive Conformation: Conformational Selection Highlights a New Concept in Allosteric Inhibitor Design*

    PubMed Central

    Badireddy, Suguna; Yunfeng, Gao; Ritchie, Mark; Akamine, Pearl; Wu, Jian; Kim, Choel W.; Taylor, Susan S.; Qingsong, Lin; Swaminathan, Kunchithapadam; Anand, Ganesh S.

    2011-01-01

    The regulatory (R) subunit of protein kinase A serves to modulate the activity of protein kinase A in a cAMP-dependent manner and exists in two distinct and structurally dissimilar, end point cAMP-bound “B” and C-subunit-bound “H”-conformations. Here we report mechanistic details of cAMP action as yet unknown through a unique approach combining x-ray crystallography with structural proteomics approaches, amide hydrogen/deuterium exchange and ion mobility mass spectrometry, applied to the study of a stereospecific cAMP phosphorothioate analog and antagonist((Rp)-cAMPS). X-ray crystallography shows cAMP-bound R-subunit in the B form but surprisingly the antagonist Rp-cAMPS-bound R-subunit crystallized in the H conformation, which was previously assumed to be induced only by C-subunit-binding. Apo R-subunit crystallized in the B form as well but amide exchange mass spectrometry showed large differences between apo, agonist and antagonist-bound states of the R-subunit. Further ion mobility reveals the apo R-subunit as an ensemble of multiple conformations with collisional cross-sectional areas spanning both the agonist and antagonist-bound states. Thus contrary to earlier studies that explained the basis for cAMP action through “induced fit” alone, we report evidence for conformational selection, where the ligand-free apo form of the R-subunit exists as an ensemble of both B and H conformations. Although cAMP preferentially binds the B conformation, Rp-cAMPS interestingly binds the H conformation. This reveals the unique importance of the equatorial oxygen of the cyclic phosphate in mediating conformational transitions from H to B forms highlighting a novel approach for rational structure-based drug design. Ideal inhibitors such as Rp-cAMPS are those that preferentially “select” inactive conformations of target proteins by satisfying all “binding” constraints alone without inducing conformational changes necessary for activation. PMID:21081668

  8. Involvement of cyclic AMP systems in morphine physical dependence in mice: prevention of development of morphine dependence by rolipram, a phosphodiesterase 4 inhibitor

    PubMed Central

    Mamiya, Takayoshi; Noda, Yukihiro; Ren, Xiuhai; Hamdy, Moustafa; Furukawa, Shoei; Kameyama, Tsutomu; Yamada, Kiyofumi; Nabeshima, Toshitaka

    2001-01-01

    In this study, we examined whether morphine dependence was inhibited by rolipram, a cyclic AMP selective phosphodiesterase inhibitor in mice, since a role for the cyclic AMP systems in the development of morphine dependence has been reported. Mice, which received morphine (10 mg kg−1 s.c.) twice a day for 5 days showed withdrawal syndromes such as jumping, rearing and forepaw tremor following naloxone challenge (5 mg kg−1 i.p.) on the 6th day. Such mice exhibited a significant elevation of cyclic AMP levels in the thalamus compared to control mice. However, co-administration of rolipram (1 mg kg−1 i.p.) with morphine for 5 days significantly attenuated the severity of the withdrawal syndrome and the increase in the cyclic AMP levels after the administration of naloxone. In naïve mice, acute morphine treatment (10 mg kg−1 s.c.) decreased cyclic AMP levels in the thalamus and cerebral cortex 10 min later. The decrease of cyclic AMP levels induced by acute morphine treatment was blocked by co-administration of rolipram (1 mg kg−1 i.p.). However, acute rolipram did not affect the naloxone-precipitated morphine withdrawal syndrome. These results suggest that the elevation of the cyclic AMP levels is involved in the development of morphine withdrawal syndrome and that blockade of the morphine-induced reduction of cyclic AMP levels by chronic rolipram inhibits the development of dependence and the behavioural and biochemical changes induced by naloxone. Furthermore, rolipram may be a useful drug for attenuating the development of morphine dependence. PMID:11226142

  9. Action of 50 Hz magnetic fields on cyclic AMP and intercellular communication in monolayers and spheroids of mammalian cells

    SciTech Connect

    Schimmelpfeng, J.; Stein, J.C.; Dertinger, H.

    1995-12-31

    To investigate the influence of physiological parameters such as cell density and three-dimensional cell contact on the biological action of a 2mT/50 Hz magnetic field, mouse fibroblasts were exposed as monolayers and as multicellular spheroids. Changes in cyclic AMP content of cells and alterations in gap junction-mediated intercellular communication were measured immediately after 5 min of exposure to the field. In monolayers of intermediate cell density (1 {times} 10{sup 5} cells/cm{sup 2}), the field treatment caused an increase in cAMP to 121% of the control level, whereas, at 3 {times} 10{sup 5} cells/cm{sup 2} (near confluence), a decrease to 88% of the unexposed cells was observed. Furthermore, field exposure stimulated gap-junction communication to 160% of the control level as determined by Lucifer yellow dye exchange. In spheroids, alterations in the radial profile of cellular cAMP were observed that were due both to field-induced local cAMP changes and to increased gap-junction permeability for this second messenger, the latter causing radial cAMP gradients to be flattened. The results indicate a strong dependence of field action on physiological parameters of the system exposed.

  10. 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

  11. 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

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

    PubMed Central

    Alqurashi, May; Gehring, Chris; Marondedze, Claudius

    2016-01-01

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

  13. Cyclic AMP-dependent phosphorylation modifies the gating properties of L-type Ca2+ channels in bovine adrenal chromaffin cells.

    PubMed

    Doupnik, C A; Pun, R Y

    1992-01-01

    We investigated the effects of cAMP-dependent phosphorylation on the voltage- and time-dependent gating properties of Ca2+ channel currents recorded from bovine adrenal chromaffin cells under whole-cell voltage clamp. Extracellular perfusion with the membrane-permeant activator of cAMP-dependent protein kinase, 8-bromo(8-Br)-cAMP (1 mM), caused a 49%, 29%, and 21% increase in Ca2+ current (ICa) amplitudes evoked by voltage steps to 0, +10, and +20 mV respectively (mean values from eight cells, p less than or equal to 0.05). Analysis of voltage-dependent steady-state activation (m infinity) curves revealed a 0.70 +/- 0.27 charge increase in the activation-gate valency (zm) following 8-Br-cAMP perfusion. Similar responses were observed when Ba2+ was the charge carrier, where zm was increased by 1.33 +/- 0.34 charges (n = 8). The membrane potential for half activation (V1/2) was also significantly shifted 6 mV more negative for IBa (mean, n = 8). The time course for IBa (and ICa) activation was well described by second-order m2 kinetics. The derived time constant for activation (tau m) was voltage-dependent, and the tau m/V relation shifted negatively after 8-Br-cAMP treatment. Ca2+ channel gating rates were derived from the tau m and m infinity 2 values according to a Hodgkin-Huxley type m2 activation process. The forward rate (alpha m) for channel activation was increased by 8-Br-cAMP at membrane potentials greater than or equal to 0 mV, and the backward rate (beta m) decreased at potentials less than or equal to + 10 mV. Time-dependent inactivation of ICa consisted of a slowly decaying component (tau h approximately 300 ms) and a "non-inactivating" steady-state component. The currents contributed by the two inactivation processes displayed different voltage dependences, the effects of 8-Br-cAMP being exclusively on the slowly inactivating L-type component.

  14. Temporal and spatial regulation of cAMP signaling in disease: role of cyclic nucleotide phosphodiesterases.

    PubMed

    Otero, Carolina; Peñaloza, Juan P; Rodas, Paula I; Fernández-Ramires, Ricardo; Velasquez, Luis; Jung, Juan E

    2014-12-01

    Since its discovery, cAMP has been proposed as one of the most versatile second messengers. The remarkable feature of cAMP to tightly control highly diverse physiological processes, including metabolism, homeostasis, secretion, muscle contraction, cell proliferation and migration, immune response, and gene transcription, is reflected by millions of different articles worldwide. Compartmentalization of cAMP in space and time, maintained by mainly phosphodiesterases, contributes to the maintenance of equilibrium inside the cell where one signal can trigger many different events. Novel cAMP sensors seem to carry out certain unexpected signaling properties of cAMP and thereby to permit delicate adaptations of biologic responses. Measuring space and time events with biosensors will increase our current knowledge on the pathophysiology of diseases, such as chronic obstructive pulmonary disease, asthma, cognitive impairment, cancer, and renal and heart failure. Further insights into the cAMP dynamics will help to optimize the pharmacological treatment for these diseases.

  15. Temporal and spatial regulation of cAMP signaling in disease: role of cyclic nucleotide phosphodiesterases.

    PubMed

    Otero, Carolina; Peñaloza, Juan P; Rodas, Paula I; Fernández-Ramires, Ricardo; Velasquez, Luis; Jung, Juan E

    2014-12-01

    Since its discovery, cAMP has been proposed as one of the most versatile second messengers. The remarkable feature of cAMP to tightly control highly diverse physiological processes, including metabolism, homeostasis, secretion, muscle contraction, cell proliferation and migration, immune response, and gene transcription, is reflected by millions of different articles worldwide. Compartmentalization of cAMP in space and time, maintained by mainly phosphodiesterases, contributes to the maintenance of equilibrium inside the cell where one signal can trigger many different events. Novel cAMP sensors seem to carry out certain unexpected signaling properties of cAMP and thereby to permit delicate adaptations of biologic responses. Measuring space and time events with biosensors will increase our current knowledge on the pathophysiology of diseases, such as chronic obstructive pulmonary disease, asthma, cognitive impairment, cancer, and renal and heart failure. Further insights into the cAMP dynamics will help to optimize the pharmacological treatment for these diseases. PMID:24750474

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

    PubMed

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

    2006-12-15

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

  17. Cyclic AMP pathway modifies memory through neural cell adhesion molecule alterations in the rat hippocampus.

    PubMed

    Razmi, Ali; Sahebgharani, Mousa; Khani, Mohammad Hossein; Paylakhi, Seyed Hassan; Faizi, Mehrdad; Zarrindast, Mohammad-Reza

    2014-01-01

    Neural Cell Adhesion Molecules (NCAMs) are known to influence memory by affecting neural cell-cell and cell-extracellular matrix junctions. This study investigated the possible role of cAMP pathway in the expression of hippocampal NCAM and its polysialylated derivative (PSA-NCAM). The following pharmacological tools were employed for manipulation of cAMP pathway: a) forskolin; the activator of adenylyl cyclase (AC), b) 8-Br-cAMP; a protein kinase A (PKA) agonist, c) 8-pCPT-2'-O-Me-cAMP; a selective enhancer of exchange protein activated by cAMP (Epac) and d) Rp-cAMP; a PKA inhibitor. Memory acquisition was tested by passive avoidance paradigm after injecting the above compounds for three consecutive days into the CA1 region of dorsal hippocampus of rats. Forskolin and 8-Br-cAMP enhanced memory retrieval while Rp-cAMP significantly reduced memory and NCAM levels. 8-pCPT-2'-O-Me-cAMP failed to alter memory performance or NCAM levels as compared to vehicle. We observed no significant changes in PSA-NCAM, however the expression of St8sia4 and St8sia2 (the polysialyltransferase isoforms) were altered. The mRNA levels of St8sia4 was down-regulated by 8-Br-cAMP, Rp-cAMP and 8-pCPT while forskolin led to almost 3 and 5 fold increase in mRNAs of St8sia2 and St8sia4, respectively. The current insight might endorse the predominant role of PKA as compared to Epac in cAMP pathway in expression of NCAM and memory function. PMID:24901853

  18. Identification of a cyclic-AMP-responsive element within the rat somatostatin gene.

    PubMed Central

    Montminy, M R; Sevarino, K A; Wagner, J A; Mandel, G; Goodman, R H

    1986-01-01

    We have examined the regulation of somatostatin gene expression by cAMP in PC12 rat pheochromocytoma cells transfected with the rat somatostatin gene. Forskolin at 10 microM caused a 4-fold increase in somatostatin mRNA levels within 4 hr of treatment in stably transfected cells. Chimeric genes containing the somatostatin gene promoter fused to the bacterial reporter gene encoding chloramphenicol acetyltransferase were also induced by cAMP in PC12 cells. To delineate the sequences required for response to cAMP, we constructed a series of promoter deletion mutants. Our studies defined a region between 60 and 29 base pairs upstream from the transcriptional initiation site that conferred cAMP responsiveness when placed adjacent to the simian virus 40 promoter. Within the cAMP-responsive element of the somatostatin gene, we observed an 8-base palindrome, 5'-TGACGTCA-3', which is highly conserved in many other genes whose expression is regulated by cAMP. cAMP responsiveness was greatly reduced when the somatostatin fusion genes were transfected into the mutant PC12 line A126-1B2, which is deficient in cAMP-dependent protein kinase 2. Our studies indicate that transcriptional regulation of the somatostatin gene by cAMP requires protein kinase 2 activity and may depend upon a highly conserved promoter element. Images PMID:2875459

  19. Cyclic AMP Affects Oocyte Maturation and Embryo Development in Prepubertal and Adult Cattle

    PubMed Central

    Bernal-Ulloa, Sandra Milena; Heinzmann, Julia; Herrmann, Doris; Hadeler, Klaus-Gerd; Aldag, Patrick; Winkler, Sylke; Pache, Dorit; Baulain, Ulrich; Lucas-Hahn, Andrea; Niemann, Heiner

    2016-01-01

    High cAMP levels during in vitro maturation (IVM) have been related to improved blastocyst yields. Here, we employed the cAMP/cGMP modulators, forskolin, IBMX, and cilostamide, during IVM to unravel the role of high cAMP in early embryonic development produced from prepubertal and adult bovine oocytes. Oocytes were collected via transvaginal aspiration and randomly assigned to three experimental groups: TCM24 (24h IVM/control), cAMP30 (2h pre-IVM (forskolin-IBMX), 30h IVM-cilostamide), and DMSO30 (Dimethyl Sulfoxide/vehicle control). After IVM, oocytes were fertilized in vitro and zygotes were cultured in vitro to blastocysts. Meiotic progression, cAMP levels, mRNA abundance of selected genes and DNA methylation were evaluated in oocytes. Blastocysts were used for gene expression or DNA methylation analyses. Blastocysts from the cAMP30 groups were transferred to recipients. The cAMP elevation delayed meiotic progression, but developmental rates were not increased. In immature oocytes, mRNA abundance of PRKACA was higher for cAMP30 protocol and no differences were found for PDE3A, SMAD2, ZAR1, PRDX1 and SLC2A8. EGR1 gene was up-regulated in prepubertal cAMP30 immature oocytes and down-regulated in blastocysts from all in vitro treatments. A similar gene expression profile was observed for DNMT3b, BCL2L1, PRDX1 and SLC2A8 in blastocysts. Satellite DNA methylation profiles were different between prepubertal and adult oocytes and blastocysts derived from the TCM24 and DMSO30 groups. Blastocysts obtained from prepubertal and adult oocytes in the cAMP30 treatment displayed normal methylation profiles and produced offspring. These data indicate that cAMP regulates IVM in prepubertal and adult oocytes in a similar manner, with impact on the establishment of epigenetic marks and acquisition of full developmental competency. PMID:26926596

  20. A possible signal-coupling role for cyclic AMP during endocytosis in Amoeba proteus.

    PubMed

    Prusch, R D; Roscoe, J C

    1993-01-01

    Cytoplasmic levels of cAMP in Amoeba proteus were measured utilizing radioimmunoassays under control conditions and when stimulated by inducers of either pinocytosis or phagocytosis. In control cells, cytoplasmic cAMP levels were approximately 0.39 pM/mg cells. When exposed to either chemotactic peptide or mannose which stimulate phagocytosis in the amoeba, there is a rapid doubling of the cAMP level within 45 sec of stimulation which then returns to the control level within 3-5 min. Theophylline prolongs the elevation of cytoplasmic cAMP in stimulated cells and is also capable of eliciting food vacuole formation in the amoeba. In addition isoproterenol also causes food vacuole formation in the amoeba as well as a large and prolonged increase in cytoplasmic cAMP levels. Inducers of pinocytosis (BSA and Na Cl) also elicit changes in cytoplasmic cAMP in the amoeba, but the response appears to differ from that elicited by inducers of phagocytosis in that the peak cAMP levels are broader and biphasic. It is concluded that cAMP plays a signal-coupling role during the early phases of both forms of endocytosis in Amoeba proteus.

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

    PubMed

    Hockberger, P; Connor, J A

    1983-02-18

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

  2. 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.

  3. The Phosphorylation Status of a Cyclic AMP-Responsive Activator Is Modulated via a Chromatin-Dependent Mechanism

    PubMed Central

    Michael, Laura F.; Asahara, Hiroshi; Shulman, Andrew I.; Kraus, W. Lee; Montminy, Marc

    2000-01-01

    Cyclic AMP (cAMP) stimulates the expression of numerous genes via the protein kinase A (PKA)-mediated phosphorylation of CREB at Ser133. Ser133 phosphorylation, in turn, promotes recruitment of the coactivator CREB binding protein and its paralog p300, histone acetyltransferases (HATs) that have been proposed to mediate target gene activation, in part, by destabilizing promoter bound nucleosomes and thereby allowing assembly of the transcriptional apparatus. Here we show that although histone deacetylase (HDAC) inhibitors potentiate target gene activation via cAMP, they do not stimulate transcription over the early burst phase, during which CREB phosphorylation and CBP/p300 recruitment are maximal. Rather, HDAC inhibitors augment CREB activity during the late attenuation phase by prolonging CREB phosphorylation on chromosomal but, remarkably, not on extrachromosomal templates. In reconstitution studies, assembly of periodic nucleosomal arrays on a cAMP-responsive promoter template potently inhibited CREB phosphorylation by PKA, and acetylation of these template-bound nucleosomes by p300 partially rescued CREB phosphorylation by PKA. Our results suggest a novel regulatory mechanism by which cellular HATs and HDACs modulate the phosphorylation status of nuclear activators in response to cellular signals. PMID:10669737

  4. Direct transcriptional control of the plasminogen activator gene of Yersinia pestis by the cyclic AMP receptor protein.

    PubMed

    Kim, Tae-Jong; Chauhan, Sadhana; Motin, Vladimir L; Goh, Ee-Been; Igo, Michele M; Young, Glenn M

    2007-12-01

    Horizontal gene transfer events followed by proper regulatory integration of a gene drive rapid evolution of bacterial pathogens. A key event in the evolution of the highly virulent plague bacterium Yersinia pestis was the acquisition of plasmid pPCP1, which carries the plasminogen activator gene, pla. This promoted the bubonic form of the disease by increasing bacterial dissemination from flea bite sites and incidentally enhanced replication in respiratory airways during pneumonic infection. We determined that expression of pla is controlled by the global regulator cyclic AMP (cAMP) receptor protein (Crp). This transcription factor is well conserved among distantly related bacteria, where it acts as a soluble receptor for the ubiquitous signaling molecule cAMP and controls a global network of metabolic and stress-protective genes. Crp has a similar physiological role in Y. pestis since loss of its function resulted in an inability to metabolize a variety of nonglucose substrates. Activation of pla expression requires a transcription activation element of the pla promoter that serves as a Crp binding site. Crp interaction with this site was demonstrated to occur only in the presence of cAMP. Alteration of the Crp binding site nucleotide sequence prevented in vitro formation of Crp-DNA complexes and inhibited in vivo expression of pla. The placement of pla under direct regulatory control of Crp highlights how highly adapted pathogens integrate laterally acquired genes to coordinate virulence factor expression with global gene networks to maintain homeostasis through the infectious life cycle.

  5. Modulation of a human lymphoblastoid B cell line by cyclic AMP. Ig secretion and phosphatidylcholine metabolism

    SciTech Connect

    Shearer, W.T.; Patke, C.L.; Gilliam, E.B.; Rosenblatt, H.M.; Barron, K.S.; Orson, F.M.

    1988-09-01

    A transformed human B cell line, LA350, was found to be sensitive to cAMP-elevating agents by responding with rapid (0 to 2 h) severalfold elevations of intracellular cAMP to treatment with cholera toxin, isobutylmethylxanthine (IBMX), forskolin, and dibutyryl cAMP (all p less than 0.001). These cAMP-elevating agents also produced significant inhibitions of subsequent (48 to 72 h) Ig secretion by the same B cells as measured by a reverse hemolytic plaque assay and an enzyme-linked immunoadsorbent assay for IgM (both p less than 0.001). PMA- and IBMX-treated cells were particularly responsive to the effects of cholera toxin, showing a doubling of cAMP content and profound decrease in Ig production (p less than 0.001). Because our previous studies had correlated activation of the metabolic turnover of the phosphatidylcholine (PC) fraction of membrane phospholipids with enhanced Ig secretion, we examined the sensitivity of PC metabolism to cAMP in control and PMA-stimulated cells. Formation of PC was found to be inhibited by forskolin and IBMX (both p less than 0.002) but breakdown of PC was stimulated (p less than 0.001). These findings imply that as the enzymatic products of PC, choline phosphate and diacylglycerol, are depleted due to the combined effects of cAMP upon synthesis and turnover of PC, there is a decrease in Ig secretion. Since diacylglycerol activates protein kinase C, it appears reasonable that Ig secretion is at least partially regulated by cAMP-responsive alterations in PC metabolism produced by protein kinase C-induced phosphorylation. We conclude that the early cAMP-sensitive changes in PC metabolism in this activated B cell line may signal for subsequent alterations in Ig secretion.

  6. Synthesis of glial fibrillary acidic protein in rat C6 glioma in chemically defined medium: cyclic AMP-dependent transcriptional and translational regulation.

    PubMed

    Messens, J; Slegers, H

    1992-06-01

    Glial fibrillary acidic protein (GFA) expression was induced in rat C6 glioma in chemically defined medium by the addition of N6, O2'-dibutyryl cyclic AMP (dbcAMP). Induction was dependent on the increase in intracellular cyclic AMP (cAMP), which was linearly correlated with added dbcAMP. Contrary to GFA mRNA synthesis, which can be obtained by cAMP-dependent and -independent pathways, translation of mRNA into GFA was observed only above a cellular cAMP concentration of approximately 0.2 fmol/cell. dbcAMP stimulation did not affect the vimentin concentration, which remained at a low level, but changed the cellular morphology from a bipolar to a stellate shape. A similar morphological change was observed after stimulation of C6 with lipopolysaccharide (LPS). However, LPS did not significantly increase the intracellular concentration of cAMP and the LPS-induced mRNA was not translated into GFA. Our results indicate that GFA synthesis is regulated at the mRNA level and at the translational level and that a cAMP-dependent mechanism determines the ultimate synthesis of GFA by a yet unknown mechanism.

  7. Downregulation of the Escherichia coli guaB promoter by upstream-bound cyclic AMP receptor protein.

    PubMed

    Husnain, Seyyed I; Busby, Stephen J W; Thomas, Mark S

    2009-10-01

    The Escherichia coli guaB promoter (P(guaB)) is responsible for directing transcription of the guaB and guaA genes, which specify the biosynthesis of the nucleotide GMP. P(guaB) is subject to growth rate-dependent control (GRDC) and possesses an UP element that is required for this regulation. In addition, P(guaB) contains a discriminator, three binding sites for the nucleoid-associated protein FIS, and putative binding sites for the regulatory proteins DnaA, PurR, and cyclic AMP receptor protein (CRP). Here we show that the CRP-cyclic AMP (cAMP) complex binds to a site located over 100 bp upstream of the guaB transcription start site, where it serves to downregulate P(guaB). The CRP-mediated repression of P(guaB) activity increases in media that support lower growth rates. Inactivation of the crp or cyaA gene or ablation/translocation of the CRP site relieves repression by CRP and results in a loss of GRDC of P(guaB). Thus, GRDC of P(guaB) involves a progressive increase in CRP-mediated repression of the promoter as the growth rate decreases. Our results also suggest that the CRP-cAMP complex does not direct GRDC at P(guaB) and that at least one other regulatory factor is required for conferring GRDC on this promoter. However, PurR and DnaA are not required for this regulatory mechanism.

  8. [Adrenaline and cyclic AMP stimulation of ketopentose and sedoheptulose formation in rat liver homogenates].

    PubMed

    Kolotilova, A I; Glushankov, E P; Epifanova, Iu E

    1976-01-01

    Formation of sedoheptulose-7-phosphate and ketopentose phosphate was studied in vitro as affected by epinephrine and cAMP. No effect of epinephrine on the activity of transketolase was found with ribose-5-phosphate as a substrate of the nonoxidative reactions of the pentose phosphate ccyle. Epinephrine and cAMP enhance the formation of ketopentoses and sedoheptulose with glycogen as a main carbohydrate source, which is most pronounced in the experiments with cold preincubation. The phosphorylase system mediate influence of epinephrine and cAMP on the nonoxidative reactions products may be assumed.

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

    PubMed

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

    2009-10-01

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

  10. Role of cyclo-oxygenase-2 induction in interleukin-1β induced attenuation of cultured human airway smooth muscle cell cyclic AMP generation in response to isoprenaline

    PubMed Central

    Pang, Linhua; Holland, Elaine; Knox, Alan J

    1998-01-01

    Airway smooth muscle (ASM) in human asthma shows reduced relaxation and cyclic AMP generation in response to β-adrenoceptor agonists. IL-β attenuates cyclic AMP generation but the underlying mechanism is unclear. We have reported that IL-1β induces cyclo-oxygenase-2 (COX-2) in human ASM cells and results in a marked increase in prostanoid generation with PGE2 and PGI2 as the major products.We investigated the role of COX-2 induction and prostanoid release (measured as PGE2) in IL-1β induced attenuation of cyclic AMP generation in response to the β-adrenoceptor agonist isoprenaline (ISO).Pre-treatment of human ASM cells with IL-1β significantly attenuated cyclic AMP generation in response to high concentrations of ISO (1.0–10.0 μM) in a time- and concentration-dependent manner. The effect was accompanied by a high concentration of PGE2 release. The non-selective COX inhibitor indomethacin (Ind), the selective COX-2 inhibitor NS-398, the protein synthesis inhibitors cycloheximide (CHX) and actinomycin D and the steroid dexamethasone (Dex) all abolished the PGE2 release and prevented the attenuated cyclic AMP generation.COX substrate arachidonic acid time- and concentration-dependently mimicked IL-1β induced attenuation and the effect was prevented by the non-selective COX inhibitors Ind and flurbiprofen, but not by NS-398, CHX and Dex.In contrast to IL-1β, TNFα and IFNγ, which are ineffective in inducing COX-2 and releasing PGE2 from human ASM cells, did not affect the cyclic AMP formation.Our study demonstrates that COX-2 induction and the consequent release of prostanoids plays a crucial role in IL-1β induced attenuation of human ASM cell cyclic AMP response to ISO. PMID:9863663

  11. 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.

  12. 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

  13. Cyclic-AMP Mediated Regulation of ABCB mRNA Expression in Mussel Haemocytes

    PubMed Central

    Franzellitti, Silvia; Fabbri, Elena

    2013-01-01

    Background The multixenobiotic resistance system (MXR) allows aquatic organisms to cope with their habitat despite high pollution levels by over-expressing membrane and intracellular transporters, including the P-glycoprotein (Pgp). In mammals transcription of the ABCB1 gene encoding Pgp is under cAMP/PKA-mediated regulation; whether this is true in mollusks is not fully clarified. Methodology/Principal Findings cAMP/PKA regulation and ABCB mRNA expression were assessed in haemocytes from Mediterranean mussels (Mytilus galloprovincialis) exposed in vivo for 1 week to 0.3 ng/L fluoxetine (FX) alone or in combination with 0.3 ng/L propranolol (PROP). FX significantly decreased cAMP levels and PKA activity, and induced ABCB mRNA down-regulation. FX effects were abolished in the presence of PROP. In vitro experiments using haemocytes treated with physiological agonists (noradrenaline and serotonin) and pharmacological modulators (PROP, forskolin, dbcAMP, and H89) of the cAMP/PKA system were performed to obtain clear evidence about the involvement of the signaling pathway in the transcriptional regulation of ABCB. Serotonin (5-HT) decreased cAMP levels, PKA activity and ABCB mRNA expression but increased the mRNA levels for a putative 5-HT1 receptor. Interestingly, 5-HT1 was also over-expressed after in vivo exposures to FX. 5-HT effects were counteracted by PROP. Forskolin and dbcAMP increased PKA activity as well as ABCB mRNA expression; the latter effect was abolished in the presence of the PKA inhibitor H89. Conclusions This study provides the first direct evidence for the cAMP/PKA-mediated regulation of ABCB transcription in mussels. PMID:23593491

  14. Positive Effect of Carbon Sources on Natural Transformation in Escherichia coli: Role of Low-Level Cyclic AMP (cAMP)-cAMP Receptor Protein in the Derepression of rpoS

    PubMed Central

    Guo, Mengyue; Wang, Huanyu; Xie, Nengbin

    2015-01-01

    ABSTRACT Natural plasmid transformation of Escherichia coli is a complex process that occurs strictly on agar plates and requires the global stress response factor σS. Here, we showed that additional carbon sources could significantly enhance the transformability of E. coli. Inactivation of phosphotransferase system genes (ptsH, ptsG, and crr) caused an increase in the transformation frequency, and the addition of cyclic AMP (cAMP) neutralized the promotional effect of carbon sources. This implies a negative role of cAMP in natural transformation. Further study showed that crp and cyaA mutations conferred a higher transformation frequency, suggesting that the cAMP-cAMP receptor protein (CRP) complex has an inhibitory effect on transformation. Moreover, we observed that rpoS is negatively regulated by cAMP-CRP in early log phase and that both crp and cyaA mutants show no transformation superiority when rpoS is knocked out. Therefore, it can be concluded that both the crp and cyaA mutations derepress rpoS expression in early log phase, whereby they aid in the promotion of natural transformation ability. We also showed that the accumulation of RpoS during early log phase can account for the enhanced transformation aroused by additional carbon sources. Our results thus demonstrated that the presence of additional carbon sources promotes competence development and natural transformation by reducing cAMP-CRP and, thus, derepressing rpoS expression during log phase. This finding could contribute to a better understanding of the relationship between nutrition state and competence, as well as the mechanism of natural plasmid transformation in E. coli. IMPORTANCE Escherichia coli, which is not usually considered to be naturally transformable, was found to spontaneously take up plasmid DNA on agar plates. Researching the mechanism of natural transformation is important for understanding the role of transformation in evolution, as well as in the transfer of pathogenicity and

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

    PubMed

    Motani, Kou; Ito, Shinji; Nagata, Shigekazu

    2015-05-15

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

  16. 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

    2015-06-18

    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.

  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. 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

  19. The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas.

    PubMed

    Du, Qingyou; Schilde, Christina; Birgersson, Elin; Chen, Zhi-hui; McElroy, Stuart; Schaap, Pauline

    2014-02-01

    Amoebas survive environmental stress by differentiating into encapsulated cysts. As cysts, pathogenic amoebas resist antibiotics, which particularly counteracts treatment of vision-destroying Acanthamoeba keratitis. Limited genetic tractability of amoeba pathogens has left their encystation mechanisms unexplored. The social amoeba Dictyostelium discoideum forms spores in multicellular fruiting bodies to survive starvation, while other dictyostelids, such as Polysphondylium pallidum can additionally encyst as single cells. Sporulation is induced by cAMP acting on PKA, with the cAMP phosphodiesterase RegA critically regulating cAMP levels. We show here that RegA is deeply conserved in social and pathogenic amoebas and that deletion of the RegA gene in P. pallidum causes precocious encystation and prevents cyst germination. We heterologously expressed and characterized Acanthamoeba RegA and performed a compound screen to identify RegA inhibitors. Two effective inhibitors increased cAMP levels and triggered Acanthamoeba encystation. Our results show that RegA critically regulates Amoebozoan encystation and that components of the cAMP signalling pathway could be effective targets for therapeutic intervention with encystation.

  20. 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

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

    PubMed

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

    2012-10-01

    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.

  2. 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

  3. Cyclic AMP-mediated endocytosis of intestinal epithelial NHE3 requires binding to synaptotagmin 1

    PubMed Central

    Musch, Mark W.; Arvans, Donna L.; Wang, Yunwei; Nakagawa, Yasushi; Solomaha, Elena

    2010-01-01

    The apical membrane Na+-H+ exchanger (NHE)3 is regulated by cAMP-dependent phosphorylation, which inhibits its activity through membrane endocytosis. The clathrin complex adaptor protein synaptotagmin 1 (Syt 1) appears to be essential to this process, but little is known about its expression in intestinal epithelial cells or interaction with NHE3. The intestinal epithelial expression and apical location of Syt 1 were determined by Syt 1 mRNA profiling and immunolocalization. Tandem mass spectrometry was used for protein identification. Bis(sulfosuccinimidyl) suberate (BS3) cross linking suggested that NHE3 and Syt 1 were in a membrane complex following cAMP stimulation of Caco2BBE (Brush Border Expressions) cells. To investigate the regulation of NHE3 appearance in a Syt 1-containing membrane compartment, doxycycline-inducible hemaglutinin (HA)-tagged NHE3 was expressed in Caco2BBE cells. HA-NHE3 correctly targeted to the apical membrane, where, upon cAMP stimulation, it was internalized with a Syt 1-containing compartment. Site-directed mutagenesis of NHE3 showed that serine 605 (S605) was pivotal to NHE3 and Syt 1 association and internalization. Direct Syt 1 interaction with NHE3 was suggested by fluorescence resonance energy transfer (FRET) analysis. The physiological role of S552 was less clear. By FRET, this serine residue appeared to be involved in cAMP-induced Syt 1 binding of NHE3. However, when HA-tagged NHE3 S552A was expressed in Caco2 cells, the mutated construct was not inserted into the apical membrane. We conclude that intestinal epithelial Syt 1 plays an important role in cAMP-stimulated endocytosis of apical NHE3 through cAMP-dependent phosphorylation of S605 that is required for NHE3 and Syt 1 association. PMID:19926819

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

    SciTech Connect

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

    1982-01-01

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

  5. Prostaglandin E2 and F2 alpha inhibit growth of human gastric carcinoma cell line KATO III with simultaneous stimulation of cyclic AMP production.

    PubMed

    Nakamura, A; Chiba, T; Yamatani, T; Yamaguchi, A; Inui, T; Morishita, T; Kadowaki, S; Fujita, T

    1989-01-01

    The effects of prostaglandins (PGs) on the growth of human gastric carcinoma cell line KATO III were investigated. PGE2 as well as PGF2 alpha significantly and dose-dependently inhibited the growth of this gastric carcinoma cell line (PGE2 greater than PGF2 alpha). This inhibition of cell growth by the PGs was associated with the increase in cyclic AMP production (PGE2 greater than PGF2 alpha), whereas inositol-phospholipid turnover was not affected by either PGE2 or PGF2 alpha as assessed by the formation of 3H-inositol phosphates. Furthermore, the proliferation of these gastric carcinoma cells was also suppressed by the administration of forskolin as well as of dibutyryl cyclic AMP. These results suggest that PGE2 and PGF2 alpha inhibit the growth of cultured human gastric carcinoma cells KATO III via stimulation of cyclic AMP production. PMID:2536452

  6. Regulation of laminin and entactin mRNA levels by retinoic acid and dibutyryl cyclic AMP

    SciTech Connect

    Durkin, M.E.; Phillips, S.L.; Carlin, B.E.; Merlie, J.P.; Chung, A.E.

    1986-05-01

    Retinoic acid and dibutyryl cAMP induced F9 embryonal carcinoma cells to differentiate to parietal endoderm; the morphological changes were accompanied by the increased synthesis of the basement membrane glycoproteins laminin and entactin. cDNA clones have been isolated for the A (400 kD), B1 (220 kD), and B2 (205 kD) chains of laminin. Northern blot analysis indicated that the A, B1, and B2 chains were encoded by RNA species of 9.8, 6.0, and 8.0 kb, respectively. The kinetics of induction of the laminin mRNAs were studied by dot-blotting dilutions of RNA extracted from F9 cells cultured in retinoic acid and dibutyryl cAMP for increasing amounts of time and hybridizing to /sup 32/P-labeled recombinant plasmids. Very low levels of the A and B chain RNAs were found in uninduced cells, and a large increase occurred between 48 and 72 hr of growth in retinoic acid and dibutyryl cAMP. A cDNA clone was also obtained for entactin, a 150 kD glycoprotein that forms a complex with laminin. Retinoic acid and dibutyryl cAMP treatment also increased the amount of entactin RNA in F9 cells. These results suggested that a common mechanism may exist for the coordinate regulation of the 4 basement membrane protein genes during differentiation.

  7. Release of prostaglandins from the isolated frog ventricle and associated changes in endogenous cyclic nucleotide levels.

    PubMed Central

    Flitney, F W; Singh, J

    1980-01-01

    reached: thus, 8-bromo-3'5'-cyclic GMP accelerates the decline in contractility and depresses the steady-state level, whereas dibutyryl 3'5'-cyclic AMP delays the development of hypodynamic depression, and elevates the final twitch tension. The effects of both 3'5' cyclic nucleotide derivatives are dose-dependent. 7. The possible involvement of prostaglandins and 3'5'-cyclic nucleotides as causal agents in the mechanism of hypodynamic depression is discussed. The biochemical basis for the implied antangonistic effects of 3'5'-cyclic AMP and 3'5'-cyclic GMP in regulating ventricular contractility is considered in the following paper (Flitney & Singh, 1980). PMID:6255139

  8. Modification by phosphate of PTH-dependent renal cyclic AMP response.

    PubMed

    Guillemant, J; Guillemant, S

    1993-04-01

    The PTH response and the renal cAMP response obtained after oral administration of either tricalcium phosphate or calcium gluconolactate were compared in 12 young adult males. Each subject was studied during a control period of two hours before and during an experimental period of four hours after ingestion of a single oral dose of calcium salt. The respective dosages (1.2 g of calcium plus 0.6 g phosphorus for tricalcium phosphate; 0.5 g of calcium for gluconolactate calcium) were chosen to provide similar significant (p = 0.0001) increases in serum ionized calcium (from 1.23 to 1.29 mmol/l vs from 1.23 to 1.28 mmol/l). After tricalcium phosphate a modest (10%) but significant (p < 0.001) rise in serum phosphate was observed. In both series of experiments similar inhibitory effects on PTH circulating levels were obtained (from 22.6 to 12.4 pg/ml after tricalcium phosphate and from 24.1 to 10.6 pg/ml after calcium gluconolactate). After ingestion of calcium gluconolactate the renal secretion of cAMP fell from 12.68 to 8.64 nmol/l GF (p < 0.001), whereas no significant alterations of the mean values of nephrogenous cAMP were detected after ingestion of tricalcium phosphate. In accordance with the role of cAMP as a second messenger, after calcium gluconolactate we obtained a significant increase in tubular maximal reabsorption of phosphate (p < 0.0001) contrasting with the absence of significant effect after tricalcium phosphate. The present results confirm that suppression of PTH secretion only depends on the rise of serum ionized calcium and suggest that additional phosphate administration could have a decoupling effect between PTH and renal cAMP secretion. PMID:8390394

  9. 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

  10. 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.

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

    PubMed

    Cowley, B D

    2008-02-01

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

  12. 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

  13. Export of cyclic AMP by avian red cells and inhibition by prostaglandin A/sub 1/

    SciTech Connect

    Heasley, L.E.

    1985-01-01

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

  14. 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

  15. Molecular basis for the specific recognition of the metazoan cyclic GMP-AMP by the innate immune adaptor protein STING.

    PubMed

    Shi, Heping; Wu, Jiaxi; Chen, Zhijian J; Chen, Chuo

    2015-07-21

    Cyclic GMP-AMP containing a unique combination of mixed phosphodiester linkages (2'3'-cGAMP) is an endogenous second messenger molecule that activates the type-I IFN pathway upon binding to the homodimer of the adaptor protein STING on the surface of endoplasmic reticulum membrane. However, the preferential binding of the asymmetric ligand 2'3'-cGAMP to the symmetric dimer of STING represents a physicochemical enigma. Here we show that 2'3'-cGAMP, but not its linkage isomers, adopts an organized free-ligand conformation that resembles the STING-bound conformation and pays low entropy and enthalpy costs in converting into the active conformation. Our results demonstrate that analyses of free-ligand conformations can be as important as analyses of protein conformations in understanding protein-ligand interactions.

  16. 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

  17. 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

  18. Identification, Characterization, and Structure Analysis of the Cyclic di-AMP-binding PII-like Signal Transduction Protein DarA*

    PubMed Central

    Gundlach, Jan; Dickmanns, Achim; Schröder-Tittmann, Kathrin; Neumann, Piotr; Kaesler, Jan; Kampf, Jan; Herzberg, Christina; Hammer, Elke; Schwede, Frank; Kaever, Volkhard; Tittmann, Kai; Stülke, Jörg; Ficner, Ralf

    2015-01-01

    The cyclic dimeric AMP nucleotide c-di-AMP is an essential second messenger in Bacillus subtilis. We have identified the protein DarA as one of the prominent c-di-AMP receptors in B. subtilis. Crystal structure analysis shows that DarA is highly homologous to PII signal transducer proteins. In contrast to PII proteins, the functionally important B- and T-loops are swapped with respect to their size. DarA is a homotrimer that binds three molecules of c-di-AMP, each in a pocket located between two subunits. We demonstrate that DarA is capable to bind c-di-AMP and with lower affinity cyclic GMP-AMP (3′3′-cGAMP) but not c-di-GMP or 2′3′-cGAMP. Consistently the crystal structure shows that within the ligand-binding pocket only one adenine is highly specifically recognized, whereas the pocket for the other adenine appears to be promiscuous. Comparison with a homologous ligand-free DarA structure reveals that c-di-AMP binding is accompanied by conformational changes of both the fold and the position of the B-loop in DarA. PMID:25433025

  19. Identification, characterization, and structure analysis of the cyclic di-AMP-binding PII-like signal transduction protein DarA.

    PubMed

    Gundlach, Jan; Dickmanns, Achim; Schröder-Tittmann, Kathrin; Neumann, Piotr; Kaesler, Jan; Kampf, Jan; Herzberg, Christina; Hammer, Elke; Schwede, Frank; Kaever, Volkhard; Tittmann, Kai; Stülke, Jörg; Ficner, Ralf

    2015-01-30

    The cyclic dimeric AMP nucleotide c-di-AMP is an essential second messenger in Bacillus subtilis. We have identified the protein DarA as one of the prominent c-di-AMP receptors in B. subtilis. Crystal structure analysis shows that DarA is highly homologous to PII signal transducer proteins. In contrast to PII proteins, the functionally important B- and T-loops are swapped with respect to their size. DarA is a homotrimer that binds three molecules of c-di-AMP, each in a pocket located between two subunits. We demonstrate that DarA is capable to bind c-di-AMP and with lower affinity cyclic GMP-AMP (3'3'-cGAMP) but not c-di-GMP or 2'3'-cGAMP. Consistently the crystal structure shows that within the ligand-binding pocket only one adenine is highly specifically recognized, whereas the pocket for the other adenine appears to be promiscuous. Comparison with a homologous ligand-free DarA structure reveals that c-di-AMP binding is accompanied by conformational changes of both the fold and the position of the B-loop in DarA. PMID:25433025

  20. 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.

  1. Cyclic AMP and c-KIT Signaling in Familial Testicular Germ Cell Tumor Predisposition

    PubMed Central

    Azevedo, Monalisa F.; Horvath, Anelia; Bornstein, Ethan R.; Almeida, Madson Q.; Xekouki, Paraskevi; Faucz, Fabio R.; Gourgari, Evgenia; Nadella, Kiran; Remmers, Elaine F.; Quezado, Martha; de Alexandre, Rodrigo Bertollo; Kratz, Christian P.; Nesterova, Maria; Greene, Mark H.

    2013-01-01

    Background: Familial testicular germ cell tumors (FTGCTs) are hypothesized to result from the combined interaction of multiple low-penetrance genes. We reported inactivating germline mutations of the cAMP-binding phosphodiesterase 11A (PDE11A) as modifiers of FTGCT risk. Recent genome-wide association studies have identified single-nucleotide polymorphisms in the KITLG gene, the ligand for the cKIT tyrosine kinase receptor, as strong modifiers of susceptibility to both familial and sporadic testicular germ cell tumors. Design: We studied 94 patients with FTGCTs and 50 at-risk male relatives from 63 unrelated kindreds, in whom the PDE11A gene had been sequenced by investigating the association between KITLG genome-wide association study single-nucleotide polymorphisms rs3782179 and rs4474514 and FTGCT risk in these patients and in 692 controls. We also examined cAMP and c-KIT signaling in testicular tissues and cell lines and extended the studies to 2 sporadic cases, one with a PDE11A defect and one without, as a comparison. Results: We found a higher frequency of the KITLG risk alleles in FTGCT patients who also had a PDE11A sequence variant, compared with those with a wild-type PDE11A sequence. In NTERA-2 and Tcam-2 cells transfected with the mutated forms of PDE11A (R52T, F258Y, Y727C, R804H, V820M, R867G, and M878V), cAMP levels were significantly higher, and the relative phosphodiesterase activity was lower than in the wild-type cells. KITLG expression was consistently increased in the presence of PDE11A-inactivating defects, both at the RNA and protein levels, in familial testicular germ cell tumors. The 2 sporadic cases that were studied, one with a PDE11A defect and another without, agreed with the data in FTGTCT and in the cell lines. Conclusions: Patients with FTGCT and PDE11A defects also carry KITLG risk alleles more frequently. There may be an interaction between cAMP and c-KIT signaling in predisposition to testicular germ cell tumors. PMID:23771924

  2. Serratia marcescens Cyclic AMP Receptor Protein Controls Transcription of EepR, a Novel Regulator of Antimicrobial Secondary Metabolites

    PubMed Central

    Stella, Nicholas A.; Lahr, Roni M.; Brothers, Kimberly M.; Kalivoda, Eric J.; Hunt, Kristin M.; Kwak, Daniel H.; Liu, Xinyu

    2015-01-01

    ABSTRACT Serratia marcescens generates secondary metabolites and secreted enzymes, and it causes hospital infections and community-acquired ocular infections. Previous studies identified cyclic AMP (cAMP) receptor protein (CRP) as an indirect inhibitor of antimicrobial secondary metabolites. Here, we identified a putative two-component regulator that suppressed crp mutant phenotypes. Evidence supports that the putative response regulator eepR was directly transcriptionally inhibited by cAMP-CRP. EepR and the putative sensor kinase EepS were necessary for the biosynthesis of secondary metabolites, including prodigiosin- and serratamolide-dependent phenotypes, swarming motility, and hemolysis. Recombinant EepR bound to the prodigiosin and serratamolide promoters in vitro. Together, these data introduce a novel regulator of secondary metabolites that directly connects the broadly conserved metabolism regulator CRP with biosynthetic genes that may contribute to competition with other microbes. IMPORTANCE This study identifies a new transcription factor that is directly controlled by a broadly conserved transcription factor, CRP. CRP is well studied in its role to help bacteria respond to the amount of nutrients in their environment. The new transcription factor EepR is essential for the bacterium Serratia marcescens to produce two biologically active compounds, prodigiosin and serratamolide. These two compounds are antimicrobial and may allow S. marcescens to compete for limited nutrients with other microorganisms. Results from this study tie together the CRP environmental nutrient sensor with a new regulator of antimicrobial compounds. Beyond microbial ecology, prodigiosin and serratamolide have therapeutic potential; therefore, understanding their regulation is important for both applied and basic science. PMID:25897029

  3. 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

  4. Cyclic AMP control measured in two compartments in HEK293 cells: phosphodiesterase K(M) is more important than phosphodiesterase localization.

    PubMed

    Matthiesen, Karina; Nielsen, Jacob

    2011-01-01

    The intracellular second messenger cyclic AMP (cAMP) is degraded by phosphodiesterases (PDE). The knowledge of individual families and subtypes of PDEs is considerable, but how the different PDEs collaborate in the cell to control a cAMP signal is still not fully understood. In order to investigate compartmentalized cAMP signaling, we have generated a membrane-targeted variant of the cAMP Bioluminiscence Resonance Energy Transfer (BRET) sensor CAMYEL and have compared intracellular cAMP measurements with it to measurements with the cytosolic BRET sensor CAMYEL in HEK293 cells. With these sensors we observed a slightly higher cAMP response to adenylyl cyclase activation at the plasma membrane compared to the cytosol, which is in accordance with earlier results from Fluorescence Resonance Energy Transfer (FRET) sensors. We have analyzed PDE activity in fractionated lysates from HEK293 cells using selective PDE inhibitors and have identified PDE3 and PDE10A as the major membrane-bound PDEs and PDE4 as the major cytosolic PDE. Inhibition of membrane-bound or cytosolic PDEs can potentiate the cAMP response to adenylyl cyclase activation, but we see no significant difference between the potentiation of the cAMP response at the plasma membrane and in cytosol when membrane-bound and cytosolic PDEs are inhibited. When different levels of stimulation were tested, we found that PDEs 3 and 10 are mainly responsible for cAMP degradation at low intracellular cAMP concentrations, whereas PDE4 is more important for control of cAMP at higher concentrations.

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  6. 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

  7. On the mechanism of action of lead in the testis: in vitro suppression of FSH receptors, cyclic AMP and steroidogenesis. [Rats

    SciTech Connect

    Wiebe, J.P.; Salhanick, A.I.; Myers, K.I.

    1983-04-25

    The purpose of the present study was to determine by in vitro methods, if Pb acts by interfering directly with hormone binding, cyclic AMP production and steroidogenic enzyme activity. Sertoli cells were isolated from testes of prepubertal rats and cultured in the presence of 2.64 x 10/sup -4/ M of either NaAc (control) or PbAc for 1, 4, 24, 48, 96 or 144 hr. There was no reduction in FSH binding and in FSH-induced cyclic AMP after a 1-4 hr exposure to Pb. After a 24-hr exposure to Pb, the cells exhibited a 10-20% decrease in FSH binding and cyclic AMP production and after 96 hr there was a 75% decrease in these 2 parameters. The inhibition was greater in cells from 16 day old than from 20 day old rats, so that in the former, after a 144 hr exposure the FSH-induced cyclic AMP of the Pb exposed cells was only 3% of the amount produced by the NaAc exposed cells (i.e. a 97% inhibition). After in vitro exposure to Pb for 48 hr, the steroidogenic activity (progesterone conversion to steroid metabolites) of Sertoli cells was significantly reduced and their steroidogenesis was no longer stimulated by FSH. A crude testicular enzyme preparation containing 3..beta..-hydroxysteroid dehydrogenase (3..beta..-HSD) exhibited approximately 25% reduction in activity if the assay buffer contained PbCl/sub 2/ instead of the equivalent in NaCl. Prolonged in vivo exposure to Pb resulted in approximately 50% reduction in 3..beta..-HSD activity. This is the first indication that in the testis Pb may act directly (immediate effect) by suppressing enzyme activities, and indirectly (long term effect) by reducing gonadotropin-receptor binding and the resultant cyclic AMP production.

  8. Hormone-induced intercellular signal transfer dissociates cyclic AMP- dependent protein kinase

    PubMed Central

    1984-01-01

    We used co-cultures of porcine ovarian granulosa cells and mouse adrenocortical tumor cells (Y-1) to examine the kinetics of contact- dependent intercellular signal transfer and to assess the molecular mechanisms employed by this process. Exposure to follicle-stimulating hormone (FSH) caused cAMP-dependent protein kinase dissociation in granulosa cells and, with time, in Y-1 cells if, and only if, they contacted a responding granulosa cell. Y-1 cells close to a granulosa cell but not touching it failed to respond similarly. In reciprocal experiments, co-cultures were stimulated with adrenocorticotropic hormone (ACTH). Y-1 cells dissociated protein kinase as did granulosa cells in contact with Y-1 cells; however, granulosa cells that were not in contact with Y-1 cells failed to respond to the hormone. Fluorogenic steroids were secreted by Y-1 cells cultured alone and stimulated with ACTH, but were not secreted by cultures exposed to FSH. Neither hormone caused fluorogenic steroid production by granulosa cells. On the other hand these steroids were secreted in co-cultures stimulated with ACTH and to a lesser degree in co-cultures exposed to FSH. Autoradiography revealed that I125-FSH bound only to granulosa cells, never to Y-1 cells, even if they were in contact with an ovarian cell. The possibility of cell fusion was tested by experiments in which Y-1 cell membranes were labeled with cationized ferritin. These cells were then placed in co-culture with ovarian granulosa cells that had previously been allowed to ingest latex spheres. At regions of gap junctions between Y-1 and granulosa cells ferritin remained attached to the adrenal cell membrane and was never observed to migrate to the granulosa cell membrane. From these data, we conclude that hormone specific stimulation of one cell type leads to protein kinase dissociation in heterotypic partners only if they contact a hormone responsive cell. This signal transfer is bidirectional, exhibits temporal kinetics and

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

    PubMed

    Petersen, T S; Kristensen, S G; Jeppesen, J V; Grøndahl, M L; Wissing, M L; Macklon, K T; Andersen, C Y

    2015-03-01

    The concentration of the important second messenger cAMP is regulated by phosphodiesterases (PDEs) and hence an attractive drug target. However, limited human data are available about the PDEs in the ovary. The aim of the present study was to describe and characterise the PDEs in the human ovary. Results were obtained by analysis of mRNA microarray data from follicles and granulosa cells (GCs), combined RT-PCR and enzymatic activity analysis in GCs, immunohistochemical analysis of ovarian sections and by studying the effect of PDE inhibitors on progesterone production from cultured GCs. We found that PDE3, PDE4, PDE7 and PDE8 are the major families present while PDE11A was not detected. PDE8B was differentially expressed during folliculogenesis. In cultured GCs, inhibition of PDE7 and PDE8 increased basal progesterone secretion while PDE4 inhibition increased forskolin-stimulated progesterone secretion. In conclusion, we identified PDE3, PDE4, PDE7 and PDE8 as the major PDEs in the human ovary.

  10. Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27Kip1) of cyclin-dependent kinase 4 activation.

    PubMed

    Kato, J Y; Matsuoka, M; Polyak, K; Massagué, J; Sherr, C J

    1994-11-01

    Cyclic AMP (cAMP) blocks the mitogenic effects of colony-stimulating factor 1 (CSF-1) in macrophages, inducing cell cycle arrest in mid-G1 phase. Complexes between cyclin D1 and cyclin-dependent kinase 4 (cdk4) assemble in growth arrested cells, but cdk4 is not phosphorylated in vivo by the cdk-activating kinase (CAK) and remains inactive. Although undetectable in lysates of cAMP-treated cells, active CAK is recovered after antibody precipitation, indicating that it is not the direct target of inhibition. Levels of the cdk inhibitor p27Klp1 increase in cAMP-treated cells, and its immunodepletion from inhibitory lysates restores CAK-mediated cdk4 activation. Kip1 does not bind to CAK, but its association with cyclin D-cdk4 prevents CAK from phosphorylating and activating the holoenzyme. PMID:7954814

  11. Unraveling the Mechanism of the Photodeprotection Reaction of 8-Bromo- and 8-Chloro-7-hydroxyquinoline Caged Acetates

    PubMed Central

    Ma, Jiani; Rea, Adam C; An, Huiying; Ma, Chensheng; Guan, Xiangguo; Li, Ming-De; Su, Tao; Yeung, Chi Shun; Harris, Kyle T; Zhu, Yue; Nganga, Jameil L; Fedoryak, Olesya D; Dore, Timothy M; Phillips, David Lee

    2012-01-01

    Abstract Photoremovable protecting groups (PPGs) when conjugated to biological effectors forming “caged compounds” are a powerful means to regulate the action of physiologically active messengers in vivo through 1-photon excitation (1PE) and 2-photon excitation (2PE). Understanding the photodeprotection mechanism is important for their physiological use. We compared the quantum efficiencies and product outcomes in different solvent and pH conditions for the photolysis reactions of (8-chloro-7-hydroxyquinolin-2-yl)methyl acetate (CHQ-OAc) and (8-bromo-7-hydroxyquinolin-2-yl)methyl acetate (BHQ-OAc), representatives of the quinoline class of phototriggers for biological use, and conducted nanosecond time-resolved spectroscopic studies using transient emission (ns-EM), transient absorption (ns-TA), transient resonance Raman (ns-TR2), and time-resolved resonance Raman (ns-TR3) spectroscopies. The results indicate differences in the photochemical mechanisms and product outcomes, and reveal that the triplet excited state is most likely on the pathway to the product and that dehalogenation competes with release of acetate from BHQ-OAc, but not CHQ-OAc. A high fluorescence quantum yield and a more efficient excited-state proton transfer (ESPT) in CHQ-OAc compared to BHQ-OAc explain the lower quantum efficiency of CHQ-OAc relative to BHQ-OAc. PMID:22511356

  12. 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.

  13. 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 +/.

  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. 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

  16. Seventeen Sxy-Dependent Cyclic AMP Receptor Protein Site-Regulated Genes Are Needed for Natural Transformation in Haemophilus influenzae

    PubMed Central

    Mell, Joshua C.; Redfield, Rosemary J.

    2012-01-01

    Natural competence is the ability of bacteria to actively take up extracellular DNA. This DNA can recombine with the host chromosome, transforming the host cell and altering its genotype. In Haemophilus influenzae, natural competence is induced by energy starvation and the depletion of nucleotide pools. This induces a 26-gene competence regulon (Sxy-dependent cyclic AMP receptor protein [CRP-S] regulon) whose expression is controlled by two regulators, CRP and Sxy. The role of most of the CRP-S genes in DNA uptake and transformation is not known. We have therefore created in-frame deletions of each CRP-S gene and studied their competence phenotypes. All but one gene (ssb) could be deleted. Although none of the remaining CRP-S genes were required for growth in rich medium or survival under starvation conditions, DNA uptake and transformation were abolished or reduced in most of the mutants. Seventeen genes were absolutely required for transformation, with 14 of these genes being specifically required for the assembly and function of the type IV pilus DNA uptake machinery. Only five genes were dispensable for both competence and transformation. This is the first competence regulon for which all genes have been mutationally characterized. PMID:22821979

  17. Prostaglandin E2 regulates Th17 cell differentiation and function through cyclic AMP and EP2/EP4 receptor signaling.

    PubMed

    Boniface, Katia; Bak-Jensen, Kristian S; Li, Ying; Blumenschein, Wendy M; McGeachy, Mandy J; McClanahan, Terrill K; McKenzie, Brent S; Kastelein, Robert A; Cua, Daniel J; de Waal Malefyt, René

    2009-03-16

    Prostaglandins, particularly prostaglandin E2 (PGE2), play an important role during inflammation. This is exemplified by the clinical use of cyclooxygenase 2 inhibitors, which interfere with PGE2 synthesis, as effective antiinflammatory drugs. Here, we show that PGE2 directly promotes differentiation and proinflammatory functions of human and murine IL-17-producing T helper (Th17) cells. In human purified naive T cells, PGE2 acts via prostaglandin receptor EP2- and EP4-mediated signaling and cyclic AMP pathways to up-regulate IL-23 and IL-1 receptor expression. Furthermore, PGE2 synergizes with IL-1beta and IL-23 to drive retinoic acid receptor-related orphan receptor (ROR)-gammat, IL-17, IL-17F, CCL20, and CCR6 expression, which is consistent with the reported Th17 phenotype. While enhancing Th17 cytokine expression mainly through EP2, PGE2 differentially regulates interferon (IFN)-gamma production and inhibits production of the antiinflammatory cytokine IL-10 in Th17 cells predominantly through EP4. Furthermore, PGE2 is required for IL-17 production in the presence of antigen-presenting cells. Hence, the combination of inflammatory cytokines and noncytokine immunomodulators, such as PGE2, during differentiation and activation determines the ultimate phenotype of Th17 cells. These findings, together with the altered IL-12/IL-23 balance induced by PGE2 in dendritic cells, further highlight the crucial role of the inflammatory microenvironment in Th17 cell development and regulation.

  18. Ca/sup + +/- and cyclic AMP-induced changes in intact cell phosphorylation of ileal microvillus membrane proteins

    SciTech Connect

    Sharp, G.W.G.; Hannah, C.M.; Cohen, M.; Donowitz, M.

    1986-03-05

    Pieces of rabbit distal ileal mucosa, with the muscularis propria and serosa removed, were incubated for 90 minutes in Krebs-Ringer bicarborate buffer (KRB) with /sup 32/PO/sub 4/ to label the intracellular nucleotide pools. After rinsing, the mucosal pieces were transferred to KRB in the absence and presence of 10 ..mu..M A23187 or 10 mM theophylline. After a further 10 minutes the cells were scraped off and microvillus membranes prepared. The membranes were solubilized, subjected to two dimensional gel electrophoresis and autoradiography, and analyzed by densitometry. A23187 increased the phosphorylation of four microvillus membrane proteins with M/sub r/ of 32, 52, 110 and 116K. Increased phosphorylation of the 52 and 116K proteins has also been detected in microvillus membranes subjected to Ca/sup + +/ and calmodulin in the presence of ..gamma..-/sup 32/P-ATP. Theophylline increased the phosphorylation of the same 32 and 52K proteins and, additionally, of a second 32K peptide. While any of these proteins could be involved in the control of electrolyte transport, it is noteworthy that increased Ca/sup + +/, and increased cyclic AMP levels exert similar effects upon intestinal electrolyte transport. That A23187 and theophylline both increase the phosphorylation of the 32 and 52K proteins increases the possibility that these are involved in ion transport.

  19. 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

  20. 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

  1. 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…

  2. Trafficking and Gating of Hyperpolarization-activated Cyclic Nucleotide-gated Channels Are Regulated by Interaction with Tetratricopeptide Repeat-containing Rab8b-interacting Protein (TRIP8b) and Cyclic AMP at Distinct Sites*

    PubMed Central

    Han, Ye; Noam, Yoav; Lewis, Alan S.; Gallagher, Johnie J.; Wadman, Wytse J.; Baram, Tallie Z.; Chetkovich, Dane M.

    2011-01-01

    Ion channel trafficking and gating are often influenced by interactions with auxiliary subunits. Tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) is an auxiliary subunit for neuronal hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. TRIP8b interacts directly with two distinct sites of HCN channel pore-forming subunits to control channel trafficking and gating. Here we use mutagenesis combined with electrophysiological studies to define and distinguish the functional importance of the HCN/TRIP8b interaction sites. Interaction with the last three amino acids of the HCN1 C terminus governed the effect of TRIP8b on channel trafficking, whereas TRIP8b interaction with the HCN1 cyclic nucleotide binding domain (CNBD) affected trafficking and gating. Biochemical studies revealed that direct interaction between TRIP8b and the HCN1 CNBD was disrupted by cAMP and that TRIP8b binding to the CNBD required an arginine residue also necessary for cAMP binding. In accord, increasing cAMP levels in cells antagonized the up-regulation of HCN1 channels mediated by a TRIP8b construct binding the CNBD exclusively. These data illustrate the distinct roles of the two TRIP8b-HCN interaction domains and suggest that TRIP8b and cAMP may directly compete for binding the HCN CNBD to control HCN channel gating, kinetics, and trafficking. PMID:21504900

  3. 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

  4. A cyclic AMP receptor protein mutant that constitutively activates an Escherichia coli promoter disrupted by an IS5 insertion.

    PubMed

    Podolny, V; Lin, E C; Hochschild, A

    1999-12-01

    Previously an Escherichia coli mutant that had acquired the ability to grow on propanediol as the sole carbon and energy source was isolated. This phenotype is the result of the constitutive expression of the fucO gene (in the fucAO operon), which encodes one of the enzymes in the fucose metabolic pathway. The mutant was found to bear an IS5 insertion in the intergenic regulatory region between the divergently oriented fucAO and fucPIK operons. Though expression of the fucAO operon was constitutive, the fucPIK operon became noninducible such that the mutant could no longer grow on fucose. A fucose-positive revertant which was found to contain a suppressor mutation in the crp gene was selected. Here we identify this crp mutation, which results in a single amino acid substitution (K52N) that has been proposed previously to uncover a cryptic activating region in the cyclic AMP receptor protein (CRP). We show that the mutant CRP constitutively activates transcription from both the IS5-disrupted and the wild-type fucPIK promoters, and we identify the CRP-binding site that is required for this activity. Our results show that the fucPIK promoter, a complex promoter which ordinarily depends on both CRP and the fucose-specific regulator FucR for its activation, can be activated in the absence of FucR by a mutant CRP that uses three, rather than two, activating regions to contact RNA polymerase. For the IS5-disrupted promoter, which retains a single CRP-binding site, the additional activating region of the mutant CRP evidently compensates for the lack of upstream regulatory sequences. PMID:10601201

  5. Interleukin-6- and Cyclic AMP-Mediated Signaling Potentiates Neuroendocrine Differentiation of LNCaP Prostate Tumor Cells

    PubMed Central

    Deeble, Paul D.; Murphy, Daniel J.; Parsons, Sarah J.; Cox, Michael E.

    2001-01-01

    Neuroendocrine (NE) differentiation in prostatic adenocarcinomas has been reported to be an early marker for development of androgen independence. Secretion of mitogenic peptides from nondividing NE cells is thought to contribute to a more aggressive disease by promoting the proliferation of surrounding tumor cells. We undertook studies to determine whether the prostate cancer cell line LNCaP could be induced to acquire NE characteristics by treatment with agents that are found in the complex environment in which progression of prostate cancer towards androgen independence occurs. We found that cotreatment of LNCaP cells with agents that signal through cyclic AMP-dependent protein kinase (PKA), such as epinephrine and forskolin, and with the cytokine interleukin-6 (IL-6) promoted the acquisition of an NE morphological phenotype above that seen with single agents. Convergent IL-6 and PKA signaling also resulted in potentiated mitogen-activated protein kinase (MAPK) activation without affecting the level of signal transducer and activator of transcription or PKA activation observed with these agents alone. Cotreatment with epinephrine and IL-6 synergistically increased c-fos transcription as well as transcription from the β4 nicotinic acetylcholine receptor subunit promoter. Potentiated transcription from these elements was shown to be dependent on the MAPK pathway. Most importantly, cotreatment with PKA activators and IL-6 resulted in increased secretion of mitogenic neuropeptides. These results indicate that PKA and IL-6 signaling participates in gene transcriptional changes that reflect acquisition of an NE phenotype by LNCaP cells and suggest that similar signaling mechanisms, particularly at sites of metastasis, may be responsible for the increased NE content of many advanced prostate carcinomas. PMID:11713282

  6. Reversal of radiation-induced cisplatin resistance in murine fibrosarcoma cells by selective modulation of the cyclic GMP-dependent transduction pathway.

    PubMed Central

    Eichholtz-Wirth, H.

    1995-01-01

    Cisplatin resistance, induced in murine fibrosarcoma cells (SSK) in vitro or in vivo by low-dose irradiation, can be overcome by activation of the cyclic GMP(cGMP)-dependent transduction pathway. This is mediated either by stimulating cGMP formation with sodium nitroprusside or by replacing cGMP with a selective activator of the cGMP-dependent protein kinase, 8-bromo-cGMP. The cyclic AMP-dependent transduction pathway is not involved in cisplatin resistance. Instead, activation of cAMP sensitises both parental and resistant SSK cells equally to the action of cisplatin. There is a 1.8 to 2.5-fold increase in drug toxicity, depending on the activating agent. Enhancement of cisplatin sensitivity is induced by specific inhibition of cAMP hydrolysis, increase in cAMP formation or by increasing the activation potential to cAMP-dependent protein kinase by specific cAMP analogues. Cells that have lost cisplatin resistance respond to cGMP- or cAMP-elevating agents in the same way as the parental SSK cells. The radiation sensitivity is unchanged in all cell lines, even after activation of cAMP or cGMP. These results suggest that specific DNA repair pathways are altered by radiation but affected only in cisplatin damage repair, which is regulated by cGMP. Although there is ample cooperativity and interaction between the cAMP- and the cGMP-dependent transduction pathways, specific substrate binding by cGMP appears to play an important role in radiation-induced cisplatin resistance. PMID:7640207

  7. 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

  8. A mechanism for the auto-inhibition of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel opening and its relief by cAMP.

    PubMed

    Akimoto, Madoka; Zhang, Zaiyong; Boulton, Stephen; Selvaratnam, Rajeevan; VanSchouwen, Bryan; Gloyd, Melanie; Accili, Eric A; Lange, Oliver F; Melacini, Giuseppe

    2014-08-01

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels control neuronal and cardiac electrical rhythmicity. There are four homologous isoforms (HCN1-4) sharing a common multidomain architecture that includes an N-terminal transmembrane tetrameric ion channel followed by a cytoplasmic "C-linker," which connects a more distal cAMP-binding domain (CBD) to the inner pore. Channel opening is primarily stimulated by transmembrane elements that sense membrane hyperpolarization, although cAMP reduces the voltage required for HCN activation by promoting tetramerization of the intracellular C-linker, which in turn relieves auto-inhibition of the inner pore gate. Although binding of cAMP has been proposed to relieve auto-inhibition by affecting the structure of the C-linker and CBD, the nature and extent of these cAMP-dependent changes remain limitedly explored. Here, we used NMR to probe the changes caused by the binding of cAMP and of cCMP, a partial agonist, to the apo-CBD of HCN4. Our data indicate that the CBD exists in a dynamic two-state equilibrium, whose position as gauged by NMR chemical shifts correlates with the V½ voltage measured through electrophysiology. In the absence of cAMP, the most populated CBD state leads to steric clashes with the activated or "tetrameric" C-linker, which becomes energetically unfavored. The steric clashes of the apo tetramer are eliminated either by cAMP binding, which selects for a CBD state devoid of steric clashes with the tetrameric C-linker and facilitates channel opening, or by a transition of apo-HCN to monomers or dimer of dimers, in which the C-linker becomes less structured, and channel opening is not facilitated.

  9. 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.

  10. Macrophage growth arrest by cyclic AMP defines a distinct checkpoint in the mid-G1 stage of the cell cycle and overrides constitutive c-myc expression.

    PubMed

    Rock, C O; Cleveland, J L; Jackowski, S

    1992-05-01

    Proliferation of a murine macrophage cell line (BAC1.2F5) in response to colony-stimulating factor 1 (CSF-1) is inhibited by prostaglandin E2 (PGE2)-mediated elevation of intracellular cyclic AMP (cAMP). When BAC1.2F5 cells were growth arrested in early G1 by CSF-1 starvation and stimulated to synchronously enter the cell cycle by readdition of growth factor, PGE2 inhibited [3H]thymidine incorporation when added before mid-G1, but its addition at later times did not block the onset of S phase. Reversible cell cycle arrest mediated by a cAMP analog required the presence of CSF-1 for cells to initiate DNA synthesis, whereas cells released from an aphidicolin block at the G1/S boundary entered S phase in the absence of CSF-1. PGE2 or cAMP analogs did not block the initial induction of c-myc mRNA by CSF-1 but abolished the CSF-1-dependent expression of c-myc mRNA in the mid-G1 stage of the cell cycle. The cAMP-mediated reduction in c-myc RNA levels was due to decreased c-myc transcription. However, CSF-1-dependent BAC1.2F5 clones infected with a c-myc retrovirus were growth arrested by cAMP analogs despite constitutive c-myc expression. Therefore, the reduction of endogenous c-myc expression by cAMP is neither necessary nor sufficient for growth inhibition.

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

    PubMed

    Rosenberg, P A; Li, Y

    1995-09-18

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

  12. Serum-stimulated cyclic-AMP production in S49 lymphoma cells grown in serum-free medium.

    PubMed

    Darfler, F J; Mullen, M D; Insel, P A

    1984-03-23

    Growth of S49 lymphoma cells with horse serum leads to an increase in cellular cAMP phosphodiesterase activity and a resultant loss of hormone- and cholera-toxin-stimulated cAMP accumulation. We now show that the serum requires protein synthesis to produce these effects. Further, we show that acute addition of serum to wild-type S49 cells, grown in serum-free medium, rapidly (under 2 min) and transiently (under 30 min) stimulates cellular cAMP, 10-fold over basal levels. This 'acute' effect of serum was not observed in UNC S49 cells, suggesting that a functional Ns, the guanine nucleotide regulatory component that mediates stimulation of adenylate cyclase, is required for the serum-mediated stimulation of cellular cAMP. Serum added acutely to wild-type S49 cells also augmented cAMP accumulation in response to isoproterenol and forskolin. The half-maximally effective concentrations of horse serum that acutely stimulated or more slowly decreased the cAMP accumulation were approx. 0.2% and 2.0%, respectively. Preliminary attempts to characterize further the serum factor indicate that it has a high (250 000-300 000) molecular weight and is insensitive to boiling; chromatography on Sepharose CL-6B yields a 100-fold purification. Thus, the serum contains one or more components that activate adenylate cyclase, increase cellular cAMP levels and ultimately induce cAMP phosphodiesterase in S49 lymphoma cells. PMID:6322858

  13. Intracellular cyclic AMP not calcium, determines the direction of vesicle movement in melanophores: direct measurement by fluorescence ratio imaging

    PubMed Central

    1992-01-01

    Intracellular movement of vesiculated pigment granules in angelfish melanophores is regulated by a signalling pathway that triggers kinesin and dyneinlike microtubule motor proteins. We have tested the relative importance of intracellular Ca2+ ([Ca2+]i) vs cAMP ([cAMP]i) in the control of such motility by adrenergic agonists, using fluorescence ratio imaging and many ways to artificially stimulate or suppress signals in these pathways. Fura-2 imaging reported a [Ca2+]i elevation accompanying pigment aggregation, but this increase was not essential since movement was not induced with the calcium ionophore, ionomycin, nor was movement blocked when the increases were suppressed by withdrawal of extracellular Ca2+ or loading of intracellular BAPTA. The phosphatase inhibitor, okadaic acid, blocked aggregation and induced dispersion at concentrations that suggested that the protein phosphatase PP-1 or PP-2A was continuously turning phosphate over during intracellular motility. cAMP was monitored dynamically in single living cells by microinjecting cAMP-dependent kinase in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine respectively (Adams et al., 1991. Nature (Lond.). 349:694- 697). Ratio imaging of F1CRhR showed that the alpha 2-adrenergic receptor-mediated aggregation was accompanied by a dose-dependent decrease in [cAMP]i. The decrease in [cAMP]i was both necessary and sufficient for aggregation, since cAMP analogs or microinjected free catalytic subunit of A kinase-blocked aggregation or caused dispersal, whereas the cAMP antagonist RpcAMPs or the microinjection of the specific kinase inhibitor PKI5-24 amide induced aggregation. Our conclusion that cAMP, not calcium, controls bidirectional microtubule dependent motility in melanophores might be relevant to other instances of non-muscle cell motility. PMID:1348251

  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.

  15. 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

  16. 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.

  17. Cyclic AMP suppresses expression of v-rasH oncogene linked to the mouse mammary tumor virus promoter.

    PubMed

    Najam, N; Clair, T; Bassin, R H; Cho-Chung, Y S

    1986-01-14

    Clone 433.3 of NIH 3T3 cells is a stable carrier of the MMTV LTR:v-rasH chimeric DNA. Only in the presence of dexamethasone (a synthetic glucocorticoid), 433.3 cells exhibit an induced level of p21 transforming protein and phenotypic transformation. N6,O2'-dibutyryl cAMP (DBcAMP) antagonized the effect of dexamethasone in a time - and concentration - dependent manner. DBcAMP (5 X 10(-4)M) added 18 hr prior to the addition of dexamethasone (10(-7)M) almost completely blocked the hormone effect: cells contained levels of p21 20% of that in the cells treated with dexamethasone alone, and formed flat, contact inhibited monolayers. On the basis of these results together with our previous data on mammary carcinomas in vivo, we postulate that cAMP may be an intracellular suppressor acting at a regulatory locus of both cellular and viral ras genes.

  18. Mutations That Affect Transcription and Cyclic Amp-Crp Regulation of the Adenylate Cyclase Gene (Cya) of Salmonella Typhimurium

    PubMed Central

    Fandl, J. P.; Thorner, L. K.; Artz, S. W.

    1990-01-01

    We studied the expression of the cya promoter(s) in cya-lac fusion strains of Salmonella typhimurium and demonstrated cAMP receptor protein (CRP)-dependent repression by cAMP. Expression of cya was reduced about fourfold in cultures grown in acetate minimal medium as compared to cultures grown in glucose-6-phosphate minimal medium. Expression of cya was also reduced about fourfold by addition of 5 mM cAMP to cultures grown in glucose minimal medium. We constructed in vitro deletion and insertion mutations altering a major cya promoter (P2) and a putative CRP binding site overlapping P2. These mutations were recombined into the chromosome by allele replacement with M13mp::cya recombinant phages and the regulation of the mutant promoters was analyzed. A 4-bp deletion of the CRP binding site and a 4-bp insertion in this site nearly eliminated repression by cAMP. A mutant with the P2 promoter and the CRP binding site both deleted exhibited an 80% reduction in cya expression; the 20% residual expression was insensitive to cAMP repression. This mutant retained a Cya(+) phenotype. Taken together, the results establish that the cya gene is transcribed from multiple promoters one of which, P2, is negatively regulated by the cAMP-CRP complex. Correction for the contribution to transcription by the cAMP-CRP nonregulated cya promoters indicates that the P2 promoter is repressed at least eightfold by cAMP-CRP. PMID:2168849

  19. Downregulation of steroidogenic acute regulatory protein (StAR) gene expression by cyclic AMP in cultured Schwann cells.

    PubMed

    Benmessahel, Yasmina; Troadec, Jean-Denis; Cadepond, Françoise; Guennoun, Rachida; Hales, Dale Buchanan; Schumacher, Michael; Groyer, Ghislaine

    2004-02-01

    Steroidogenic acute regulatory protein (StAR) plays a key role in the availability of cholesterol to the inner mitochondrial membrane, where the first step of steroidogenesis, its conversion to pregnenolone, takes place. Here, we demonstrate for the first time that the StAR gene is also expressed in the rat sciatic nerve and in cultured Schwann cells. The addition to the culture medium of the cAMP-elevating agent forskolin or of the cAMP analogue 8Br-cAMP produced a time-course extinction of StAR gene expression. An inverse relationship was demonstrated between StAR gene expression and the intracellular cAMP content. Accordingly, pharmacological inhibition of the activities of Schwann cell adenylyl cyclase or of phosphodiesterase IV resulted in modifications of StAR gene expression. Since StAR gene expression is stimulated by cAMP in classical steroidogenic cells, our work is the first demonstration of a negative regulation of StAR gene by cAMP.

  20. The cyclic AMP pathway is a sex-specific modifier of glioma risk in type I neurofibromatosis patients.

    PubMed

    Warrington, Nicole M; Sun, Tao; Luo, Jingqin; McKinstry, Robert C; Parkin, Patricia C; Ganzhorn, Sara; Spoljaric, Debra; Albers, Anne C; Merkelson, Amanda; Stewart, Douglas R; Stevenson, David A; Viskochil, David; Druley, Todd E; Forys, Jason T; Reilly, Karlyne M; Fisher, Michael J; Tabori, Uri; Allen, Jeffrey C; Schiffman, Joshua D; Gutmann, David H; Rubin, Joshua B

    2015-01-01

    Identifying modifiers of glioma risk in patients with type I neurofibromatosis (NF1) could help support personalized tumor surveillance, advance understanding of gliomagenesis, and potentially identify novel therapeutic targets. Here, we report genetic polymorphisms in the human adenylate cyclase gene adenylate cyclase 8 (ADCY8) that correlate with glioma risk in NF1 in a sex-specific manner, elevating risk in females while reducing risk in males. This finding extends earlier evidence of a role for cAMP in gliomagenesis based on results in a genetically engineered mouse model (Nf1 GEM). Thus, sexually dimorphic cAMP signaling might render males and females differentially sensitive to variation in cAMP levels. Using male and female Nf1 GEM, we found significant sex differences exist in cAMP regulation and in the growth-promoting effects of cAMP suppression. Overall, our results establish a sex-specific role for cAMP regulation in human gliomagenesis, specifically identifying ADCY8 as a modifier of glioma risk in NF1.

  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.; 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.

  2. p54nrb/NONO Regulates Cyclic AMP-Dependent Glucocorticoid Production by Modulating Phosphodiesterase mRNA Splicing and Degradation

    PubMed Central

    Lu, Jia Yang

    2015-01-01

    Glucocorticoid production in the adrenal cortex is activated in response to an increase in cyclic AMP (cAMP) signaling. The nuclear protein p54nrb/NONO belongs to the Drosophila behavior/human splicing (DBHS) family and has been implicated in several nuclear processes, including transcription, splicing, and RNA export. We previously identified p54nrb/NONO as a component of a protein complex that regulates the transcription of CYP17A1, a gene required for glucocorticoid production. Based on the multiple mechanisms by which p54nrb/NONO has been shown to control gene expression and the ability of the protein to be recruited to the CYP17A1 promoter, we sought to further define the molecular mechanism by which p54nrb/NONO confers optimal cortisol production. We show here that silencing p54nrb/NONO expression in H295R human adrenocortical cells decreases the ability of the cells to increase intracellular cAMP production and subsequent cortisol biosynthesis in response to adrenocorticotropin hormone (ACTH) stimulation. Interestingly, the expression of multiple phosphodiesterase (PDE) isoforms, including PDE2A, PDE3A, PDE3B, PDE4A, PDE4D, and PDE11A, was induced in p54nrb/NONO knockdown cells. Investigation of the mechanism by which silencing of p54nrb/NONO led to increased expression of select PDE isoforms revealed that p54nrb/NONO regulates the splicing of a subset of PDE isoforms. Importantly, we also identify a role for p54nrb/NONO in regulating the stability of PDE transcripts by facilitating the interaction between the exoribonuclease XRN2 and select PDE transcripts. In summary, we report that p54nrb/NONO modulates cAMP-dependent signaling, and ultimately cAMP-stimulated glucocorticoid biosynthesis by regulating the splicing and degradation of PDE transcripts. PMID:25605330

  3. 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.

  4. 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.

  5. Regulation of vascular smooth muscle cell calcification by extracellular pyrophosphate homeostasis: synergistic modulation by cyclic AMP and hyperphosphatemia

    PubMed Central

    Prosdocimo, Domenick A.; Wyler, Steven C.; Romani, Andrea M.; O'Neill, W. Charles

    2010-01-01

    Vascular calcification is a multifaceted process involving gain of calcification inducers and loss of calcification inhibitors. One such inhibitor is inorganic pyrophosphate (PPi), and regulated generation and homeostasis of extracellular PPi is a critical determinant of soft-tissue mineralization. We recently described an autocrine mechanism of extracellular PPi generation in cultured rat aortic vascular smooth muscle cells (VSMC) that involves both ATP release coupled to the ectophosphodiesterase/pyrophosphatase ENPP1 and efflux of intracellular PPi mediated or regulated by the plasma membrane protein ANK. We now report that increased cAMP signaling and elevated extracellular inorganic phosphate (Pi) act synergistically to induce calcification of these VSMC that is correlated with progressive reduction in ability to accumulate extracellular PPi. Attenuated PPi accumulation was mediated in part by cAMP-dependent decrease in ANK expression coordinated with cAMP-dependent increase in expression of TNAP, the tissue nonselective alkaline phosphatase that degrades PPi. Stimulation of cAMP signaling did not alter ATP release or ENPP1 expression, and the cAMP-induced changes in ANK and TNAP expression were not sufficient to induce calcification. Elevated extracellular Pi alone elicited only minor calcification and no significant changes in ANK, TNAP, or ENPP1. In contrast, combined with a cAMP stimulus, elevated Pi induced decreases in the ATP release pathway(s) that supports ENPP1 activity; this resulted in markedly reduced rates of PPi accumulation that facilitated robust calcification. Calcified VSMC were characterized by maintained expression of multiple SMC differentiation marker proteins including smooth muscle (SM) α-actin, SM22α, and calponin. Notably, addition of exogenous ATP (or PPi per se) rescued cAMP + phosphate-treated VSMC cultures from progression to the calcified state. These observations support a model in which extracellular PPi generation mediated

  6. Thyroid-stimulating hormone stimulates increases in inositol phosphates as well as cyclic AMP in the FRTL-5 rat thyroid cell line.

    PubMed Central

    Field, J B; Ealey, P A; Marshall, N J; Cockcroft, S

    1987-01-01

    Studies were conducted to determine whether thyroid-stimulating hormone (TSH; thyrotropin), a hormone known to increase cytosol concentrations of cyclic AMP, also stimulates the formation of inositol phosphates in thyroid cells. TSH and noradrenaline both stimulated [3H]inositol phosphate formation in a concentration-dependent manner in the rat thyroid cell line, FRTL-5 cells, which had been prelabelled with [3H]inositol. The threshold concentration of TSH required to stimulate inositol phosphate formation was more than 20 munits/ml, which is approx. 10(3)-fold greater than that required for cyclic AMP accumulation and growth in these cells. We also demonstrate that membranes prepared from FRTL-5 cells possess a guanine nucleotide-activatable polyphosphoinositide phosphodiesterase, which suggests that activation of inositide metabolism in these cells may be coupled to receptors by the G-protein, Gp. Our findings suggest that two second-messenger systems exist to mediate the action of TSH in the thyroid. PMID:2827631

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. Role of the human cytomegalovirus major immediate-early promoter's 19-base-pair-repeat cyclic AMP-response element in acutely infected cells.

    PubMed

    Keller, M J; Wheeler, D G; Cooper, E; Meier, J L

    2003-06-01

    Prior studies have suggested a role of the five copies of the 19-bp-repeat cyclic AMP (cAMP)-response element (CRE) in major immediate-early (MIE) promoter activation, the rate-limiting step in human cytomegalovirus (HCMV) replication. We used two different HCMV genome modification strategies to test this hypothesis in acutely infected cells. We report the following: (i) the CREs do not govern basal levels of MIE promoter activity at a high or low multiplicity of infection (MOI) in human foreskin fibroblast (HFF)- or NTera2-derived neuronal cells; (ii) serum and virion components markedly increase MIE promoter-dependent transcription at a low multiplicity of infection (MOI), but this increase is not mediated by the CREs; (iii) forskolin stimulation of the cAMP signaling pathway induces a two- to threefold increase in MIE RNA levels in a CRE-specific manner at a low MOI in both HFF- and NTera2-derived neuronal cells; and (iv) the CREs do not regulate basal levels of HCMV DNA replication at a high or low MOI in HFF. Their presence does impart a forskolin-induced increase in viral DNA replication at a low MOI but only when basal levels of MIE promoter activity are experimentally diminished. In conclusion, the 19-bp-repeat CREs add to the robust MIE promoter activity that occurs in the acutely infected stimulated cells, although the CREs' greater role may be in other settings.

  12. Influence of cyclic nucleotides (cAMP) on inositol phospholipid (InsPL) metabolism in cultured mesangial (MS) cells

    SciTech Connect

    Troyer, D.A.; Venkatachalam, M.A.; Bonventre, J.V.; Kreisberg, J.I.

    1986-03-01

    Elevation of cAMP inhibits hormone-induced contraction of MS cells, and in other cell types, reduces stimulated InsPL metabolism. The authors found that neither isobutylmethylxanthine (MIX, 0.5 mM), which increases MS cell cAMP levels 4-fold, nor forskolin (100 ..mu..M) altered vasopressin (VP, 10 nM) induced release of /sup 3/H-inositol trisphosphate from prelabelled MS cells. Also, maneuvers which elevated cAMP did not block the VP-induced rise of intracellular calcium as measured by quin-2. Further, neither MIX nor isoproterenol affected the stimulation of glycolysis by VP as measured by lactic acid production. MIX diminished VP stimulated /sup 32/P orthophosphate (/sup 32/P) incorporation into phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 4-phosphate and phosphatidylinositol. The /sup 32/P content in phosphoinositides of cells treated with MIX and VP was 65% of that in cells treated with VP only. However, the authors found that the specific activity of /sup 32/P in ATP in the presence of MIX + VP was 74% of that with VP alone. Thus, the apparent suppression of /sup 32/P incorporation due to MIX was attributable to a concurrent diminution of the specific activity of /sup 32/P in ATP. The authors conclude that increases of cAMP interfere with contraction distal to PIP/sub 2/ hydrolysis, inositol phosphate release, calcium mobilization, and enhancement of glycolysis.

  13. Involvement of the cyclic-AMP-dependent protein kinase A pathway in thyroxine effects on calcitonin secretion from TT cells.

    PubMed

    Lu, C-C; Tsai, S-C

    2011-01-01

    Previous studies have demonstrated that plasma calcitonin is lower in hypothyroid patients and that thyroxine stimulates the human thyroid to release calcitonin. Therefore, thyroid hormones may regulate the secretion of calcitonin, but further work is needed to address this possibility in more detail. TT cells, a model of human thyroid C cells, were incubated in a medium containing vehicle, thyroxine, or thyroxine methyl-hemisuccinate-bovine serum albumin (BSA-L-T(4), thyroxine was immobilized and linked to BSA); then, the levels of secreted calcitonin (hCT), calcitonin mRNA, and cAMP were measured. To study links that connect the cAMP-dependent protein kinase A (PKA) pathway to the observed thyroxine effects, cells were treated with either vehicle or thyroxine plus SQ22536 [an adenylyl cyclase (AC) inhibitor], KT5720 (a PKA inhibitor), or 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor). The activity levels of AC and PKA, and secreted calcitonin were then measured. The results indicate that thyroxine increases calcitonin secretion, cellular cAMP accumulation, and the activities of AC and PKA, but does not increase hCT mRNA levels in TT cells. BSA-L-T(4) also increases calcitonin secretion. These effects are inhibited by SQ22536, and KT5720 and suggest that the nongenomic thyroxine effects that stimulate calcitonin secretion from TT cells involve the cAMP-dependent PKA pathway.

  14. Cyclic AMP directs inositol (1,4,5)-trisphosphate-evoked Ca2+ signalling to different intracellular Ca2+ stores

    PubMed Central

    Tovey, Stephen C.; Taylor, Colin W.

    2013-01-01

    Summary Cholesterol depletion reversibly abolishes carbachol-evoked Ca2+ release from inositol (1,4,5)-trisphosphate (IP3)-sensitive stores, without affecting the distribution of IP3 receptors (IP3R) or endoplasmic reticulum, IP3 formation or responses to photolysis of caged IP3. Receptors that stimulate cAMP formation do not alone evoke Ca2+ signals, but they potentiate those evoked by carbachol. We show that these potentiated signals are entirely unaffected by cholesterol depletion and that, within individual cells, different IP3-sensitive Ca2+ stores are released by carbachol alone and by carbachol combined with receptors that stimulate cAMP formation. We suggest that muscarinic acetylcholine receptors in lipid rafts deliver IP3 at high concentration to associated IP3R, stimulating them to release Ca2+. Muscarinic receptors outside rafts are less closely associated with IP3R and provide insufficient local IP3 to activate IP3R directly. These IP3R, probably type 2 IP3R within a discrete Ca2+ store, are activated only when their sensitivity is increased by cAMP. Sensitization of IP3R by cAMP extends the effective range of signalling by phospholipase C, allowing muscarinic receptors that are otherwise ineffective to recruit additional IP3-sensitive Ca2+ stores. PMID:23525004

  15. A Ric8/synembryn homolog promotes Gpa1 and Gpa2 activation to respectively regulate cyclic AMP and pheromone signaling in Cryptococcus neoformans.

    PubMed

    Gong, Jinjun; Grodsky, Jacob D; Zhang, Zhengguang; Wang, Ping

    2014-10-01

    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 GPA1(Q284L) 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 Gpa1(Q284L) negatively affected its interaction with Ric8, whereas the activated Gpa2(Q203L) 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.

  16. Regulation of the Subcellular Localization of Cyclic AMP-Dependent Protein Kinase in Response to Physiological Stresses and Sexual Differentiation in the Fission Yeast Schizosaccharomyces pombe▿ †

    PubMed Central

    Matsuo, Yasuhiro; McInnis, Brittney; Marcus, Stevan

    2008-01-01

    We describe regulation of the subcellular localization of cyclic AMP (cAMP)-dependent protein kinase (PKA) regulatory (Cgs1p) and catalytic (Pka1p) subunits in the fission yeast Schizosaccharomyces pombe in response to physiological stresses and during sexual differentiation as determined by fluorescence microscopy of the Cgs1-green fluorescent protein (GFP) and Pka1-GFP fusion proteins, respectively. In wild-type S. pombe cells cultured to log phase under normal growth conditions, Cgs1p and Pka1p are concentrated in the nucleus and more diffusely present in the cytoplasm. Nuclear localization of both proteins is dependent on cAMP, since in cells lacking adenylate cyclase they are detectable only in the cytoplasm. In cells lacking Cgs1p or both Cgs1p and adenylate cyclase, Pka1p is concentrated in the nucleus, demonstrating a role for Cgs1p in the nuclear exclusion of Pka1p. Nuclear-cytoplasmic redistribution of Cgs1p and Pka1p is triggered by growth in glucose-limited or hyperosmotic media and in response to stationary-phase growth. In addition, both proteins are excluded from the nucleus in mating cells undergoing karyogamy and subsequently concentrated in postmeiotic spores. Cgs1p is required for subcellular redistribution of Pka1p induced by growth in glucose-limited and hyperosmotic media and during karyogamy but is not required for Pka1p redistribution triggered by stationary-phase growth or for the enrichment of Pka1p in spores. Our results demonstrate that PKA localization is regulated by cAMP and regulatory subunit-dependent and -independent mechanisms in S. pombe. PMID:18621924

  17. 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

  18. 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

  19. 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

  20. Aldo-keto reductase 1b7, a novel marker for renin cells, is regulated by cyclic AMP signaling

    PubMed Central

    Lin, Eugene E.; Pentz, Ellen S.; Sequeira-Lopez, Maria Luisa S.

    2015-01-01

    We previously identified aldo-keto reductase 1b7 (AKR1B7) as a marker for juxtaglomerular renin cells in the adult mouse kidney. However, the distribution of renin cells varies dynamically, and it was unknown whether AKR1B7 maintains coexpression with renin in response to different developmental, physiological, and pathological situations, and furthermore, whether similar factor(s) simultaneously regulate both proteins. We show here that throughout kidney development, AKR1B7 expression—together with renin—is progressively restricted in the kidney arteries toward the glomerulus. Subsequently, when formerly renin-expressing cells reacquire renin expression, AKR1B7 is reexpressed as well. This pattern of coexpression persists in extreme pathological situations, such as deletion of the genes for aldosterone synthase or Dicer. However, the two proteins do not colocalize within the same organelles: renin is found in the secretory granules, whereas AKR1B7 localizes to the endoplasmic reticulum. Interestingly, upon deletion of the renin gene, AKR1B7 expression is maintained in a pattern mimicking the embryonic expression of renin, while ablation of renin cells resulted in complete abolition of AKR1B7 expression. Finally, we demonstrate that AKR1B7 transcription is controlled by cAMP. Cultured cells of the renin lineage reacquire the ability to express both renin and AKR1B7 upon elevation of intracellular cAMP. In vivo, deleting elements of the cAMP-response pathway (CBP/P300) results in a stark decrease in AKR1B7- and renin-positive cells. In summary, AKR1B7 is expressed within the renin cell throughout development and perturbations to homeostasis, and AKR1B7 is regulated by cAMP levels within the renin cell. PMID:26180185

  1. 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.

  2. Conformational analysis of PKI(5-22)amide, the active inhibitory fragment of the inhibitor protein of the cyclic AMP-dependent protein kinase.

    PubMed

    Reed, J; De Ropp, J S; Trewhella, J; Glass, D B; Liddle, W K; Bradbury, E M; Kinzel, V; Walsh, D A

    1989-12-01

    Fourier-transform i.r. spectroscopy, 1H-n.m.r. spectroscopy and X-ray scattering were used to study the conformation and shape of the peptide PKI(5-22)amide, which contains the active site of the inhibitor protein of the cyclic AMP-dependent protein kinase [Cheng, Van Pattern, Smith & Walsh (1985) Biochem. J. 231, 655-661]. The X-ray-scattering solution studies show that the peptide has a compact structure with Rg 0.9 nm (9.0 A) and a linear maximum dimension of 2.5 nm (25A). Compatible with this, Fourier-transform i.r. and n.m.r. determinations indicate that the peptide contains approx. 26% alpha-helix located in the N-terminal one-third of the molecule. This region contains the phenylalanine residue that is one essential recognition determinant for high-affinity binding to the protein kinase catalytic site.

  3. Release of Ca2+ by inositol 1,4,5-trisphosphate in platelet membrane vesicles is not dependent on cyclic AMP-dependent protein kinase.

    PubMed Central

    O'Rourke, F; Zavoico, G B; Feinstein, M B

    1989-01-01

    In contrast with previous reports, it was found that membrane-protein phosphorylation by the catalytic subunit (CS) of cyclic AMP-dependent protein kinase had no effect on Ca2+ uptake into platelet membrane vesicles or on subsequent Ca2+ release by inositol 1,4,5-trisphosphate (IP3). Furthermore, IP-20, a highly potent synthetic peptide inhibitor of CS, which totally abolished membrane protein phosphorylation by endogenous or exogenous CS, also had no effect on either Ca2+ uptake or release by IP3. Commercial preparations of protein kinase inhibitor protein (PKI) usually had no effect, but one preparation partially inhibited Ca2+ uptake, which is attributable to the gross impurity of the commercial PKI preparation. IP3-induced release of Ca2+ was also unaffected by the absence of ATP from the medium, supporting the conclusion that Ca2+ release by IP3 does not require the phosphorylation of membrane protein. Images Fig. 3. PMID:2784669

  4. Differential activation of the 21-base-pair enhancer element of human T-cell leukemia virus type I by its own trans-activator and cyclic AMP.

    PubMed Central

    Nakamura, M; Niki, M; Ohtani, K; Sugamura, K

    1989-01-01

    A transcriptional trans-acting factor p40tax of human T-cell leukemia virus type I (HTLV-I) functions as an inducer for expression of HTLV-I provirus via activation of the enhancer in the long terminal repeat of HTLV-I. In addition to p40tax and a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, we report here that forskolin, an activator of adenyl cyclase, also induces function of the HTLV-I enhancer. Experiments with mutants of the HTLV-I enhancer revealed that TPA-induced activation was not mediated by solely a 21-base-pair (bp) sequence that is repeated three times in the enhancer, whereas the 21-bp enhancer element can act as a sufficient cis-acting sequence for activation by both p40tax and forskolin. In addition, we found that nuclear factor(s) like the cyclic AMP-responsive element (CRE) binding factor could bind to the HTLV-I 21-bp enhancer element. However, a difference was found in sequences required for activation by p40tax and forskolin. A CRE related sequence present in the 21-bp enhancer element was enough for forskolin-induced activation. On the other hand, p40tax required a much longer sequence that is overlapping but not identical to the CRE related sequence, suggesting that the forskolin-induced cyclic AMP pathway may be partly involved in, but not sufficient for p40tax-mediating trans-activation of the HTLV-I enhancer. Images PMID:2548156

  5. Dibutyryl cyclic AMP stimulation of a monocyte-like cell line, U937: a model for monocyte chemotaxis and Fc receptor-related functions.

    PubMed Central

    Sheth, B; Dransfield, I; Partridge, L J; Barker, M D; Burton, D R

    1988-01-01

    Treatment of the U937 cell line with 1 mM dibutyryl cyclic AMP (Bt2cAMP) resulted in a reduction in cell size and inhibition of DNA synthesis, and morphologically the cells appeared similar to macrophages. Electron micrographs indicated an increase in intracellular apparatus, whilst histochemical studies revealed smaller, denser nuclei and a greater intensity of non-specific esterase staining. Ia-like antigens (HLA-DR and HLA-DC) and complement receptor CR1 were not detected on U937 cells by monoclonal antibodies, nor were they induced by Bt2cAMP. CR3 was present in small amounts on U937 cells, and stimulation with Bt2cAMP increased the expression of this molecule in the cytoplasm and on the cell surface. Leu M3, a monocyte-specific antibody, was weakly reactive on both unstimulated and stimulated cells, whereas transferrin receptors, present on 90% of U937 cells, were lost after 48-hr stimulation with Bt2cAMP. JW6 and NH6, two monoclonal antibodies raised in our laboratory and found to be against immature monocytic antigens, showed decreased expression on stimulation. Monomer IgG binding via Fc receptors decreased on stimulated cells, and a monoclonal antibody (32.2) specific for FcRI confirmed this to be due to a decrease in the number of high-affinity receptors, rather than a decrease in IgG-binding affinity. In contrast, expression of the low-affinity FcRII, monitored by monoclonal antibody IV3, increased dramatically after stimulation. Other functional changes included the production of superoxide anions and the induction of non-specific phagocytosis. Two dimensional gel analysis, of detergent soluble proteins from unstimulated and 48-hr stimulated U937 cells, showed many differences in protein expression. A detailed investigation of these changes will facilitate a better understanding of the molecular mechanisms involved in the differentiation of U937 cells. Images Figure 2 Figure 3 Figure 6 PMID:2832314

  6. Membrane physical properties do not explain increased cyclic AMP production in hepatocytes from rats fed menhaden oil.

    PubMed

    Bizeau, M E; Hazel, J R

    2000-06-01

    To study the effect of altering plasma membrane fatty acid composition on the glucagon signal transduction pathway, cAMP accumulation was measured in hepatocytes from rats fed diets containing either menhaden oil (MO) or coconut oil (CO). Hepatocytes from MO-fed animals produced significantly more cAMP in response to glucagon and forskolin compared to CO-fed animals. Glucagon receptor number and affinity were similar in MO- and CO-fed rats. Liver plasma membranes from MO-fed animals were enriched in long-chain n-3 fatty acids and contained significantly lower amounts of saturated C10-C16 and 18:1n-9 than CO-fed animals. Membrane physical properties were examined using both Fourier transform infrared spectroscopy (FTIR) and the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). FTIR analysis revealed that below 34 degrees C, CO membranes were more ordered than MO membranes. However, as assay temperature approached 37 degrees C, MO and CO membranes became similarly ordered. DPH polarization values indicated no differences in membrane order at 37 degrees C, whereas membrane order was decreased in CO-fed animals at 25 degrees C. These data indicate the importance of assay temperature in assessing the influence of membrane physical properties on the activity of signal transduction pathways. Whereas increased signal transduction activity has been correlated to reduced membrane order in MO-fed animals, these data indicate that at physiological temperatures membrane order did not vary between groups. Enhanced cAMP accumulation in response to forskolin indicates that adenylate cyclase activity or content may be elevated in MO- vs. CO-fed rats. Enhanced adenylate cyclase activity may result, in part, from changes in specific fatty acids in hepatocyte plasma membranes without demonstrable changes in membrane physical properties.

  7. Influence of cyclic AMP, agmatine, and a novel protein encoded by a flanking gene on speB (agmatine ureohydrolase) in Escherichia coli.

    PubMed Central

    Szumanski, M B; Boyle, S M

    1992-01-01

    The speB gene of Escherichia coli encodes agmatine ureohydrolase (AUH), a putrescine biosynthetic enzyme. The speB gene is transcribed either from its own promoter or as a polycistronic message from the promoter of the speA gene encoding arginine decarboxylase. Two open reading frames (ORF1 and ORF2) are present on the strand complementary to speB; approximately 90% of ORF2 overlaps the speB coding region. Analysis of transcriptional and translational fusions of ORF1 or ORF2 to lacZ revealed that ORF1 encoded a novel protein while ORF2 was not transcribed. Deletion of ORF1 from a plasmid containing ORF1, ORF2, and speB reduced the activity of AUH by 83%. In contrast, the presence of plasmid-encoded ORF1 caused an 86% increase in chromosomally encoded AUH activity. ORF1 did not stimulate alkaline phosphatase expressed from a phi(speB-phoA) transcriptional fusion encoded on the same plasmid. Western analysis (immunoblot) of a phi(ORF1-lacZ) translational fusion revealed that ORF1 encodes a 25.3-kDa protein. Agmatine induced transcription of phi(speB-phoA) but not phi(speA-phoA) fusions. Consequently, agmatine affects selection between the monocistronic and the polycistronic modes of speB transcription. In contrast, cyclic AMP (cAMP) repressed AUH activity of chromosomally encoded AUH but had no effect on plasmid-borne speB nor phi(speB-phoA). It is concluded that ORF1 encodes a protein which is a posttranscriptional regulator of speB, agmatine induces speB independent of speA, and cAMP regulates speB indirectly. Images PMID:1310091

  8. Cyclic AMP-dependent constitutive expression of gal operon: use of repressor titration to isolate operator mutations.

    PubMed Central

    Irani, M; Orosz, L; Busby, S; Taniguchi, T; Adhya, S

    1983-01-01

    When the gal operator region is present in a multicopy plasmid it binds to all ("titrates") the gal repressor and "induces" the chromosomal gal operon. To make operator mutations (Oa) with reduced affinity toward the repressor, plasmid DNA was irradiated with UV light and mutant derivatives were isolated that were unable to release the chromosomal gal genes from repression. Then with such an Oa plasmid operator revertants were isolated that had reacquired the ability to release repression. Both sets of mutations have been localized by DNA sequence analysis. When the Oa mutations were transferred from the plasmid to the chromosome by recombination these mutant operators were found to make gal expression constitutive (independent of repressor) but still dependent on cAMP, whereas the previously reported gal operator mutants (Oc) are constitutive both in the presence and in the absence of cAMP. The titration method of isolating mutants enables the isolation of strains with operator mutations that also affect normal promoter activity, and it provides an easy way to isolate revertants of operator mutations. Images PMID:6308647

  9. 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

  10. Insights into GPCR pharmacology from the measurement of changes in intracellular cyclic AMP; advantages and pitfalls of differing methodologies

    PubMed Central

    Hill, Stephen J; Williams, Christine; May, Lauren T

    2010-01-01

    It is clear that the G protein-coupled receptor family play a key role in the pharmaceutical industry, with a significant proportion of approved drugs targeting this protein class. While our growing understanding of the complexity of G protein-coupled receptor pharmacology is playing a key role in the future success of these endeavours, with allosteric mechanisms now well integrated into the industrial community and G protein-independent signalling mechanisms establishing themselves as novel phenomenon to be exploited, it is still possible to underestimate the complexity of G protein signal transduction mechanisms and the impact that inappropriate study of these mechanisms can have on data interpretation. In this manuscript we review different approaches to measuring the cAMP signal transduction pathway, with particular emphasis on key parameters influencing the data quality and biological relevance. PMID:21049583

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

    PubMed

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

    2016-07-15

    Propofol inhibits myocardial contraction in a dose dependent manner. The present study is designed to examine the effect of propofol on PKA mediated myocardial contraction in the absence of adrenoreceptor agonist. The contraction of isolated rat heart was measured in the presence or absence of PKA inhibitor H89 or propofol, using a pressure transducer. The levels of cAMP and PKA kinase activity were detected by ELISA. The mRNA and total protein or phosphorylation level of PKA and downstream proteins were tested in the presence or absence of PKA inhibitor H89 or propofol, using RT-PCR, QPCR and western blotting. The phosphorylation level of PKA was examined thoroughly using immunofluorescence and PKA activity non-radioactive detection kit. Propofol induced a dose-dependent negative contractile response on the rat heart. The inhibitory effect of high concentration propofol (50μM) with 45% decease of control could be partly reversed by the PKA inhibitor H89 (10μM) and the depressant effect of propofol decreased from 45% to 10%. PKA kinase activity was inhibited by propofol in a dose-dependent manner. Propofol also induced a decrease in phosphorylation of PKA, which was also inhibited by H89, but did not alter the production of cAMP and the mRNA levels of PKA. The downstream proteins of PKA, PLN and RyR2 were phosphorylated to a lesser extent with propofol or H89 than control. These results demonstrated that propofol induced a negative myocardial contractile response partly by mediating the PKA phosphorylation pathway. PMID:27495954

  12. Effect of Serum from Chickens Treated with Clenbuterol on Myosin Accumulation, Beta-Adrenergic Receptor Population, and Cyclic AMP Synthesis in Embryonic Chicken Skeletal Muscle Cell Cultures

    NASA Technical Reports Server (NTRS)

    Young, Ronald B.; Bridge, Kristin Y.; Wuethrich, Andrew J.; Hancock, Deana L.

    2002-01-01

    Broiler chickens at 35 d of age were fed 1 ppm clenbuterol for 14 d. This level of dietary clenbuterol led to 5-7% increases in the weights of leg and breast muscle tissue. At the end of the 14-d period, serum was prepared from both control and clenbuterol-treated chickens, and was then employed as a component of cell culture media at a final concentration of 20% (v/v). Muscle cell cultures were prepared from both the leg and the breast muscle groups of 12-d chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 uM clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 d, beginning on the seventh d in culture. Neither the percent fusion nor the number of nuclei in myotubes was significantly affected by any of the treatments. The quantity of myosin heavy chains (MHCs) was not increased by serum from clenbuterol-treated chickens in either breast or leg muscle cultures; however, the MHC quantity was 50-150% higher in cultures grown in control chicken serum to which 10 and 50 nM clenbuterol had also been added. The B-adrenergic receptor (betaAR) population was 4000-7000 betaARs per cell in cultures grown in chicken serum with leg muscle cultures having approximately 25-30% more receptors than breast muscle Culture. Receptor population was not significantly affected by the presence of clenbuterol or by the presence of serum from clenbuterol-treated chickens. In contrast, the betaAR Population in leg and breast muscle cultures grown in the presence of 10% horse serum was 16,000-18,000 betaARs per cell. Basal concentration of cyclic adenosine 3':5'monophosphate (cAMP) was not significantly affected by the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 uM isoproterenol, limited increases of 12-20% in cAMP Concentration above the. basal levels were observed. However, when cultures grown in the presence of horse serum were

  13. Volume-activated Na/H exchange activity in fetal and adult pig red cells: inhibition by cyclic AMP.

    PubMed

    Sergeant, S; Sohn, D H; Kim, H D

    1989-08-01

    Hyposmotic swelling of pig red cells leads to a selective increase in K permeability, whereas hyperosmotic cell shrinkage augments the Na permeability. In this regard, the ouabain-resistant (OR) Na flux of red cells of newborn and adult pigs is characterized in detail. A reduction in cell volume by approximately 18% leads to an increase in the OR Na efflux of fetal and adult cells by 15- and fourfold, respectively. The OR Na influx in both cell types is equally influenced by cell shrinkage. Depletion of cellular K does not influence the volume-activated OR Na efflux. Nor does OR Na influx require external K. Both OR Na efflux and influx activated by shrinkage are inhibited by the diuretics furosemide and amiloride. The rank order of decreasing anion sensitivity for diuretic-sensitive Na efflux was acetate greater than chloride greater than gluconate greater than nitrate. Cell shrinkage induced by the addition of hypertonic salts results in an acidification of the unbuffered and CO2-free media, provided that both Na and DIDS are present. The acidification process can be reversed by either of the diuretic agents. These findings suggest that the shrinkage-activated OR Na flux is primarily mediated by a Na/H exchanger rather than by a Na/K/Cl cotransporter. Once loaded with either cAMP or cGMP, cell swelling can no longer activate the Na/H exchanger. The Na/H exchanger activity is detectable in the fetal cells of normal volume but quiescent in adult cells, indicating that the exchanger undergoes a developmental change during the transition from the fetal to adult stage. PMID:2552123

  14. 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.

  15. 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

  16. One-electron reduction of 8-bromo-2-aminoadenosine in the aqueous phase: radiation chemical and DFT studies of the mechanism.

    PubMed

    Kaloudis, Panagiotis; D'Angelantonio, Mila; Guerra, Maurizio; Gimisis, Thanasis; Mulazzani, Quinto G; Chatgilialoglu, Chryssostomos

    2008-04-24

    Two tautomeric forms of one-electron oxidized 2-aminoadenosine (2AA) have been produced by reactions of hydrated electrons (e aq-) with 8-bromo-2-aminoadenosine (8-Br-2AA) at natural pH, whereas only one tautomer is formed by oxidation of 2AA. Tailored experiments by pulse radiolysis and time-dependent DFT (TD-B3LYP/6-311G**//B1B95/6-31+G**) calculations allowed the definition of the reaction mechanism in some detail. The electron adducts of 8-Br-2AA protonated at C8 eject Br- and produce the two short-lived tautomers (8 and 9). The first observable species decays by first-order kinetics to produce the second intermediate, which is also obtained by oxidation of 2AA by SO4*-. The rate of tautomerization (k taut = 4.5 x 104 s-1) is strongly accelerated by phosphate and is retarded in D2O (kinetic isotope effect 7). B1B95/6-31+G** calculations showed that the tautomerization is a water-assisted process. In acidic or basic solutions, the "instantaneous" formation of one-electron oxidized 2AA or its deprotonated forms has been produced by reactions of e aq- with 8-Br-2AA. gamma-Radiolysis of 8-Br-2AA in aqueous solutions followed by product studies led to the formation of 2AA as a single product.

  17. Cyclic AMP and AKAP-mediated targeting of protein kinase A regulates lactate dehydrogenase subunit A mRNA stability.

    PubMed

    Jungmann, Richard A; Kiryukhina, Olga

    2005-07-01

    Expression of the lactate dehydrogenase A subunit (ldh-A) gene is controlled through transcriptional as well as post-transcriptional mechanisms. Both mechanisms involve activation of protein kinase A (PKA) into its subunits and subsequent phosphorylation and activation of several key regulatory factors. In rat C6 glioma cells, post-transcriptional gene regulation occurs through PKA-mediated stabilization of LDH-A mRNA and subsequent increase of intracellular LDH-A mRNA levels. Previous studies have demonstrated a cAMP-stabilizing region (CSR) located in the LDH-A 3'-untranslated region which, in combination with several phosphorylated CSR-binding proteins (CSR-BP), regulates the PKA-mediated stabilization of LDH-A mRNA. However, the mechanistic details of interaction of CSR with proteins as they pertain to mRNA stabilization by PKA are so far largely unknown. In this study we tested the hypothesis that ribosomal protein extracts (RSW) from glioma cells contain PKA regulatory (RII) and catalytic (C) subunits that, in combination with a protein kinase A anchoring protein (AKAP 95) and CSR-BPs participate in forming CSR-protein complexes that are responsible for mRNA stability regulation. To demonstrate the importance of CSR-protein complex formation, the PKA subunits and AKAP 95 were removed from the RSW by immunoprecipitation, and the antigen-deleted RSW were subjected to CSR binding analysis using gel mobility shift and UV cross-linking. It was shown that AKAP 95 as well as RII formed a direct linkage with CSR during CSR-protein complex formation. In contrast, the catalytic subunit formed part of the CSR-protein complex but did not bind to CSR directly in a covalent linkage. To determine whether formation of CSR complexes that included C, RII, and AKAP 95 constituted a functional event and was necessary for mRNA stabilization, cell-free decay reactions were carried out with RSW extracts, and the kinetics of decay of LDH-A mRNA was determined. Depletion of PKA

  18. Cyclic AMP and AKAP-mediated targeting of protein kinase A regulates lactate dehydrogenase subunit A mRNA stability.

    PubMed

    Jungmann, Richard A; Kiryukhina, Olga

    2005-07-01

    Expression of the lactate dehydrogenase A subunit (ldh-A) gene is controlled through transcriptional as well as post-transcriptional mechanisms. Both mechanisms involve activation of protein kinase A (PKA) into its subunits and subsequent phosphorylation and activation of several key regulatory factors. In rat C6 glioma cells, post-transcriptional gene regulation occurs through PKA-mediated stabilization of LDH-A mRNA and subsequent increase of intracellular LDH-A mRNA levels. Previous studies have demonstrated a cAMP-stabilizing region (CSR) located in the LDH-A 3'-untranslated region which, in combination with several phosphorylated CSR-binding proteins (CSR-BP), regulates the PKA-mediated stabilization of LDH-A mRNA. However, the mechanistic details of interaction of CSR with proteins as they pertain to mRNA stabilization by PKA are so far largely unknown. In this study we tested the hypothesis that ribosomal protein extracts (RSW) from glioma cells contain PKA regulatory (RII) and catalytic (C) subunits that, in combination with a protein kinase A anchoring protein (AKAP 95) and CSR-BPs participate in forming CSR-protein complexes that are responsible for mRNA stability regulation. To demonstrate the importance of CSR-protein complex formation, the PKA subunits and AKAP 95 were removed from the RSW by immunoprecipitation, and the antigen-deleted RSW were subjected to CSR binding analysis using gel mobility shift and UV cross-linking. It was shown that AKAP 95 as well as RII formed a direct linkage with CSR during CSR-protein complex formation. In contrast, the catalytic subunit formed part of the CSR-protein complex but did not bind to CSR directly in a covalent linkage. To determine whether formation of CSR complexes that included C, RII, and AKAP 95 constituted a functional event and was necessary for mRNA stabilization, cell-free decay reactions were carried out with RSW extracts, and the kinetics of decay of LDH-A mRNA was determined. Depletion of PKA

  19. Nerve Growth Factor is Required for Induction of c-Fos Immunoreactivity by Serum, Depolarization, Cyclic AMP or Trauma in Cultured Rat Sympathetic Neurons.

    PubMed

    Buckmaster, A; Nobes, C D; Edwards, S N; Tolkovsky, A M

    1991-01-01

    Nerve growth factor (NGF) induces transient Fos-immunoreactivity (Fos-IR) independently of any other factor, both in newly isolated rat sympathetic neurons and in established cultures after NGF deprivation. The same proportion of neurons that express Fos-IR in response to NGF also survive. In addition to direct stimulation of Fos-IR expression, the presence or recent exposure to NGF is required to obtain Fos-IR expression by other stimuli. In newly isolated neurons no Fos-IR is detected in response to stimulation by serum alone and a response to depolarization or cyclic AMP is obtained only if neurons are stimulated within a short period after ganglion excision. In established cultures none of these stimuli, nor the trauma of cutting neurites or spiking cell bodies with a microinjection needle induce Fos-IR unless NGF is present or had been removed for <8 - 16 h. The lack of response is not due to a general decrease in the rate of protein or RNA synthesis. These findings show that in regenerating sympathetic neurons NGF induces c-Fos and suggest that NGF may activate a master trigger that is required for c-Fos expression to be induced by other stimuli.

  20. 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.

  1. The Small Molecule Triclabendazole Decreases the Intracellular Level of Cyclic AMP and Increases Resistance to Stress in Saccharomyces cerevisiae

    PubMed Central

    Lee, Yong Joo; Shi, Runhua; Witt, Stephan N.

    2013-01-01

    The Ras-adenylyl cyclase-protein kinase A nutrient-sensing pathway controls metabolism, proliferation and resistance to stress in Saccharomyces cerevisiae. The genetic disruption of this pathway increases resistance to a variety of stresses. We show here that the pharmacological inhibition of this pathway by the drug triclabendazole increases resistance to oxidants, heat stress and extends the chronological life. Evidence is presented that triclabendazole decreases the intracellular level of cyclic AMP by inhibiting adenylyl cyclase and triggers the parallel rapid translocation of the stress-resistance transcription factor Msn2 from the cytosol into the nucleus, as deduced from experiments employing a strain in which MSN2 is replaced with MSN2-GFP (GFP, green fluorescent protein). Msn2 and Msn4 are responsible for activating the transcription of numerous genes that encode proteins that protect cells from stress. The results are consistent with triclabendazole either inhibiting the association of Ras with adenylyl cyclase or directly inhibiting adenylyl cyclase, which in turn triggers Msn2/4 to enter the nucleus and activate stress-responsible element gene expression. PMID:23667708

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

    PubMed

    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

    2015-04-01

    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 non-ribosomal peptide synthetase, FmpE. Overexpression of the regulatory gene fmpR using the Tet(On) 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 HPLC-HRESI-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.

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

    PubMed

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

    2016-06-14

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

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

    PubMed

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

    1996-09-01

    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.

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

    PubMed

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

    2016-06-14

    The innate immune system is the first line of defense against Neisseria gonorrhoeae (GC). Exposure of cells to GC lipooligosaccharides induces a strong immune response, leading to type I interferon (IFN) production via TLR4/MD-2. In addition to living freely in the extracellular space, GC can invade the cytoplasm to evade detection and elimination. Double-stranded DNA introduced into the cytosol binds and activates the enzyme cyclic-GMP-AMP synthase (cGAS), which produces 2'3'-cGAMP and triggers STING/TBK-1/IRF3 activation, resulting in type I IFN expression. Here, we reveal a cytosolic response to GC DNA that also contributes to type I IFN induction. We demonstrate that complete IFN-β induction by live GC depends on both cGAS and TLR4. Type I IFN is detrimental to the host, and dysregulation of iron homeostasis genes may explain lower bacteria survival in cGAS(-/-) and TLR4(-/-) cells. Collectively, these observations reveal cooperation between TLRs and cGAS in immunity to GC infection. PMID:27264171

  6. 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.

  7. Rat hippocampal neurons express genes for both rod retinal and olfactory cyclic nucleotide-gated channels: novel targets for cAMP/cGMP function.

    PubMed Central

    Kingston, P A; Zufall, F; Barnstable, C J

    1996-01-01

    Cyclic nucleotide-gated (CNG) channels are Ca(2+)-permeable, nonspecific cation channels that can be activated through direct interaction with cAMP and/or cGMP. Recent electrophysiological evidence for these channels in cultured hippocampal neurons prompted us to investigate the expression of CNG channel genes in hippocampus. PCR amplification detected the expression of transcripts for subunit 1 of both the rod photoreceptor (RCNGC1) and the olfactory receptor cell (OCNGC1) subtype of CNG channel in adult rat hippocampus. In situ hybridization detected expression of both channel subtypes in most principal neurons, including pyramidal cells of the CA1 through CA3 regions and granule cells of the dentate gyrus. From the hybridization patterns, we conclude that the two genes are colocalized in individual neurons. Comparison of the patterns of expression of type 1 cGMP-dependent protein kinase and the CNG channels suggests that hippocampal neurons can respond to changes in cGMP levels with both rapid changes in CNG channel activity and slower changes induced by phosphorylation. Future models of hippocampal function should include CNG channels and their effects on both electrical responses and intracellular Ca2+ levels. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8816819

  8. Binding of Cyclic Di-AMP to the Staphylococcus aureus Sensor Kinase KdpD Occurs via the Universal Stress Protein Domain and Downregulates the Expression of the Kdp Potassium Transporter

    PubMed Central

    Moscoso, Joana A.; Schramke, Hannah; Tosi, Tommaso; Dehbi, Amina; Jung, Kirsten

    2015-01-01

    ABSTRACT Nucleotide signaling molecules are important intracellular messengers that regulate a wide range of biological functions. The human pathogen Staphylococcus aureus produces the signaling nucleotide cyclic di-AMP (c-di-AMP). This molecule is common among Gram-positive bacteria and in many organisms is essential for survival under standard laboratory growth conditions. In this study, we investigated the interaction of c-di-AMP with the S. aureus KdpD protein. The sensor kinase KdpD forms a two-component signaling system with the response regulator KdpE and regulates the expression of the kdpDE genes and the kdpFABC operon coding for the Kdp potassium transporter components. Here we show that the S. aureus KdpD protein binds c-di-AMP specifically and with an affinity in the micromolar range through its universal stress protein (USP) domain. This domain is located within the N-terminal cytoplasmic region of KdpD, and amino acids of a conserved SXS-X20-FTAXY motif are important for this binding. We further show that KdpD2, a second KdpD protein found in some S. aureus strains, also binds c-di-AMP, and our bioinformatics analysis indicates that a subclass of KdpD proteins in c-di-AMP-producing bacteria has evolved to bind this signaling nucleotide. Finally, we show that c-di-AMP binding to KdpD inhibits the upregulation of the kdpFABC operon under salt stress, thus indicating that c-di-AMP is a negative regulator of potassium uptake in S. aureus. IMPORTANCE Staphylococcus aureus is an important human pathogen and a major cause of food poisoning in Western countries. A common method for food preservation is the use of salt to drive dehydration. This study sheds light on the regulation of potassium uptake in Staphylococcus aureus, an important aspect of this bacterium's ability to tolerate high levels of salt. We show that the signaling nucleotide c-di-AMP binds to a regulatory component of the Kdp potassium uptake system and that this binding has an inhibitory

  9. Partial characterization of cyclic AMP-dependent protein kinases in guinea-pig lung employing the synthetic heptapeptide substrate, kemptide. In vitro sensitivity of the soluble enzyme to isoprenaline, forskolin, methacholine and leukotriene D4.

    PubMed

    Giembycz, M A; Diamond, J

    1990-04-15

    This paper describes the partial characterization of soluble cyclic AMP-dependent protein kinase (A-kinase) in guinea-pig lung using Kemptide, a synthetic serine-containing heptapeptide, and examines the sensitivity of this enzyme to drugs which are reported to increase and to decrease the intracellular concentration of cyclic AMP. Differential centrifugation of lung homogenates revealed that 78% of A-kinase was present in the 31,000 gmax x 15 min supernatant fraction. Both basal and cyclic AMP-stimulated phosphotransferase activity of this 'soluble' enzyme were abolished by the heat-stable inhibitor of A-kinase. Soluble A-kinase was Mg2(+)-dependent (apparent Km and and Kact 8.6 and 2.6 mM, respectively) and was stimulated nine-fold by saturating concentrations of both cyclic AMP (Kact: 131 nM) and cyclic GMP (Kact: 28.7 microM) at a protein (enzyme) concentration of 1.3 micrograms. Kinetic analysis of the effect of Kemptide and ATP revealed linear, Hanes plots with Michealis constants of ca. 12 and 13 microM, respectively. Chromatography of the soluble enzyme over DEAE-cellulose resolved three peaks of catalytic activity when fractions were assayed in the presence of cyclic AMP (10 microM): (i) free catalytic subunits (5%), (ii) Type I isoenzyme (5%) and (iii) Type II isoenzyme (90%). The A-kinase activity ratio was markedly increased in lung pre-treated with the smooth muscle relaxants isoprenaline and forskolin. This biochemical effect was both time- and concentration-dependent and was temporally associated with the ability of these drugs to reduce lung parenchymal tone. In contrast, the contractile agonists, methacholine (Mch) and leukotriene (LT) D4 exerted opposite effects on A-kinase activity. Thus, Mch significantly reduced cyclic AMP levels and lowered basal A-kinase activity whilst the converse was true for LTD4. For both drugs this biochemical effect accompanied contraction of the lung. Pre-treatment of lung tissue with flurbiprofen, an irreversible

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

    PubMed

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

    2013-12-01

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

  11. Evidence for a cyclic GMP-independent mechanism in the anti-platelet action of S-nitrosoglutathione

    PubMed Central

    Gordge, M P; Hothersall, J S; Noronha-Dutra, A A

    1998-01-01

    We have measured the ability of a range of NO donor compounds to stimulate cyclic GMP accumulation and inhibit collagen-induced aggregation of human washed platelets. In addition, the rate of spontaneous release of NO from each donor has been measured spectrophotometrically by the oxidation of oxyhaemoglobin to methaemoglobin. The NO donors used were five s-nitrosothiol compounds: S-nitrosoglutathione (GSNO), S-nitrosocysteine (cysNO), S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitroso-N-acetyl-cysteine (SNAC), S-nitrosohomocysteine (homocysNO), and two non-nitrosothiol compounds: diethylamine NONOate (DEANO) and sodium nitroprusside (SNP).Using 10 μM of each donor compound, mean±s.e.mean rate of NO release ranged from 0.04±0.001 nmol min−1 (for SNP) to 3.15±0.29 nmol min−1 (for cysNO); cyclic GMP accumulation ranged from 0.43±0.05 pmol per 108 platelets (for SNP) to 2.67±0.31 pmol per 108 platelets (for cysNO), and inhibition of platelet aggregation ranged from 40±6.4% (for SNP) to 90±3.8% (for SNAC).There was a significant positive correlation between the rate of NO release and the ability of the different NO donors to stimulate intra-platelet cyclic GMP accumulation (r=0.83; P=0.02). However, no significant correlation was observed between the rate of NO release and the inhibition of platelet aggregation by the different NO donors (r=−0.17), nor was there a significant correlation between cyclic GMP accumulation and inhibition of aggregation by the different NO donor compounds (r=0.34).Comparison of the dose-response curves obtained with GSNO, DEANO and 8-bromo cyclic GMP showed DEANO to be the most potent stimulator of intraplatelet cyclic GMP accumulation (P<0.001 vs both GSNO and 8-bromo cyclic GMP), but GSNO to be the most potent inhibitor of platelet aggregation (P<0.01 vs DEANO, and P<0.001 vs 8-bromo cyclic GMP).The rate of NO release from GSNO, and its ability both to stimulate intra-platelet cyclic GMP accumulation and to

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

    PubMed

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

    2015-06-26

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

  13. 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

  14. Species differences in the effects of prostaglandins on inositol trisphosphate accumulation, phosphatidic acid formation, myosin light chain phosphorylation and contraction in iris sphincter of the mammalian eye: interaction with the cyclic AMP system.

    PubMed

    Yousufzai, S Y; Chen, A L; Abdel-Latif, A A

    1988-12-01

    Comparative studies on the effects of prostaglandins (PGs) on 1,2-diacylglycerol, measured as phosphatidic acid (PA), and inositol trisphosphate (IP3) production, cyclic AMP (cAMP) formation, myosin light chain (MLC) phosphorylation and contraction in the iris sphincter smooth muscle of rabbit, bovine and other mammalian species were undertaken and functional and biochemical relationships between the IP3-Ca++ and cAMP second messenger systems were demonstrated. The findings obtained from these studies can be summarized as follows: 1) all PGs investigated, including PGE2, PGF2 alpha, PGF2 alpha-ester, PGE1 and PGA2 increased IP3 accumulation and PA formation, and the extent of stimulation was dependent on the animal species. Thus, PGF2 alpha-ester (1 microM), the most potent of the PGs, increased IP3 accumulation in rabbit and bovine sphincters by 33 and 58%, respectively, and increased PA formation by 67 and 56%, respectively. The PG increased IP3 accumulation in both rabbit and bovine sphincters very rapidly (T1/2 values about 26 sec) and in a dose-dependent manner. 2) The PG had no effect on MLC phosphorylation in the rabbit sphincter, but it increased that of the bovine by 36%. 3) The PG increased cAMP formation by 75% in the rabbit sphincter but it had no effect on that of the bovine. 4) The PG induced a maximal contractile response in the bovine sphincter but it had no effect on that of the rabbit. 5) In the bovine, PGA2 induced IP3 accumulation and contraction, without an effect on cAMP formation; however, in the rabbit, cat and dog it increased cAMP formation and had no effect on IP3 accumulation and contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

  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. 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.

  17. Cyclooxygenase-2 expression in lipopolysaccharide-stimulated human monocytes is modulated by cyclic AMP, prostaglandin E(2), and nonsteroidal anti-inflammatory drugs.

    PubMed

    Hinz, B; Brune, K; Pahl, A

    2000-11-30

    Using human blood monocytes (for determination of cyclooxygenase-2 (COX-2) mRNA by RT-PCR) and human whole blood (for prostanoid determination), the present study investigates the influence of the second messenger cAMP on lipopolysaccharide (LPS)-induced COX-2 expression with particular emphasis on the role of prostaglandin E(2) (PGE(2)) in this process. Elevation of intracellular cAMP with a cell-permeable cAMP analogue (dibutyryl cAMP), an adenylyl cyclase activator (cholera toxin), or a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine) substantially enhanced LPS-induced PGE(2) formation and COX-2 mRNA expression, but did not modify COX-2 enzyme activity. Moreover, up-regulation of LPS-induced COX-2 expression was caused by PGE(2), butaprost (selective agonist of the adenylyl cyclase-coupled EP(2) receptor) and 11-deoxy PGE(1) (EP(2)/EP(4) agonist), whereas sulprostone (EP(3)/EP(1) agonist) left COX-2 expression unaltered. Abrogation of LPS-induced PGE(2) synthesis with the selective COX-2 inhibitor NS-398 caused a decrease in COX-2 mRNA levels that was restored by exogenous PGE(2) and mimicked by S(+)-flurbiprofen and ketoprofen. Overall, these results indicate a modulatory role of cAMP in the regulation of COX-2 expression. PGE(2), a cAMP-elevating final product of the COX-2 pathway, may autoregulate COX-2 expression in human monocytes via a positive feedback mechanism.

  18. H2O2-induced filamin redistribution in endothelial cells is modulated by the cyclic AMP-dependent protein kinase pathway.

    PubMed

    Hastie, L E; Patton, W F; Hechtman, H B; Shepro, D

    1997-09-01

    Hypoxia/reoxygenation injury in vitro causes endothelial cell cytoskeletal rearrangement that is related to increased monolayer permeability. Nonmuscle filamin (ABP-280) promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. Human umbilical vein endothelial cell monolayers are exposed to H2O2 (100 microM) for 1-60 min, with or without modulators of cAMP-dependent second-messenger pathways, and evaluated for changes in filamin distribution, cAMP levels, and the formation of gaps at interendothelial junctions. Filamin translocates from the membrane-cytoskeletal interface to the cytosol within 1 min of exposure to H2O2. This is associated with a decrease in endothelial cell cAMP levels from 83 pmoles/mg protein to 15 pmoles/mg protein. Intercellular gaps form 15 min after H2O2 treatment and progressively increase in number and diameter through 60 min. Both filamin redistribution and actin redistribution are associated with decreased phosphorylation of filamin and are prevented by activation of the cAMP-dependent protein kinase pathway. A synthetic peptide corresponding to filamin's C-terminal, cAMP-dependent, protein kinase phosphorylation site effectively induces filamin translocation and intercellular gap formation, which suggests that decreased phosphorylation of filamin at this site causes filamin redistribution and destabilization of junctions. These data indicate that H2O2-induced filamin redistribution and interendothelial cell gap formation result from inhibition of the cAMP-dependent protein kinase pathway.

  19. Isolation and characterization of cAMP-resistant mutants of the H-4 rat hepatoma cells

    SciTech Connect

    Liu, A.Y.; Lin, Z.

    1987-05-01

    H-4 rat hepatoma cells were mutagenized with ethyl methane-sulfonate and the frequency of emergence of cAMP resistant mutant cells were evaluated by cloning the EMS-treated cells in a semi-solid agar medium that contained either 1-3 mM 8-bromo-cAMP plus 1 mM 3-isobutyl-1-methyl xanthine or 5 ..mu..g/ml cholera toxin plus 1 mM IBMX. cAMP resistant mutants emerged at a frequency of 8 x 10/sup -5/. 15 colonies were isolated, recloned, grown in mass culture, and cell extracts were prepared. Analysis of cAMP-dependent protein kinase demonstrated that: (1) the type II enzyme is the only cAMP-dependent protein kinase detected in extracts of the hepatoma cells; (2) of the 15 cAMP resistant clonal cell lines examined, only one (H/sub 4/M/sub 18/) was found to be devoid of cAMP-dependent protein kinase activity. In another cell line (H/sub 4/M/sub 10/) the activity was 30% of that of the parental H-4 cells; (3) there was an increase (130-300%) in cAMP-dependent protein kinase activity in 13/15 of the mutant cell lines over that of the parental H-4 cells. Analysis of cAMP-phosphodiesterase demonstrated significant increases (150-370%) in the enzyme activity in extracts of the mutants over that of the H-4 parental line. Their results suggest that while a deficiency in cAMP-dependent protein kinase may confer resistance to the hepatoma cells against the cytostatic effects of 8-bromo-cAMP and cholera toxin, other events such as overexpression of phosphodiesterase may contribute to this phenotype.

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

    PubMed

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

    1989-01-01

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

  1. Follicle-stimulating hormone and cyclic AMP induce transcription from the human urokinase promoter in primary cultures of mouse Sertoli cells.

    PubMed

    Rossi, P; Grimaldi, P; Blasi, F; Geremia, R; Verde, P

    1990-06-01

    The hormonal regulation of the human urokinase type plasminogen activator (uPA) gene has been studied by introducing into mouse and rat Sertoli cell primary cultures a recombinant plasmid, in which the transcription regulatory elements of the cloned human uPA gene drive the expression of the bacterial chloramphenicol-acetyl-transferase gene. It was found to be expressed and regulated by FSH and (Bu)2cAMP in the mouse cells only, in agreement with data on the expression of the endogenous gene in rat and mouse gonads. The stimulation of transcription by FSH was evident in cultures from 13-day-old but not from 18-day-old mice, even though (Bu)2cAMP induction could be observed at both ages. Phorbol-myristate acetate was found to activate the human uPA promoter in Sertoli cell cultures from mice of both ages, even though the effect was less evident in cultures of 18-day-old animals. Deletion analysis of the human uPA 5'-flanking region showed that the distal enhancer element is not needed for (Bu)2cAMP induction, and that at least two promoter regions are involved in (Bu)2cAMP induced transcription. One of these cAMP responsive regions lies between nucleotides -72 and -29 from the CAP site. The sequence of this region would suggest the binding of transcription factor AP-2, a cell-specific mediator of both cAMP and phorbol esters action on gene expression. However, these sequences do not mediate phorbol ester activation of human uPA promoter in mouse Sertoli cells.

  2. Pleiotrophin mediates the neurotrophic effect of cyclic AMP on dopaminergic neurons: analysis of suppression-subtracted cDNA libraries and confirmation in vitro.

    PubMed

    Mourlevat, Sophie; Debeir, Thomas; Ferrario, Juan E; Delbe, Jean; Caruelle, Daniele; Lejeune, Olivier; Depienne, Christel; Courty, José; Raisman-Vozari, Rita; Ruberg, Merle

    2005-07-01

    To better understand the particular vulnerability of mesencephalic dopaminergic neurons to toxins or gene mutations causing parkinsonism, we have taken advantage of a primary cell culture system in which these neurons die selectively. Antimitotic agents, such as cytosine arabinoside or cAMP, prevent the death of the neurons by arresting astrocyte proliferation. To identify factors implicated in either the death of the dopaminergic neurons or in the neuroprotective effect of cAMP, we constructed cDNA libraries enriched by subtractive hybridization and suppressive PCR in transcripts that are preferentially expressed in either control or cAMP-treated cultures. Differentially expressed transcripts were identified by hybridization of the enriched cDNAs with a commercially available cDNA expression array. The proteoglycan receptors syndecan-3 and the receptor protein tyrosine phosphatase zeta/beta were found among the transcripts preferentially expressed under control conditions, and their ligand, the cytokine pleiotrophin, was highly represented in the cDNA libraries for both conditions. Since pleiotrophin is expressed during embryonic and perinatal neural development and following lesions in the adult brain, we investigated its role in our cell culture model. Pleiotrophin was not responsible for the death of dopaminergic neurons under control conditions, or for their survival in cAMP-treated cultures. It was, however, implicated in the initial and cAMP-dependent enhancement of the differentiation of the dopaminergic neurons in our cultures. In addition, our experiments have provided evidence for a cAMP-dependent regulatory pathway leading to protease activation, and the identification of pleiotrophin as a target of this pathway.

  3. 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

  4. 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

  5. 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...

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

    NASA Astrophysics Data System (ADS)

    Habara, Yoshiaki

    1989-06-01

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

  7. 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...

  8. 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

  9. 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…

  10. 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

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

    SciTech Connect

    Lund, Kaleb C. Wallace, Kendall B.

    2008-01-01

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

  12. 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

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

    PubMed

    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 Ca(2+) concentration ([Ca(2+)]c); glucose evoked an immediate elevation of [Ca(2+)]c, which was followed by a decrease in [Ca(2+)]c, and after a certain lag period it induced large oscillatory elevations of [Ca(2+)]c. Initial rapid peak and subsequent reduction of [Ca(2+)]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 Ca(2+), 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 [Ca(2+)]c and [cAMP]c by activating the cell-surface glucose-sensing receptor. Hence, glucose generates rapid intracellular signals by activating the cell-surface receptor.

  14. Prostaglandin E2 induces vascular endothelial growth factor secretion in prostate cancer cells through EP2 receptor-mediated cAMP pathway.

    PubMed

    Wang, Xingya; Klein, Russell D

    2007-11-01

    Prostaglandin E2 (PGE2) has been shown to induce expression of vascular endothelial growth factor (VEGF) and other signaling molecules in several cancers. PGE2 elicits its functions though four G-protein coupled membrane receptors (EP1-4). In this study, we investigated the role of EP receptors in PGE2-induced molecular events in prostate cancer cells. qRT-PCR analysis revealed that PC-3 cells express a substantially higher level of EP2 and moderately higher EP4 than DU145 and LNCaP cells. LNCaP cells had virtually no detectable EP2 mRNA. EP1 and EP3 mRNAs were not detected in these cells. Treatment of prostate cancer cells with PGE2 (1 nM-10 microM) increased both VEGF secretion and cyclic adenosine monophosphate (cAMP) production. Levels of induction in PC-3 cells were greater than in DU145 and LNCaP cells. The selective EP2 agonist CAY10399 also significantly increased VEGF secretion and cAMP production in PC-3 cells, but not in DU145 and LNCaP cells. Moreover, PGE2 and CAY10399 increased mitogen activated protein kinase/extracellular signal regulated kinase (MAPK/Erk) and Akt phosphorylation in PC-3 and DU145 cells, but not in LNCaP cells. However, neither the MAPK/Erk inhibitor U0126 nor the PI3K/Akt inhibitor LY294002 abolished PGE2-induced VEGF secretion in PC-3 cells. We further demonstrated that the adenylate cyclase activator forskolin and the cAMP anologue 8-bromo-cAMP mimicked the effects of PGE2 on VEGF secretion in PC-3 cells. Meanwhile, the adenylate cyclase inhibitor 2'5'-dideoxyadenosine, at concentrations that inhibited PGE2-induced cAMP, significantly blocked PGE2-induced VEGF secretion in PC-3 cells. We conclude that PGE2-induced VEGF secretion in prostate cancer cells is mediated through EP2-, and possibly EP4-, dependent cAMP signaling pathways. PMID:17427962

  15. Stimulation of oxidative phosphorylation by calcium in cardiac mitochondria is not influenced by cAMP and PKA activity.

    PubMed

    Covian, Raul; French, Stephanie; Kusnetz, Heather; Balaban, Robert S

    2014-12-01

    Cardiac oxidative ATP generation is finely tuned to match several-fold increases in energy demand. Calcium has been proposed to play a role in the activation of ATP production via PKA phosphorylation in response to intramitochondrial cAMP generation. We evaluated the effect of cAMP, its membrane permeable analogs (dibutyryl-cAMP, 8-bromo-cAMP), and the PKA inhibitor H89 on respiration of isolated pig heart mitochondria. cAMP analogs did not stimulate State 3 respiration of Ca2 +-depleted mitochondria (82.2 ± 3.6% of control), in contrast to the 2-fold activation induced by 0.95 μM free Ca2 +, which was unaffected by H89. Using fluorescence and integrating sphere spectroscopy, we determined that Ca2 + increased the reduction of NADH (8%), and of cytochromes bH (3%), c1 (3%), c (4%), and a (2%), together with a doubling of conductances for Complex I + III and Complex IV. None of these changes were induced by cAMP analogs nor abolished by H89. In Ca2 +-undepleted mitochondria, we observed only slight changes in State 3 respiration rates upon addition of 50 μM cAMP (85 ± 9.9%), dibutyryl-cAMP (80.1 ± 5.2%), 8-bromo-cAMP (88.6 ± 3.3%), or 1 μM H89 (89.7 ± 19.9%) with respect to controls. Similar results were obtained when measuring respiration in heart homogenates. Addition of exogenous PKA with dibutyryl-cAMP or the constitutively active catalytic subunit of PKA to isolated mitochondria decreased State 3 respiration by only 5–15%. These functional studies suggest that alterations in mitochondrial cAMP and PKA activity do not contribute significantly to the acute Ca2 + stimulation of oxidative phosphorylation

  16. Phospholipid turnover and ultrastructural correlates during spontaneous germinal vesicle breakdown of the bovine oocyte: Effects of a cyclic AMP phosphodiesterase inhibitor

    SciTech Connect

    Homa, S.T.; Webster, S.D.; Russell, R.K. )

    1991-08-01

    The turnover of (32P)orthophosphate in bovine oocyte phospholipids was studied during the early stages of spontaneous meiotic maturation, and during inhibition of this process by the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). Radioactive lipids were separated by TLC and the meiotic stage was determined cytogenetically. Ultrastructure of the nuclear membrane was examined using transmission EM. During the commitment period to meiotic resumption, which precedes germinal vesicle breakdown (GVBD), small localized convolutions appeared in the intact nuclear membrane. This was accompanied by a decrease in (32P)phosphatidic acid (PA) and an increase in (32P)-phosphatidylcholine (PC). This was followed by extensive convolutions, and subsequent dissociation, of the nuclear membrane, concomitant with a tremendous surge in (32P)PC and (32P)phosphatidylethanolamine (PE). The cAMP-mediated maintenance of meiotic arrest involved retention of entire nuclear envelope integrity and total inhibition of the surge in (32P)PC and (32P)PE which accompanied GVBD. The increase in (32P)phosphatidylinositol (PI) associated with all stages of early meiotic resumption was unaffected by IBMX. Microinjection of heparin inhibited GVBD, and injection of inositol 1,4,5-trisphosphate (IP3) overrode IBMX-maintained meiotic arrest in almost 40% of the oocytes. The results suggest that there may be several functions for phospholipid turnover in the regulation of spontaneous meiotic resumption in the bovine oocyte. The first precedes the commitment period, and involves IP3 generation to serve as the primary signal for meiotic resumption. The second occurs concomitant with the commitment period, is unaffected by the level of intracellular cAMP, and is associated with the general turnover of phospholipid.

  17. {alpha}MSH and Cyclic AMP elevating agents control melanosome pH through a protein kinase A-independent mechanism.

    PubMed

    Cheli, Yann; Luciani, Flavie; Khaled, Mehdi; Beuret, Laurent; Bille, Karine; Gounon, Pierre; Ortonne, Jean-Paul; Bertolotto, Corine; Ballotti, Robert

    2009-07-10

    Melanins are synthesized in melanocytes within specialized organelles called melanosomes. Numerous studies have shown that the pH of melanosome plays a key role in the regulation of melanin synthesis. However, until now, acute regulation of melanosome pH by a physiological stimulus has never been demonstrated. In the present study, we show that the activation of the cAMP pathway by alphaMSH or forskolin leads to an alkalinization of melanosomes and a concomitant regulation of vacuolar ATPases and ion transporters of the solute carrier family. The solute carrier family members include SLC45A2, which is mutated in oculocutaneous albinism type IV, SLC24A4 and SLC24A5, proteins implicated in the control of eye, hair, and skin pigmentation, and the P protein, encoded by the oculocutaneous albinism type II locus. Interestingly, H89, a pharmacological inhibitor of protein kinase A (PKA), prevents the cAMP-induced pigmentation and induces acidification of melanosomes. The drastic depigmenting effect of H89 is not due to an inhibition of tyrosinase expression. Indeed, H89 blocks the induction of melanogenesis induced by LY294002, a potent inhibitor of the PI 3-kinase pathway, without any effect on tyrosinase expression. Furthermore, PKA is not involved in the inhibition of pigmentation promoted by H89 because LY294002 induces pigmentation independently of PKA. Also, other PKA inhibitors do not affect pigmentation. Taken together, our results strengthen the support for a key role of melanosome pH in the regulation of melanin synthesis and, for the first time, demonstrate that melanosome pH is regulated by cAMP and alphaMSH. Notably, these are both mediators of the response to solar UV radiation, the main physiological stimulus of skin pigmentation.

  18. 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

  19. Histamine H(2) -like receptors in chick cerebral cortex: effects on cyclic AMP synthesis and characterization by [(3) H]tiotidine binding.

    PubMed

    Zawilska, Jolanta B; Woldan-Tambor, Agata; Nowak, Jerzy Z

    2002-06-01

    In this study, histamine (HA) receptors in chick cerebral cortex were characterized using two approaches: (1) analysis of the effects of HA-ergic drugs on the cAMP-generating system, and (2) radioreceptor binding of [(3) H]tiotidine, a selective H(2) antagonist. HA was a weak activator of adenylyl cyclase in a crude membrane preparation of chick cerebrum. On the other hand, HA (0.1-1000 microm) potently and concentration dependently stimulated cAMP production in [(3) H]adenine pre-labelled slices of chick cerebral cortex, displaying an EC(50) value (concentration that produces 50% of maximum response) of 2.65 microm. The effect of HA was mimicked by agonists of HA receptors with the following rank order of potency: HA >or= 4-methylHA (H(2)) >or= N alpha,N alpha-dimethylHA (H(3) > H(2) = H(1)) > 2-methylHA (H(1)) > 2-thiazolylethylamine (H(1)) >or= R alpha-methylHA (H(3)) > amthamine, dimaprit (H(2)), immepip (H(3), H(4)). The HA-evoked increase in cAMP production in chick cerebral cortex was antagonized by selective H(2) receptor blockers (aminopotentidine >or= tiotidine > ranitidine > zolantidine), and not significantly affected by mepyramine and thioperamide, selective H(1) and H(3) /H(4) receptor blockers, respectively. A detailed analysis of the antagonistic action of aminopotentidine (vs. HA) revealed a non-competitive mode of action. The binding of [(3) H]tiotidine to chick cortical membranes was rapid, stable and reversible. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of receptor binding site with high affinity [equilibrium dissociation constant (K (d)) = 4.42 nm] and high capacity [maximum number of binding sites (B (max) ) = 362 fmol/mg protein]. The relative rank order of HA-ergic drugs to inhibit [(3) H]tiotidine binding to chick cerebrum was: antagonists - tiotidine > aminopotentidine = ranitidine >or= zolantadine > thioperamide - triprolidine; agonists - HA >or= 4-methylHA > 2-methylHA >or=R alpha

  20. Single Nucleotide Polymorphisms That Cause Structural Changes in the Cyclic AMP Receptor Protein Transcriptional Regulator of the Tuberculosis Vaccine Strain Mycobacterium bovis BCG Alter Global Gene Expression without Attenuating Growth▿

    PubMed Central

    Hunt, Debbie M.; Saldanha, José W.; Brennan, John F.; Benjamin, Pearline; Strom, Molly; Cole, Jeffrey A.; Spreadbury, Claire L.; Buxton, Roger S.

    2008-01-01

    Single nucleotide polymorphisms (SNPs) are present in the global transcriptional regulator cyclic AMP (cAMP) receptor protein (CRP) of the attenuated vaccine strain Mycobacterium bovis, bacillus Calmette-Guérin (BCG). We have found that these SNPs resulted in small but significant changes in the expression of a number of genes in M. tuberculosis when a deletion of the Rv3676 CRP was complemented by the BCG allele, compared to complementation by the M. tuberculosis allele. We can explain these changes in gene expression by modeling the structure of the mycobacterial protein on the known structure of CRP from Escherichia coli. Thus, the SNP change in the DNA-binding domain, Lys178, is predicted to form a hydrogen bond with the phosphate backbone of the DNA, as does the equivalent residue in E. coli, whereas Glu178 in M. tuberculosis/M. bovis does not, thus explaining the stronger binding reported for CRP of BCG to CRP-binding sites in mycobacterial DNA. In contrast, the SNP change in the nucleotide binding domain (Leu47Pro) is predicted to result in the loss of one hydrogen bond, which is accommodated by the structure, and would not therefore be expected to cause any change in function relating to cAMP binding. The BCG allele fully complemented the growth defect caused by the deletion of the Rv3676 protein in M. tuberculosis, both in vitro and in macrophage and mouse infections, suggesting that these SNPs do not play any role in the attenuation of BCG. However, they may have allowed BCG to grow better under the in vitro-selective conditions used in its derivation from the M. bovis wild type. PMID:18332206

  1. A functional cyclic AMP response element plays a crucial role in neuroendocrine cell type-specific expression of the secretory granule protein chromogranin A.

    PubMed Central

    Wu, H; Mahata, S K; Mahata, M; Webster, N J; Parmer, R J; O'Connor, D T

    1995-01-01

    Chromogranin A, a soluble acidic protein, is a ubiquitous component of secretory vesicles throughout the neuroendocrine system. We reported previously the cloning and initial characterization of the mouse chromogranin A gene promoter, which showed that the promoter contains both positive and negative domains and that a proximal promoter spanning nucleotides -147 to +42 bp relative to the transcriptional start site is sufficient for neuroendocrine cell type-specific expression. The current study was undertaken to identify the particular elements within this proximal promoter that control tissue-specific expression. We found that deletion or point mutations in the potential cAMP response element (CRE) site at -68 bp virtually abolished promoter activity specifically in neuroendocrine (PC12 chromaffin or AtT20 corticotrope) cells, with little effect on activity in control (NIH3T3 fibroblast) cells; thus, the CRE box is necessary for neuroendocrine cell type-specific activity of the chromogranin A promoter. Furthermore, the effect of the CRE site is enhanced in the context of intact (wild-type) promoter sequences between -147 and -100 bp. DNase I footprint analysis showed that these regions (including the CRE box) bind nuclear proteins present in both neuroendocrine (AtT20) and control (NIH3T3) cells. In AtT20 cells, electrophoretic mobility shift assays and factor-specific antibody supershifts showed that an oligonucleotide containing the chromogranin A CRE site formed a single, homogeneous protein-DNA complex containing the CRE-binding protein CREB. However, in control NIH3T3 cells we found evidence for an additional immunologically unrelated protein in this complex. A single copy of this oligonucleotide was able to confer neuroendocrine-specific expression to a heterologous (thymidine kinase) promoter, albeit with less fold selectivity than the full proximal chromogranin A promoter. Hence, the CRE site was partially sufficient to explain the neuroendocrine cell type

  2. 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

  3. Rat neuropeptide Y precursor gene expression. mRNA structure, tissue distribution, and regulation by glucocorticoids, cyclic AMP, and phorbol ester.

    PubMed

    Higuchi, H; Yang, H Y; Sabol, S L

    1988-05-01

    Rat brain neuropeptide Y precursor (prepro-NPY) cDNA clones were isolated and sequenced in order to study regulation of the prepro-NPY gene. Rat prepro-NPY (98 amino acid residues) contains a 36-residue NPY sequence, followed by a proteolysis/amidation site Gly-Lys-Arg, followed by a 30-residue COOH-terminal sequence. The strong evolutionary conservation of rat and human sequences of NPY (100%) and COOH-terminal peptide (93%) suggests that both peptides have important biological functions. In the rat central nervous system, prepro-NPY mRNA (800 bases) is most abundant in the striatum and cortex and moderately abundant in the hippocampus, hypothalamus, and spinal cord. The rat adrenal, spleen, heart, and lung have significant levels of prepro-NPY mRNA. Regulation of the prepro-NPY mRNA abundance was studied in several rodent neural cell lines. PC12 rat pheochromocytoma and N18TG-2 mouse neuroblastoma cells possess low basal levels of prepro-NPY mRNA, while NG108-15 hybrid cells possess high levels. Treatment of PC12 cells with a glucocorticoid such as dexamethasone or elevation of cAMP by forskolin increased the prepro-NPY mRNA level 2-3-fold or 3-10-fold, respectively. In N18TG-2 cells dexamethasone and forskolin synergistically increased prepro-NPY mRNA 7-fold. Treatment of PC12 cells with the protein kinase C activator phorbol 12-myristate 13-acetate alone elevated prepro-NPY mRNA marginally, but the phorbol ester plus forskolin elicited 20-70-fold increases, which were further enhanced to over 200-fold by dexamethasone and the calcium ionophore A23187. These results indicate that NPY gene expression can be positively regulated by synergistic actions of glucocorticoids, cAMP elevation, and protein kinase C activation.

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

    PubMed Central

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

    2014-01-01

    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

  5. GPR26-deficient mice display increased anxiety- and depression-like behaviors accompanied by reduced phosphorylated cyclic AMP responsive element-binding protein level in central amygdala.

    PubMed

    Zhang, L-L; Wang, J-J; Liu, Y; Lu, X-B; Kuang, Y; Wan, Y-H; Chen, Y; Yan, H-M; Fei, J; Wang, Z-G

    2011-11-24

    Anxiety disorders are among the most common and well studied psychiatric disorders in humans. A number of animal models have been established to study the mechanisms of anxiety and to test putative anxiolytic drugs. Gpr26 belongs to the G-protein-coupled receptor family and is exclusively expressed in brain tissue. To investigate the biological function of Gpr26 in vivo, we have generated Gpr26 knockout mice. The mutant mice grew and developed normally but displayed increased levels of anxiety-like behaviors in the open field and elevated plus maze tests, as well as a higher level of depression-like behaviors in the forced-swim and tail-suspension tests. Meanwhile, no significant alteration in spatial learning and memory abilities were found for Gpr26-deficient mice in the Morris water maze test. Previous studies demonstrated that lower protein kinase A (PKA)-cAMP responsive element-binding protein (CREB)-neuropeptide Y (NPY) signaling in the amygdala is linked to higher anxiety and excessive alcohol-drinking behaviors in rats. Therefore, we further examined the phosphorylated CREB (pCREB) and CREB levels in the brains of Gpr26-deficient mice. Reduced pCREB levels were observed in the central amygdala but not in the other regions, while total CREB levels remained comparable between wild-type and mutant mice. Combined, our data indicate that Gpr26 is important for emotion regulation in mice, a function probably mediated by the phosphorylation of CREB in the central amygdala.

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

    PubMed

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

    2015-11-01

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

  7. NDE1 and GSK3β Associate with TRAK1 and Regulate Axonal Mitochondrial Motility: Identification of Cyclic AMP as a Novel Modulator of Axonal Mitochondrial Trafficking.

    PubMed

    Ogawa, Fumiaki; Murphy, Laura C; Malavasi, Elise L V; O'Sullivan, Shane T; Torrance, Helen S; Porteous, David J; Millar, J Kirsty

    2016-05-18

    Mitochondria are essential for neuronal function, providing the energy required to power neurotransmission, and fulfilling many important additional roles. In neurons, mitochondria must be efficiently transported to sites, including synapses, where their functions are required. Neurons, with their highly elongated morphology, are consequently extremely sensitive to defective mitochondrial trafficking which can lead to neuronal ill-health/death. We recently demonstrated that DISC1 associates with mitochondrial trafficking complexes where it associates with the core kinesin and dynein adaptor molecule TRAK1. We now show that the DISC1 interactors NDE1 and GSK3β also associate robustly with TRAK1 and demonstrate that NDE1 promotes retrograde axonal mitochondrial movement. GSK3β is known to modulate axonal mitochondrial motility, although reports of its actual effect are conflicting. We show that, in our system, GSK3β promotes anterograde mitochondrial transport. Finally, we investigated the influence of cAMP elevation upon mitochondrial motility, and found a striking increase in mitochondrial motility and retrograde movement. DISC1, NDE1, and GSK3β are implicated as risk factors for major mental illness. Our demonstration that they function together within mitochondrial trafficking complexes suggests that defective mitochondrial transport may be a contributory disease mechanism in some cases of psychiatric disorder. PMID:26815013

  8. The role of the ERK1/2 pathway as an alternative to the aging-diminished cyclic AMP pathway in calcitonin-mediated chondrogenesis in human nucleus pulposus.

    PubMed

    Chen, Wei-Hong; Zeng, Rong; Lo, Wen-Cheng; Tina Chen, Szu-Yu; Lai, Tung-Yuan; Williams, David F; Deng, Win-Ping

    2012-11-01

    Human disc degeneration initiated by aging in the central nucleus pulposus (hNP) is an irreversible process and the recovery has become seriously emerging. In this study, the related mechanisms of calcitonin on the regeneration of hNP and the effects of calcitonin on the age-related alterations were examined. The harvested hNP population was designated as YhNP (from young donor, age <50) and OhNP (from old donor, age >50). Primary OhNP cells showed more hypertrophic phenotypes than YhNP. However, calcitonin (10(-8)-10(-6) M) was able to induce the same chondrogenesis in both YhNP and OhNP by elevating chondrogenic specific-mRNA and protein expressions. Their cell viabilities were increased with calcitonin treatment. No significant differences of calcitonin receptor (CTR) were expressed between YhNP and OhNP cells. Interestingly, in calcitonin-induced pathways for chondrogenesis, highly increased cyclic AMP (cAMP) was detected in YhNP but was strongly diminished by aging in OhNP after calcitonin treatment. However, to maintain the chondrogenesis, calcitonin-induced an alterative phosphorylated ERK1/2 (p-ERK) in both cells. After inhibiting ERK1/2 by PD98059, calcitonin-induced chondrogenesis in OhNP was almost restrained while YhNP cells were not affected. Our results demonstrated that the regeneration of calcitonin on hNP was maintained with aging which was satisfied by an alternative signaling pathway. Therefore, calcitonin shows great potential for clinical therapy for disc regeneration without aging considerations.

  9. 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

  10. 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

  11. 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

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

    PubMed

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

    2015-08-01

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

  14. 2'-deoxy cyclic adenosine 5'-diphosphate ribose derivatives: importance of the 2'-hydroxyl motif for the antagonistic activity of 8-substituted cADPR derivatives.

    PubMed

    Zhang, Bo; Wagner, Gerd K; Weber, Karin; Garnham, Clive; Morgan, Anthony J; Galione, Antony; Guse, Andreas H; Potter, Barry V L

    2008-03-27

    The structural features needed for antagonism at the cyclic ADP-ribose (cADPR) receptor are unclear. Chemoenzymatic syntheses of novel 8-substituted 2'-deoxy-cADPR analogues, including 8-bromo-2'-deoxy-cADPR 7, 8-amino-2'-deoxy-cADPR 8, 8- O-methyl-2'-deoxy-cADPR 9, 8-phenyl-2'-deoxy-cADPR 10 and its ribose counterpart 8-phenyl-cADPR 5 are reported, including improved syntheses of established antagonists 8-amino-cADPR 2 and 8-bromo-cADPR 3. Aplysia californica ADP-ribosyl cyclase tolerates even the bulky 8-phenyl-nicotinamide adenine 5'-dinucleotide as a substrate. Structure-activity relationships of 8-substituted cADPR analogues in both Jurkat T-lymphocytes and sea urchin egg homogenate (SUH) were investigated. 2'-OH Deletion decreased antagonistic activity (at least for the 8-amino series), showing it to be an important motif. Some 8-substituted 2'-deoxy analogues showed agonist activity at higher concentrations, among which 8-bromo-2'-deoxy-cADPR 7 was, unexpectedly, a weak but almost full agonist in SUH and was membrane-permeant in whole eggs. Classical antagonists 2 and 3 also showed previously unobserved agonist activity at higher concentrations in both systems. The 2'-OH group, without effect on the Ca (2+)-mobilizing ability of cADPR itself, is an important motif for the antagonistic activities of 8-substituted cADPR analogues. PMID:18303825

  15. 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

  16. Modification of radiosensitivity of mammalian cells by cyclic nucleotides. [Mice

    SciTech Connect

    Hess, D.; Prasad, K.N.

    1981-07-06

    Some in vitro and in vivo studies suggest that adenosine 3',5'-cyclic monophosphate (cyclic AMP) may be one of the important factors in determining the radiosensitivity of certain mammalian cells; however, the role of guanosine 3',5'-cyclic monophosphate (cyclic GMP) in radiosensitivity of mammalian cells is completely unknown. Recent data also suggest that the mechanism of radiation protection afforded by moderate hypoxia and SH-containing compounds may involve an alteration in the intracellular level of cyclic AMP. At least one in vivo study shows that cyclic AMP protects hair follicles and gut epithelial cells against radiation damage; however, it does not protect lymphosarcoma and breast carcinoma in mice. If a similar phenomenon is found in humans, an elevation of the intracellular level of cyclic AMP during radiation exposure may improve the effectiveness of radiation therapy in those cases where the radiation damage of normal tissue becomes the limiting factor for a continuation of the therapy program.

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

    PubMed Central

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

    1975-01-01

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

  18. Cyclic GMP is involved in auxin signalling during Arabidopsis root growth and development.

    PubMed

    Nan, Wenbin; Wang, Xiaomin; Yang, Lei; Hu, Yanfeng; Wei, Yuantao; Liang, Xiaolei; Mao, Lina; Bi, Yurong

    2014-04-01

    The second messenger cyclic guanosine 3',5'-monophosphate (cGMP) plays an important role in plant development and responses to stress. Recent studies indicated that cGMP is a secondary signal generated in response to auxin stimulation. cGMP also mediates auxin-induced adventitious root formation in mung bean and gravitropic bending in soybean. Nonetheless, the mechanism of the participation of cGMP in auxin signalling to affect these growth and developmental processes is largely unknown. In this report we provide evidence that indole-3-acetic acid (IAA) induces cGMP accumulation in Arabidopsis roots through modulation of the guanylate cyclase activity. Application of 8-bromo-cGMP (a cell-permeable cGMP derivative) increases auxin-dependent lateral root formation, root hair development, primary root growth, and gene expression. In contrast, inhibitors of endogenous cGMP synthesis block these processes induced by auxin. Data also showed that 8-bromo-cGMP enhances auxin-induced degradation of Aux/IAA protein modulated by the SCF(TIR1) ubiquitin-proteasome pathway. Furthermore, it was found that 8-bromo-cGMP is unable to directly influence the auxin-dependent TIR1-Aux/IAA interaction as evidenced by pull-down and yeast two-hybrid assays. In addition, we provide evidence for cGMP-mediated modulation of auxin signalling through cGMP-dependent protein kinase (PKG). Our results suggest that cGMP acts as a mediator to participate in auxin signalling and may govern this process by PKG activity via its influence on auxin-regulated gene expression and auxin/IAA degradation.

  19. Cyclic GMP is involved in auxin signalling during Arabidopsis root growth and development

    PubMed Central

    Nan, Wenbin; Wang, Xiaomin; Bi, Yurong

    2014-01-01

    The second messenger cyclic guanosine 3′,5′-monophosphate (cGMP) plays an important role in plant development and responses to stress. Recent studies indicated that cGMP is a secondary signal generated in response to auxin stimulation. cGMP also mediates auxin-induced adventitious root formation in mung bean and gravitropic bending in soybean. Nonetheless, the mechanism of the participation of cGMP in auxin signalling to affect these growth and developmental processes is largely unknown. In this report we provide evidence that indole-3-acetic acid (IAA) induces cGMP accumulation in Arabidopsis roots through modulation of the guanylate cyclase activity. Application of 8-bromo-cGMP (a cell-permeable cGMP derivative) increases auxin-dependent lateral root formation, root hair development, primary root growth, and gene expression. In contrast, inhibitors of endogenous cGMP synthesis block these processes induced by auxin. Data also showed that 8-bromo-cGMP enhances auxin-induced degradation of Aux/IAA protein modulated by the SCFTIR1 ubiquitin-proteasome pathway. Furthermore, it was found that 8-bromo-cGMP is unable to directly influence the auxin-dependent TIR1-Aux/IAA interaction as evidenced by pull-down and yeast two-hybrid assays. In addition, we provide evidence for cGMP-mediated modulation of auxin signalling through cGMP-dependent protein kinase (PKG). Our results suggest that cGMP acts as a mediator to participate in auxin signalling and may govern this process by PKG activity via its influence on auxin-regulated gene expression and auxin/IAA degradation. PMID:24591051

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

    PubMed

    Ashton, A R; Polya, G M

    1977-07-01

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

  1. 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

  2. The nitric oxide-cyclic GMP pathway and synaptic plasticity in the rat superior cervical ganglion.

    PubMed Central

    Southam, E.; Charles, S. L.; Garthwaite, J.

    1996-01-01

    1. We have investigated the possibility that nitric oxide (NO) and soluble guanylyl cyclase, an enzyme that synthesizes guanosine 3':5'-cyclic monophosphate (cyclic GMP) in response to NO, contributes to plasticity of synaptic transmission in the rat isolated superior cervical ganglion (SCG). 2. Exposure of ganglia to the NO donor, nitroprusside, caused a concentration-dependent accumulation of cyclic GMP which was augmented in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine. The compound, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of soluble guanylyl cyclase, completely blocked this cyclic GMP response. 3. As assessed by extracellular recording, nitroprusside (100 microM) and another NO donor, S-nitrosoglutathione (30 microM) increased the efficacy of ganglionic synaptic transmission in response to electrical stimulation of the preganglionic nerve, an effect that was reversible and which could be replicated by the cyclic GMP analogue, 8-bromo-cyclic GMP. Ganglionic depolarizations resulting from stimulation of nicotinic receptors with carbachol were not increased by nitroprusside. The potentiating actions of the NO donors on synaptic transmission, but not that of 8-bromo-cyclic GMP, were inhibited by ODQ. 4. Brief tetanic stimulation of the preganglionic nerve resulted in a long-term potentiation (LTP) of synaptic transmission that was unaffected by ODQ, either in the absence or presence of the NO synthase inhibitor, NG-nitro-L-arginine (L-NOARG, 100 microM). A lack of influence of L-NOARG was confirmed in intracellular recordings of LTP of the excitatory postsynaptic potential. Furthermore, under conditions where tetanically-induced LTP was saturated, nitroprusside was still able to potentiate synaptic transmission, as judged from extracellular recording. 5. We conclude that NO is capable of potentiating ganglionic neurotransmission and this effect is mediated through the stimulation of soluble guanylyl

  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. Influence of cAMP on reporter bioassays for dioxin and dioxin-like compounds

    SciTech Connect

    Kasai, Ayumi; Yao, Jian; Yamauchi, Kozue; Hiramatsu, Nobuhiko; Hayakawa, Kunihiro; Meng, Yiman; Maeda, Shuichiro; Kitamura, Masanori . E-mail: masanori@yamanashi.ac.jp

    2006-02-15

    In reporter assays for detection of dioxins, the dioxin-responsive element (DRE) is generally used as a sensor sequence. In several systems, the CYP1A1 promoter containing DREs (DRE{sup cyp}) is inserted into a part of the long terminal repeat of mouse mammary tumor virus (LTR{sup MMTV}) to improve sensitivity of assays. We found that DRE{sup cyp}-LTR{sup MMTV} responds not only to dioxins and dioxin-like compounds but also to forskolin, a cAMP-elevating agent. This effect was dose-dependent and reproduced by other cAMP-elevating agents including 8-bromo-cAMP and 3-isobutyl-methylxanthine. The cAMP response element (CRE) and CRE-like sequences were absent in DRE{sup cyp}-LTR{sup MMTV} and not involved in this process. In contrast to the effect of dioxin, the activation of DRE{sup cyp}-LTR{sup MMTV} by cAMP was independent of the aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor for DRE. Furthermore, neither DRE{sup cyp}, LTR{sup MMTV} nor the consensus sequence of DRE alone was activated in response to cAMP. These data elucidated for the first time that the combination of DRE{sup cyp} with LTR{sup MMTV} causes a peculiar response to cAMP and suggested that use of AhR antagonists is essential to exclude false-positive responses of DRE{sup cyp}-LTR{sup MMTV}-based bioassays for detection and quantification of dioxins and dioxin-like compounds.

  5. Cyclic nucleotide signalling in kidney fibrosis

    PubMed Central

    Schinner, Elisabeth; Wetzl, Veronika; Schlossmann, Jens

    2015-01-01

    Kidney fibrosis is an important factor for the progression of kidney diseases, e.g., diabetes mellitus induced kidney failure, glomerulosclerosis and nephritis resulting in chronic kidney disease or end-stage renal disease. Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were implicated to suppress several of the above mentioned renal diseases. In this review article, identified effects and mechanisms of cGMP and cAMP regarding renal fibrosis are summarized. These mechanisms include several signalling pathways of nitric oxide/ANP/guanylyl cyclases/cGMP-dependent protein kinase and cAMP/Epac/adenylyl cyclases/cAMP-dependent protein kinase. Furthermore, diverse possible drugs activating these pathways are discussed. From these diverse mechanisms it is expected that new pharmacological treatments will evolve for the therapy or even prevention of kidney failure. PMID:25622251

  6. 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

    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.

  7. 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

  8. Role of IgE immune complexes in the regulation of HIV-1 replication and increased cell death of infected U1 monocytes: involvement of CD23/Fc epsilon RII-mediated nitric oxide and cyclic AMP pathways.

    PubMed Central

    Ouaaz, F.; Ruscetti, F. W.; Dugas, B.; Mikovits, J.; Agut, H.; Debré, P.; Mossalayi, M. D.

    1996-01-01

    BACKGROUND: IgE/anti-IgE immune complexes (IgE-IC) induce the release of multiple mediators from monocytes/macrophages and the monocytic cell line U937 following the ligation of the low-affinity Fc epsilon receptors (Fc epsilon RII/CD23). These effects are mediated through an accumulation of cAMP and the generation of L-arginine-dependent nitric oxide (NO). Since high IgE levels predict more rapid progression to acquired immunodeficiency syndrome, we attempted to define the effects of IgE-IC on human immunodeficiency virus (HIV) production in monocytes. MATERIALS AND METHODS: Two variants of HIV-1 chronically infected monocytic U1 cells were stimulated with IgE-IC and virus replication was quantified. NO and cAMP involvement was tested through the use of agonistic and antagonistic chemicals of these two pathways. RESULTS: IgE-IC induced p24 production by U1 cells with low-level constitutive expression of HIV-1 mRNAs and extracellular HIV capsid protein p24 levels (U1low), upon their pretreatment with interleukin 4 (IL-4) or IL-13. This effect was due to the crosslinking of CD23, as it was reversed by blocking the IgE binding site on CD23. The IgE-IC effect could also be mimicked by crosslinking of CD23 by a specific monoclonal antibody. p24 induction by IgE-IC was then shown to be due to CD23-mediated stimulation of cAMP, NO, and tumor necrosis factor alpha (TNF alpha) generation. In another variant of U1 cells with > 1 log higher constitutive production of p24 levels (U1high), IgE-IC addition dramatically decreased all cell functions tested and accelerated cell death. This phenomenon was reversed by blocking the nitric oxide generation. CONCLUSIONS: These data point out a regulatory role of IgE-IC on HIV-1 production in monocytic cells, through CD23-mediated stimulation of cAMP and NO pathways. IgE-IC can also stimulate increased cell death in high HIV producing cells through the NO pathway. Images FIG. 1 FIG. 2 FIG. 5 PMID:8900533

  9. 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

  10. 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

  11. Advances in targeting cyclic nucleotide phosphodiesterases.

    PubMed

    Maurice, Donald H; Ke, Hengming; Ahmad, Faiyaz; Wang, Yousheng; Chung, Jay; Manganiello, Vincent C

    2014-04-01

    Cyclic nucleotide phosphodiesterases (PDEs) catalyse the hydrolysis of cyclic AMP and cyclic GMP, thereby regulating the intracellular concentrations of these cyclic nucleotides, their signalling pathways and, consequently, myriad biological responses in health and disease. Currently, a small number of PDE inhibitors are used clinically for treating the pathophysiological dysregulation of cyclic nucleotide signalling in several disorders, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication and chronic obstructive pulmonary disease. However, pharmaceutical interest in PDEs has been reignited by the increasing understanding of the roles of individual PDEs in regulating the subcellular compartmentalization of specific cyclic nucleotide signalling pathways, by the structure-based design of novel specific inhibitors and by the development of more sophisticated strategies to target individual PDE variants.

  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. Human papillomavirus E5 protein induces expression of the EP4 subtype of prostaglandin E2 receptor in cyclic AMP response element-dependent pathways in cervical cancer cells.

    PubMed

    Oh, Jung-Min; Kim, Su-Hyeong; Lee, Yun-Il; Seo, Miran; Kim, So-Young; Song, Yong-Sang; Kim, Woo-Ho; Juhnn, Yong-Sung

    2009-01-01

    Human papillomavirus (HPV) is the major cause of uterine cervical cancer, but the role of the HPV E5 in carcinogenesis is not clearly understood. Prostaglandins are known to contribute to carcinogenesis of cervical cancer, and we therefore investigated the effect of HPV16 E5 on the expression of prostaglandin E2 (PGE2) receptors and underlying mechanisms. Stable expression of the E5 induced expression of the EP4 subtype of PGE2 receptors in C33A cervical cancer cells, and transfection of E5 small interfering RNA (siRNA) decreased it. EP4 protein expression was increased in human cervical cancer tissues, and EP4 mediated E5-induced increase in anchorage-independent colony formation and vascular endothelial growth factor expression. E5 induced cyclooxygenase-2 (COX-2) expression, and COX-2 increased PGE2 secretion and EP4 expression. The induction of EP4 by PGE2 and E5 was inhibited by an EP4 antagonist, inhibitors of cyclic adenosine monophosphate-dependent protein kinase or phosphatidylinositol 3-kinase, and a cyclic adenosine monophosphate response element (CRE) decoy. E5 increased the luciferase expression controlled by a variant CRE of the EP4 promoter, and it also increased the binding of cyclic adenosine monophosphate response element binding protein (CREB) to oligonucleotides containing this CRE. We conclude that the HPV16 E5 protein induces EP4 receptor protein in cervical cancer cells and that this induction involves epidermal growth factor receptor, COX-2, PGE2, EP2 and EP4, protein kinase A, CREB and CRE.

  14. Cyclic nucleotides of canine antral smooth muscle. Effects of acetylcholine, catecholamines and gastrin.

    PubMed

    Baur, S; Grant, B; Wooton, J

    1981-01-01

    1. The effects of acetylcholine, catecholamines and gastrin on the intracellular content of cyclic AMP and cyclic GMP in antral circular muscle have been determined. 2. Acetylcholine results in a significant but transient increase in intracellular cyclic GMP. 3. Isoproterenol and norepinephrine increase intracellular cyclic AMP. Based on half-maximal effective doses, isoproterenol is 2.7-times more effective than norepinephrine. The increase in intracellular cyclic AMP by both agents is inhibited by propranolol but not phentolamine, indicating that both agents act on the muscle cell by a beta-receptor-coupled mechanism. 4. Gastrin has no demonstrable effect on either cyclic AMP or cyclic GMP. This suggests that while gastrin and acetylcholine can produce a like myoelectric response in the muscle cell, the action of gastrin is mediated by a separate receptor, presumably on the muscle cell, and not by a release of acetylcholine.

  15. Regulation of chloride self exchange by cAMP in cortical collecting tubule

    SciTech Connect

    Tago, K.; Schuster, V.L.; Stokes, J.B.

    1986-07-01

    The hormonal control of Cl transport was examined in rabbit cortical collecting tubules using the lumen-to-bath /sup 36/Cl tracer rate coefficient (K/sub Cl/, nm/s). Tracer movement via Sl-HCO/sub 3/ exchange was minimized by using HCO/sub 3/-CO/sub 2/-free solutions. The electrical driving force was minimized by treating with amiloride. Under these conditions, net Cl transport was zero, yet there was a large K/sub Cl/ that fell 88% on removing bath (trans) Cl. These results are consistent with the mechanism of tracer flux being predominantly Cl self exchange. K/sub Cl/ fell spontaneously with time in vitro; after this decline K/sub Cl/ could be stimulated with 8-bromo-cAMP. cAMP present from the onset of perfusion prevented the time-dependent fall in K/sub Cl/. When tracer movement was restricted to diffusion by eliminating Cl self exchange (0 Cl bath), cAMP had no effect on K/sub Cl/. Although both isoproterenol and vasopressin are known to stimulate adenylate cyclase in this epithelium, only isoproterenol mimicked the cAMP effect on K/sub Cl/. The isoproterenol effect was blocked by either propranolol or prostaglandin E/sub 2/. Lumen addition of the disulfonic stilbene DIDS had no effect on K/sub Cl/. Lumen addition of furosemide or trichloromethiazide had minimal or no effect. Taken together, these results indicate that Cl self exchange is regulated by ..beta..-adrenergic agents acting via cAMP. The lack of an effect of vasopressin suggests cellular heterogeneity in this response to cAMP.

  16. Cyclic Voltammetry.

    ERIC Educational Resources Information Center

    Evans, Dennis H.; And Others

    1983-01-01

    Cyclic voltammetry is a simple experiment that has become popular in chemical research because it can provide useful information about redox reactions in a form which is easily obtained and interpreted. Discusses principles of the method and illustrates its use in the study of four electrode reactions. (Author/JN)

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

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

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

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

    PubMed Central

    Boularan, Cédric; Gales, Céline

    2015-01-01

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

  19. Cyclic nucleotides in tissues during long-term hypokinesia

    NASA Technical Reports Server (NTRS)

    Makeyeva, V. F.; Komolova, G. S.; Yegorov, I. A.; Serova, L. V.; Chelnaya, N. A.

    1981-01-01

    Male Wistar rates were kept hypokinetic by placing them in small containers for 22 days. Blood plasma cAMP content was subsequently found increased, and cGMP content decreased, in the experimental animals. Liver and thymus cAMP content was similar in the control and experimental animals. There was a 20 and 38% decrease of cAMP content in the kidneys and spleen, respectively. Hypokinesia's reduction of cyclic nucleotides seems to inhibit RNA and protein synthesis.

  20. Dopaminergic tone regulates transient potassium current maximal conductance through a translational mechanism requiring D1Rs, cAMP/PKA, Erk and mTOR

    PubMed Central

    2013-01-01

    Background Dopamine (DA) can produce divergent effects at different time scales. DA has opposing immediate and long-term effects on the transient potassium current (IA) within neurons of the pyloric network, in the Panulirus interruptus stomatogastric ganglion. The lateral pyloric neuron (LP) expresses type 1 DA receptors (D1Rs). A 10 min application of 5-100 μM DA decreases LP IA by producing a decrease in IA maximal conductance (Gmax) and a depolarizing shift in IA voltage dependence through a cAMP-Protein kinase A (PKA) dependent mechanism. Alternatively, a 1 hr application of DA (≥5 nM) generates a persistent (measured 4 hr after DA washout) increase in IA Gmax in the same neuron, through a mechanistic target of rapamycin (mTOR) dependent translational mechanism. We examined the dose, time and protein dependencies of the persistent DA effect. Results We found that disrupting normal modulatory tone decreased LP IA. Addition of 500 pM-5 nM DA to the saline for 1 hr prevented this decrease, and in the case of a 5 nM DA application, the effect was sustained for >4 hrs after DA removal. To determine if increased cAMP mediated the persistent effect of 5nM DA, we applied the cAMP analog, 8-bromo-cAMP alone or with rapamycin for 1 hr, followed by wash and TEVC. 8-bromo-cAMP induced an increase in IA Gmax, which was blocked by rapamycin. Next we tested the roles of PKA and guanine exchange factor protein activated by cAMP (ePACs) in the DA-induced persistent change in IA using the PKA specific antagonist Rp-cAMP and the ePAC specific agonist 8-pCPT-2′-O-Me-cAMP. The PKA antagonist blocked the DA induced increases in LP IA Gmax, whereas the ePAC agonist did not induce an increase in LP IA Gmax. Finally we tested whether extracellular signal regulated kinase (Erk) activity was necessary for the persistent effect by co-application of Erk antagonists PD98059 or U0126 with DA. Erk antagonism blocked the DA induced persistent increase in LP IA. Conclusions These

  1. Regulation of sex steroid production and mRNAs encoding gonadotropin receptors and steroidogenic proteins by gonadotropins, cyclic AMP and insulin-like growth factor-I in ovarian follicles of rainbow trout (Oncorhynchus mykiss) at two stages of vitellogenesis.

    PubMed

    Nakamura, Ikumi; Kusakabe, Makoto; Swanson, Penny; Young, Graham

    2016-11-01

    At the completion of vitellogenesis, the steroid biosynthetic pathway in teleost ovarian follicles switches from estradiol-17β (E2) to maturational progestin production, associated with decreased follicle stimulating hormone (Fsh) and increased luteinizing hormone (Lh) signaling. This study compared effects of gonadotropins, human insulin-like growth factor-I (IGF1), and cAMP/protein kinase A signaling (forskolin) on E2 production and levels of mRNAs encoding steroidogenic proteins and gonadotropin receptors using midvitellogenic (MV) and late/postvitellogenic (L/PV) ovarian follicles of rainbow trout. Fsh, Lh and forskolin, but not IGF1, increased testosterone and E2 production in MV and L/PV follicles. Fsh increased steroidogenic acute regulatory protein (star; MV), 3β-hydroxysteroid dehydrogenase/Δ(5-4) isomerase (hsd3b; MV) and P450 aromatase (cyp19a1a; MV) transcript levels. Lh increased star mRNA levels (MV, L/PV) but reduced cyp19a1a transcripts in L/PV follicles. At both follicle stages, IGF1 reduced levels of hsd3b transcripts. In MV follicles, IGF1 decreased P450 side-chain cleavage enzyme (cyp11a1) transcripts but increased cyp19a1a transcripts. In MV follicles only, forskolin increased star and hsd3b transcripts. Forskolin reduced MV follicle cyp11a1 transcripts and reduced cyp19a1a transcripts in follicles at both stages. Fsh and Lh reduced fshr transcripts in L/PV follicles. Lh also reduced lhcgr transcripts (L/PV). IGF1 had no effect on gonadotropin receptor transcripts. Forskolin reduced MV follicle fshr transcript levels and reduced lhcgr transcripts in L/PV follicles. These results reveal hormone- and stage-specific transcriptional regulation of steroidogenic protein and gonadotropin receptor genes and suggest that the steroidogenic shift at the completion of vitellogenesis involves loss of stimulatory effects of Fsh and Igfs on cyp19a1a expression and inhibition of cyp19a1a transcription by Lh.

  2. Cyclic multiverses

    NASA Astrophysics Data System (ADS)

    Marosek, Konrad; Dąbrowski, Mariusz P.; Balcerzak, Adam

    2016-09-01

    Using the idea of regularization of singularities due to the variability of the fundamental constants in cosmology we study the cyclic universe models. We find two models of oscillating and non-singular mass density and pressure (`non-singular' bounce) regularized by varying gravitational constant G despite the scale factor evolution is oscillating and having sharp turning points (`singular' bounce). Both violating (big-bang) and non-violating (phantom) null energy condition models appear. Then, we extend this idea on to the multiverse containing cyclic individual universes with either growing or decreasing entropy though leaving the net entropy constant. In order to get an insight into the key idea, we consider the doubleverse with the same geometrical evolution of the two `parallel' universes with their physical evolution [physical coupling constants c(t) and G(t)] being different. An interesting point is that there is a possibility to exchange the universes at the point of maximum expansion - the fact which was already noticed in quantum cosmology. Similar scenario is also possible within the framework of Brans-Dicke theory where varying G(t) is replaced by the dynamical Brans-Dicke field φ(t) though these theories are slightly different.

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

    PubMed

    Jaramillo, Maritza; Olivier, Martin

    2002-12-15

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

  4. A functional CFTR protein is required for mouse intestinal cAMP-, cGMP- and Ca(2+)-dependent HCO3- secretion.

    PubMed Central

    Seidler, U; Blumenstein, I; Kretz, A; Viellard-Baron, D; Rossmann, H; Colledge, W H; Evans, M; Ratcliff, R; Gregor, M

    1997-01-01

    1. Most segments of the gastrointestinal tract secrete HCO3-, but the molecular nature of the secretory mechanisms has not been identified. We had previously speculated that the regulator for intestinal electrogenic HCO3- secretion is the cystic fibrosis transmembrane regulator (CFTR) channel. To prove this hypothesis, we have now measured HCO3- secretion by pH-stat titration, and recorded the electrical parameters of in vitro duodenum, jejunum and ileum of mice deficient in the gene for the CFTR protein ('CF-mice') and their normal littermates. 2. Basal HCO3- secretory rates were reduced in all small intestinal segments of CF mice. Forskolin, PGE2, 8-bromo-cAMP and VIP (cAMP-dependent agonists), heat-stable enterotoxin of Escherichia coli (STa), guanylin and 8-bromo-cGMP (cGMP-dependent agonists) and carbachol (Ca2+ dependent) stimulated both the short-circuit current (Isc) and the HCO3- secretory rate (JHCO3-) in all intestinal segments in normal mice, whereas none of these agonists had any effect on JHCO3- in the intestine of CF mice. 3. To investigate whether Cl(-)-HCO3- exchangers, which have been implicated in mediating the response to some of these agonists in the intestine, were similarly active in the small intestine of normal and CF mice, we studied Cl- gradient-driven 36Cl- uptake into brush-border membrane (BBM) vesicles isolated from normal and CF mouse small intestine. Both the time course and the peak value for 4,4'-diisothiocyanostilbene-2',2-disulphonic acid (DIDS)-inhibited 36Cl- uptake was similar in normal and CF mice BBM vesicles. 4. In summary, the results demonstrate that the presence of the CFTR channel is necessary for agonist-induced stimulation of electrogenic HCO3- secretion in all segments of the small intestine, and all three intracellular signal transduction pathways stimulate HCO3- secretion exclusively via activation of the CFTR channel. PMID:9423183

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    1993-01-01

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

  7. May Cyclic Nucleotides Be a Source for Abiotic RNA Synthesis?

    NASA Astrophysics Data System (ADS)

    Costanzo, Giovanna; Pino, Samanta; Botta, Giorgia; Saladino, Raffaele; di Mauro, Ernesto

    2011-12-01

    Nucleic bases are obtained by heating formamide in the presence of various catalysts. Formamide chemistry also allows the formation of acyclonucleosides and the phosphorylation of nucleosides in every possible position, also affording 2',3' and 3',5' cyclic forms. We have reported that 3',5' cyclic GMP and 3',5' cyclic AMP polymerize in abiotic conditions yielding short oligonucleotides. The characterization of this reaction is being pursued, several of its parameters have been determined and experimental caveats are reported. The yield of non-enzymatic polymerization of cyclic purine nucleotides is very low. Polymerization is strongly enhanced by the presence of base-complementary RNA sequences.

  8. The Popeye Domain Containing Genes and cAMP Signaling

    PubMed Central

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

    2016-01-01

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

  9. Control of bacterial exoelectrogenesis by c-AMP-GMP

    PubMed Central

    Nelson, James W.; Sudarsan, Narasimhan; Phillips, Grace E.; Stav, Shira; Lünse, Christina E.; McCown, Phillip J.; Breaker, Ronald R.

    2015-01-01

    Major changes in bacterial physiology including biofilm and spore formation involve signaling by the cyclic dinucleotides c-di-GMP and c-di-AMP. Recently, another second messenger dinucleotide, c-AMP-GMP, was found to control chemotaxis and colonization by Vibrio cholerae. We have identified a superregulon of genes controlled by c-AMP-GMP in numerous Deltaproteobacteria, including Geobacter species that use extracellular insoluble metal oxides as terminal electron acceptors. This exoelectrogenic process has been studied for its possible utility in energy production and bioremediation. Many genes involved in adhesion, pilin formation, and others that are important for exoelectrogenesis are controlled by members of a variant riboswitch class that selectively bind c-AMP-GMP. These RNAs constitute, to our knowledge, the first known specific receptors for c-AMP-GMP and reveal that this molecule is used by many bacteria to control specialized physiological processes. PMID:25848023

  10. Control of bacterial exoelectrogenesis by c-AMP-GMP.

    PubMed

    Nelson, James W; Sudarsan, Narasimhan; Phillips, Grace E; Stav, Shira; Lünse, Christina E; McCown, Phillip J; Breaker, Ronald R

    2015-04-28

    Major changes in bacterial physiology including biofilm and spore formation involve signaling by the cyclic dinucleotides c-di-GMP and c-di-AMP. Recently, another second messenger dinucleotide, c-AMP-GMP, was found to control chemotaxis and colonization by Vibrio cholerae. We have identified a superregulon of genes controlled by c-AMP-GMP in numerous Deltaproteobacteria, including Geobacter species that use extracellular insoluble metal oxides as terminal electron acceptors. This exoelectrogenic process has been studied for its possible utility in energy production and bioremediation. Many genes involved in adhesion, pilin formation, and others that are important for exoelectrogenesis are controlled by members of a variant riboswitch class that selectively bind c-AMP-GMP. These RNAs constitute, to our knowledge, the first known specific receptors for c-AMP-GMP and reveal that this molecule is used by many bacteria to control specialized physiological processes.

  11. Smooth muscle phosphatase is regulated in vivo by exclusion of phosphorylation of threonine 696 of MYPT1 by phosphorylation of Serine 695 in response to cyclic nucleotides.

    PubMed

    Wooldridge, Anne A; MacDonald, Justin A; Erdodi, Ferenc; Ma, Chaoyu; Borman, Meredith A; Hartshorne, David J; Haystead, Timothy A J

    2004-08-13

    Regulation of smooth muscle myosin phosphatase (SMPP-1M) is thought to be a primary mechanism for explaining Ca(2+) sensitization/desensitization in smooth muscle. Ca(2+) sensitization induced by activation of G protein-coupled receptors acting through RhoA involves phosphorylation of Thr-696 (of the human isoform) of the myosin targeting subunit (MYPT1) of SMPP-1M inhibiting activity. In contrast, agonists that elevate intracellular cGMP and cAMP promote Ca(2+) desensitization in smooth muscle through apparent activation of SMPP-1M. We show that cGMP-dependent protein kinase (PKG)/cAMP-dependent protein kinase (PKA) efficiently phosphorylates MYPT1 in vitro at Ser-692, Ser-695, and Ser-852 (numbering for human isoform). Although phosphorylation of MYPT1 by PKA/PKG has no direct effect on SMPP-1M activity, a primary site of phosphorylation is Ser-695, which is immediately adjacent to the inactivating Thr-696. In vitro, phosphorylation of Ser-695 by PKA/PKG appeared to prevent phosphorylation of Thr-696 by MYPT1K. In ileum smooth muscle, Ser-695 showed a 3-fold increase in phosphorylation in response to 8-bromo-cGMP. Addition of constitutively active recombinant MYPT1K to permeabilized smooth muscles caused phosphorylation of Thr-696 and Ca(2+) sensitization; however, this phosphorylation was blocked by preincubation with 8-bromo-cGMP. These findings suggest a mechanism of Ca(2+) desensitization in smooth muscle that involves mutual exclusion of phosphorylation, whereby phosphorylation of Ser-695 prevents phosphorylation of Thr-696 and therefore inhibition of SMPP-1M.

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

    NASA Astrophysics Data System (ADS)

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

    1982-12-01

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

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

    ERIC Educational Resources Information Center

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

    2007-01-01

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

  14. Rp-cAMPS Prodrugs Reveal the cAMP Dependence of First-Phase Glucose-Stimulated Insulin Secretion

    PubMed Central

    Schwede, Frank; Chepurny, Oleg G.; Kaufholz, Melanie; Bertinetti, Daniela; Leech, Colin A.; Cabrera, Over; Zhu, Yingmin; Mei, Fang; Cheng, Xiaodong; Manning Fox, Jocelyn E.; MacDonald, Patrick E.; Genieser, Hans-G.; Herberg, Friedrich W.

    2015-01-01

    cAMP-elevating agents such as the incretin hormone glucagon-like peptide-1 potentiate glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. However, a debate has existed since the 1970s concerning whether or not cAMP signaling is essential for glucose alone to stimulate insulin secretion. Here, we report that the first-phase kinetic component of GSIS is cAMP-dependent, as revealed through the use of a novel highly membrane permeable para-acetoxybenzyl (pAB) ester prodrug that is a bioactivatable derivative of the cAMP antagonist adenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In dynamic perifusion assays of human or rat islets, a step-wise increase of glucose concentration leads to biphasic insulin secretion, and under these conditions, 8-bromoadenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer, 4-acetoxybenzyl ester (Rp-8-Br-cAMPS-pAB) inhibits first-phase GSIS by up to 80%. Surprisingly, second-phase GSIS is inhibited to a much smaller extent (≤20%). Using luciferase, fluorescence resonance energy transfer, and bioluminescence resonance energy transfer assays performed in living cells, we validate that Rp-8-Br-cAMPS-pAB does in fact block cAMP-dependent protein kinase activation. Novel effects of Rp-8-Br-cAMPS-pAB to block the activation of cAMP-regulated guanine nucleotide exchange factors (Epac1, Epac2) are also validated using genetically encoded Epac biosensors, and are independently confirmed in an in vitro Rap1 activation assay using Rp-cAMPS and Rp-8-Br-cAMPS. Thus, in addition to revealing the cAMP dependence of first-phase GSIS from human and rat islets, these findings establish a pAB-based chemistry for the synthesis of highly membrane permeable prodrug derivatives of Rp-cAMPS that act with micromolar or even nanomolar potency to inhibit cAMP signaling in living cells. PMID:26061564

  15. Rp-cAMPS Prodrugs Reveal the cAMP Dependence of First-Phase Glucose-Stimulated Insulin Secretion.

    PubMed

    Schwede, Frank; Chepurny, Oleg G; Kaufholz, Melanie; Bertinetti, Daniela; Leech, Colin A; Cabrera, Over; Zhu, Yingmin; Mei, Fang; Cheng, Xiaodong; Manning Fox, Jocelyn E; MacDonald, Patrick E; Genieser, Hans-G; Herberg, Friedrich W; Holz, George G

    2015-07-01

    cAMP-elevating agents such as the incretin hormone glucagon-like peptide-1 potentiate glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. However, a debate has existed since the 1970s concerning whether or not cAMP signaling is essential for glucose alone to stimulate insulin secretion. Here, we report that the first-phase kinetic component of GSIS is cAMP-dependent, as revealed through the use of a novel highly membrane permeable para-acetoxybenzyl (pAB) ester prodrug that is a bioactivatable derivative of the cAMP antagonist adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In dynamic perifusion assays of human or rat islets, a step-wise increase of glucose concentration leads to biphasic insulin secretion, and under these conditions, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, 4-acetoxybenzyl ester (Rp-8-Br-cAMPS-pAB) inhibits first-phase GSIS by up to 80%. Surprisingly, second-phase GSIS is inhibited to a much smaller extent (≤20%). Using luciferase, fluorescence resonance energy transfer, and bioluminescence resonance energy transfer assays performed in living cells, we validate that Rp-8-Br-cAMPS-pAB does in fact block cAMP-dependent protein kinase activation. Novel effects of Rp-8-Br-cAMPS-pAB to block the activation of cAMP-regulated guanine nucleotide exchange factors (Epac1, Epac2) are also validated using genetically encoded Epac biosensors, and are independently confirmed in an in vitro Rap1 activation assay using Rp-cAMPS and Rp-8-Br-cAMPS. Thus, in addition to revealing the cAMP dependence of first-phase GSIS from human and rat islets, these findings establish a pAB-based chemistry for the synthesis of highly membrane permeable prodrug derivatives of Rp-cAMPS that act with micromolar or even nanomolar potency to inhibit cAMP signaling in living cells. PMID:26061564

  16. Essential role for NHERF in cAMP-mediated inhibition of the Na+-HCO3- co-transporter in BSC-1 cells.

    PubMed

    Weinman, E J; Evangelista, C M; Steplock, D; Liu, M Z; Shenolikar, S; Bernardo, A

    2001-11-01

    Prior studies have indicated a requirement for the PDZ domain-containing protein, Na(+)/H(+) Exchanger Regulatory Factor (NHERF), for protein kinase A (PKA)-mediated inhibition of the renal basolateral Na(+)-HCO(3)(-) co-transporter (NBC). The present studies explore the potential mechanisms by which NHERF transduces cAMP signals to inhibit NBC. In BSC-1 cells, cells that express NBC but lack NHERF, 8-bromo-cAMP (100 microm for 15 min) failed to inhibit transport until wild-type mNHERF-(1-355) was expressed. mNHERF-(116-355) containing PDZ II and C-terminal ezrin-binding sequences or a mutant unphosphorylated form of rabbit NHERF effectively transduced the cAMP signals that inhibited NBC. By contrast, mNHERF-(1-126) encompassing N-terminal PDZ I and mNHERF-(1-325), which lacks ezrin-binding, failed to support cAMP inhibition of NBC activity. NBC and NHERF did not associate with each other in yeast two-hybrid or co-immunoprecipitation assays, and confocal microscopy indicated distinct subcellular localization of the two proteins. NBC was phosphorylated in BSC-1 cells, but its phosphorylation was not increased by cAMP nor was immunoprecipitated NBC phosphorylated by PKA in vitro. Acute exposure of mNHERF-(1-355)-expressing BSC-1 cells to cAMP did not change cell surface expression of NBC. Although these results established an essential role for NHERF in cAMP-mediated inhibition of NBC in BSC-1 cells, they also suggest a novel mechanism for NHERF-mediated signal transduction distinct from that previously characterized from studies of other NHERF targets.

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

    PubMed

    Trevillyan, J M; Pall, M L

    1979-05-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2013-10-01

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

  20. AMPED Program Overview

    ScienceCinema

    Gur, Ilan

    2016-07-12

    An overview presentation about ARPA-E's AMPED program. AMPED projects seek to develop advanced sensing, control, and power management technologies that redefine the way we think about battery management. Energy storage can significantly improve U.S. energy independence, efficiency, and security by enabling a new generation of electric vehicles. While rapid progress is being made in new battery materials and storage technologies, few innovations have emerged in the management of advanced battery systems. AMPED aims to unlock enormous untapped potential in the performance, safety, and lifetime of today's commercial battery systems exclusively through system-level innovations, and is thus distinct from existing efforts to enhance underlying battery materials and architectures.

  1. AMPED Program Overview

    SciTech Connect

    Gur, Ilan

    2014-03-04

    An overview presentation about ARPA-E's AMPED program. AMPED projects seek to develop advanced sensing, control, and power management technologies that redefine the way we think about battery management. Energy storage can significantly improve U.S. energy independence, efficiency, and security by enabling a new generation of electric vehicles. While rapid progress is being made in new battery materials and storage technologies, few innovations have emerged in the management of advanced battery systems. AMPED aims to unlock enormous untapped potential in the performance, safety, and lifetime of today's commercial battery systems exclusively through system-level innovations, and is thus distinct from existing efforts to enhance underlying battery materials and architectures.

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

    PubMed Central

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

    2016-01-01

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

  3. Revisiting cAMP signaling in the carotid body

    PubMed Central

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

    2014-01-01

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

  4. Critical Role of Nitric Oxide-cGMP Cascade in the Formation of cAMP-Dependent Long-Term Memory

    ERIC Educational Resources Information Center

    Aonuma, Hitoshi; Mizunami, Makoto; Matsumoto, Yukihisa; Unoki, Sae

    2006-01-01

    Cyclic AMP pathway plays an essential role in formation of long-term memory (LTM). In some species, the nitric oxide (NO)-cyclic GMP pathway has been found to act in parallel and complementary to the cAMP pathway for LTM formation. Here we describe a new role of the NO-cGMP pathway, namely, stimulation of the cAMP pathway to induce LTM. We have…

  5. Crystal structure of a c-di-AMP riboswitch reveals an internally pseudo-dimeric RNA.

    PubMed

    Jones, Christopher P; Ferré-D'Amaré, Adrian R

    2014-11-18

    Cyclic diadenosine monophosphate (c-di-AMP) is a second messenger that is essential for growth and homeostasis in bacteria. A recently discovered c-di-AMP-responsive riboswitch controls the expression of genes in a variety of bacteria, including important pathogens. To elucidate the molecular basis for specific binding of c-di-AMP by a gene-regulatory mRNA domain, we have determined the co-crystal structure of this riboswitch. Unexpectedly, the structure reveals an internally pseudo-symmetric RNA in which two similar three-helix-junction elements associate head-to-tail, creating a trough that cradles two c-di-AMP molecules making quasi-equivalent contacts with the riboswitch. The riboswitch selectively binds c-di-AMP and discriminates exquisitely against other cyclic dinucleotides, such as c-di-GMP and cyclic-AMP-GMP, via interactions with both the backbone and bases of its cognate second messenger. Small-angle X-ray scattering experiments indicate that global folding of the riboswitch is induced by the two bound cyclic dinucleotides, which bridge the two symmetric three-helix domains. This structural reorganization likely couples c-di-AMP binding to gene expression. PMID:25271255

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

    PubMed

    Bodnaryk, R P

    1976-11-01

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

  7. Cyclic AMP signaling reduces sirtuin 6 expression in non-small cell lung cancer cells by promoting ubiquitin-proteasomal degradation via inhibition of the Raf-MEK-ERK (Raf/mitogen-activated extracellular signal-regulated kinase/extracellular signal-regulated kinase) pathway.

    PubMed

    Kim, Eui-Jun; Juhnn, Yong-Sung

    2015-04-10

    The cAMP signaling system regulates various cellular functions, including metabolism, gene expression, and death. Sirtuin 6 (SIRT6) removes acetyl groups from histones and regulates genomic stability and cell viability. We hypothesized that cAMP modulates SIRT6 activity to regulate apoptosis. Therefore, we examined the effects of cAMP signaling on SIRT6 expression and radiation-induced apoptosis in lung cancer cells. cAMP signaling in H1299 and A549 human non-small cell lung cancer cells was activated via the expression of constitutively active Gαs plus treatment with prostaglandin E2 (PGE2), isoproterenol, or forskolin. The expression of sirtuins and signaling molecules were analyzed by Western blotting. Activation of cAMP signaling reduced SIRT6 protein expression in lung cancer cells. cAMP signaling increased the ubiquitination of SIRT6 protein and promoted its degradation. Treatment with MG132 and inhibiting PKA with H89 or with a dominant-negative PKA abolished the cAMP-mediated reduction in SIRT6 levels. Treatment with PGE2 inhibited c-Raf activation by increasing inhibitory phosphorylation at Ser-259 in a PKA-dependent manner, thereby inhibiting downstream MEK-ERK signaling. Inhibiting ERK with inhibitors or with dominant-negative ERKs reduced SIRT6 expression, whereas activation of ERK by constitutively active MEK abolished the SIRT6-depleting effects of PGE2. cAMP signaling also augmented radiation-induced apoptosis in lung cancer cells. This effect was abolished by exogenous expression of SIRT6. It is concluded that cAMP signaling reduces SIRT6 expression by promoting its ubiquitin-proteasome-dependent degradation, a process mediated by the PKA-dependent inhibition of the Raf-MEK-ERK pathway. Reduced SIRT6 expression mediates the augmentation of radiation-induced apoptosis by cAMP signaling in lung cancer cells.

  8. [cAMP as a regulator of the phototransduction cascade].

    PubMed

    Astakhova, L A; Kapitskiĭ, S V; Govardovskiĭ, V I; Firsov, M L

    2012-11-01

    Until recently, it has generally been believed that cyclic AMP plays an important role in supporting circadian cycles in the vertebrate retina, but does not directly control the photoreceptors' phototransduction cascade. However, the cAMP levels in photoreceptors oscillate during the day/night cycle, and the cAMP turnover in photoreceptors may be light-dependent. Thus it is natural to suggest that the cAMP-dependent protein phosphorylation may be a mechanism of tuning phototransduction to lighting conditions. In the present review, we summarize available information on the structure and operation of the retinal pacemaker, role(s) of cAMP in its functioning, and identified intracellular targets that could be controlled by cAMP. We discuss our recent results that show that cAMP changes do regulate the phototransduction cascade. This regulation may substantially extend the range of photoreceptor's adaptation by increasing its sensitivity at night, and reducing the sensitivity in bright light. PMID:23431758

  9. 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.

  10. Ca sup 2+ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes

    SciTech Connect

    Mery, P-F.; Fischmeister, R. ); Lohmann, S.M.; Walter, U. )

    1991-02-15

    Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with regulation of the L-type Ca{sup 2+}-channel current (I{sub Ca}) through the opposite actions for two second messengers, cyclic AMP and cyclic GMP. While cyclic AMP stimulation of I{sub Ca} is mediated by the activation of cyclic AMP-dependent protein kinase, inhibition of I{sub Ca} by cyclic GMP in frog heart is largely mediated by activation of cyclic AMP phosphodiesterase. The present patch-clamp study reveals that, in rat ventricular cells, cyclic GMP can also regulate I{sub Ca} via activation of endogenous cyclic GMP-dependent protein kinase (cGMP-PK). Indeed, the effect of cyclic GMP on I{sub Ca} was mimicked by intracellular perfusion with the proteolytic active fragment of purified cGMP-PK. Moreover, cGMP-PK immunoreactivity was detected in pure rat ventricular myocytes by using a specific polyclonal antibody. These results demonstrate a dual mechanism for the inhibitory action of cyclic GMP in heart, as well as a physiological role for cGMP-PK in the control of mammalian heart function.

  11. Applying Mathematical Processes (AMP)

    ERIC Educational Resources Information Center

    Kathotia, Vinay

    2011-01-01

    This article provides insights into the "Applying Mathematical Processes" resources, developed by the Nuffield Foundation. It features Nuffield AMP activities--and related ones from Bowland Maths--that were designed to support the teaching and assessment of key processes in mathematics--representing a situation mathematically, analysing,…

  12. Oocyte maturation and quality: role of cyclic nucleotides.

    PubMed

    Gilchrist, R B; Luciano, A M; Richani, D; Zeng, H T; Wang, X; Vos, M De; Sugimura, S; Smitz, J; Richard, F J; Thompson, J G

    2016-11-01

    The cyclic nucleotides, cAMP and cGMP, are the key molecules controlling mammalian oocyte meiosis. Their roles in oocyte biology have been at the forefront of oocyte research for decades, and many of the long-standing controversies in relation to the regulation of oocyte meiotic maturation are now resolved. It is now clear that the follicle prevents meiotic resumption through the actions of natriuretic peptides and cGMP - inhibiting the hydrolysis of intra-oocyte cAMP - and that the pre-ovulatory gonadotrophin surge reverses these processes. The gonadotrophin surge also leads to a transient spike in cAMP in the somatic compartment of the follicle. Research over the past two decades has conclusively demonstrated that this surge in cAMP is important for the subsequent developmental capacity of the oocyte. This is important, as oocyte in vitro maturation (IVM) systems practised clinically do not recapitulate this cAMP surge in vitro, possibly accounting for the lower efficiency of IVM compared with clinical IVF. This review particularly focuses on this latter aspect - the role of cAMP/cGMP in the regulation of oocyte quality. We conclude that clinical practice of IVM should reflect this new understanding of the role of cyclic nucleotides, thereby creating a new generation of ART and fertility treatment options. PMID:27422885

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

    PubMed Central

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

    1971-01-01

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

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

    PubMed

    Yang, Haihua; Yang, Linghai

    2016-08-01

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

  15. Multiple Facets of cAMP Signalling and Physiological Impact: cAMP Compartmentalization in the Lung

    PubMed Central

    Oldenburger, Anouk; Maarsingh, Harm; Schmidt, Martina

    2012-01-01

    Therapies involving elevation of the endogenous suppressor cyclic AMP (cAMP) are currently used in the treatment of several chronic inflammatory disorders, including chronic obstructive pulmonary disease (COPD). Characteristics of COPD are airway obstruction, airway inflammation and airway remodelling, processes encompassed by increased airway smooth muscle mass, epithelial changes, goblet cell and submucosal gland hyperplasia. In addition to inflammatory cells, airway smooth muscle cells and (myo)fibroblasts, epithelial cells underpin a variety of key responses in the airways such as inflammatory cytokine release, airway remodelling, mucus hypersecretion and airway barrier function. Cigarette smoke, being next to environmental pollution the main cause of COPD, is believed to cause epithelial hyperpermeability by disrupting the barrier function. Here we will focus on the most recent progress on compartmentalized signalling by cAMP. In addition to G protein-coupled receptors, adenylyl cyclases, cAMP-specific phospho-diesterases (PDEs) maintain compartmentalized cAMP signalling. Intriguingly, spatially discrete cAMP-sensing signalling complexes seem also to involve distinct members of the A-kinase anchoring (AKAP) superfamily and IQ motif containing GTPase activating protein (IQGAPs). In this review, we will highlight the interaction between cAMP and the epithelial barrier to retain proper lung function and to alleviate COPD symptoms and focus on the possible molecular mechanisms involved in this process. Future studies should include the development of cAMP-sensing multiprotein complex specific disruptors and/or stabilizers to orchestrate cellular functions. Compartmentalized cAMP signalling regulates important cellular processes in the lung and may serve as a therapeutic target. PMID:24281338

  16. MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish

    PubMed Central

    Lundegaard, Pia R.; Anastasaki, Corina; Grant, Nicola J.; Sillito, Rowland R.; Zich, Judith; Zeng, Zhiqiang; Paranthaman, Karthika; Larsen, Anders Peter; Armstrong, J. Douglas; Porteous, David J.; Patton, E. Elizabeth

    2015-01-01

    Summary Altered phosphodiesterase (PDE)-cyclic AMP (cAMP) activity is frequently associated with anxiety disorders, but current therapies act by reducing neuronal excitability rather than targeting PDE-cAMP-mediated signaling pathways. Here, we report the novel repositioning of anti-cancer MEK inhibitors as anxiolytics in a zebrafish model of anxiety-like behaviors. PDE inhibitors or activators of adenylate cyclase cause behaviors consistent with anxiety in larvae and adult zebrafish. Small-molecule screening identifies MEK inhibitors as potent suppressors of cAMP anxiety behaviors in both larvae and adult zebrafish, while causing no anxiolytic behavioral effects on their own. The mechanism underlying cAMP-induced anxiety is via crosstalk to activation of the RAS-MAPK signaling pathway. We propose that targeting crosstalk signaling pathways can be an effective strategy for mental health disorders, and advance the repositioning of MEK inhibitors as behavior stabilizers in the context of increased cAMP. PMID:26388333

  17. Cyclic phosphonium ionic liquids

    PubMed Central

    Mukhlall, Joshua A; Romeo, Alicia R; Gohdo, Masao; Ramati, Sharon; Berman, Marc; Suarez, Sophia N

    2014-01-01

    Summary Ionic liquids (ILs) incorporating cyclic phosphonium cations are a novel category of materials. We report here on the synthesis and characterization of four new cyclic phosphonium bis(trifluoromethylsulfonyl)amide ILs with aliphatic and aromatic pendant groups. In addition to the syntheses of these novel materials, we report on a comparison of their properties with their ammonium congeners. These exemplars are slightly less conductive and have slightly smaller self-diffusion coefficients than their cyclic ammonium congeners. PMID:24605146

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

    PubMed

    Orlowski, M

    1980-06-01

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

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

    PubMed

    Ravnskjaer, Kim; Madiraju, Anila; Montminy, Marc

    2016-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  1. Cyclic Dinucleotide-Controlled Regulatory Pathways in Streptomyces Species

    PubMed Central

    2015-01-01

    The cyclic dinucleotides cyclic 3′,5′-diguanylate (c-di-GMP) and cyclic 3′,5′-diadenylate (c-di-AMP) have emerged as key components of bacterial signal transduction networks. These closely related second messengers follow the classical general principles of nucleotide signaling by integrating diverse signals into regulatory pathways that control cellular responses to changing environments. They impact distinct cellular processes, with c-di-GMP having an established role in promoting bacterial adhesion and inhibiting motility and c-di-AMP being involved in cell wall metabolism, potassium homeostasis, and DNA repair. The involvement of c-dinucleotides in the physiology of the filamentous, nonmotile streptomycetes remained obscure until recent discoveries showed that c-di-GMP controls the activity of the developmental master regulator BldD and that c-di-AMP determines the level of the resuscitation-promoting factor A(RpfA) cell wall-remodelling enzyme. Here, I summarize our current knowledge of c-dinucleotide signaling in Streptomyces species and highlight the important roles of c-di-GMP and c-di-AMP in the biology of these antibiotic-producing, multicellular bacteria. PMID:26216850

  2. Are Math Grades Cyclical?

    ERIC Educational Resources Information Center

    Adams, Gerald J.; Dial, Micah

    1998-01-01

    The cyclical nature of mathematics grades was studied for a cohort of elementary school students from a large metropolitan school district in Texas over six years (average cohort size of 8495). The study used an autoregressive integrated moving average (ARIMA) model. Results indicate that grades do exhibit a significant cyclical pattern. (SLD)

  3. Affordable Cyclic Voltammetry

    ERIC Educational Resources Information Center

    Stewart, Greg; Kuntzleman, Thomas S.; Amend, John R.; Collins, Michael J.

    2009-01-01

    Cyclic voltammetry is an important component of the undergraduate chemical curriculum. Unfortunately, undergraduate students rarely have the opportunity to conduct experiments in cyclic voltammetry owing to the high cost of potentiostats, which are required to control these experiments. By using MicroLab data acquisition interfaces in conjunction…

  4. Three-dimensional measurement of cAMP gradients using hyperspectral confocal microscopy

    NASA Astrophysics Data System (ADS)

    Rich, Thomas C.; Annamdevula, Naga; Britain, Andrea L.; Mayes, Samuel; Favreau, Peter F.; Leavesley, Silas J.

    2016-03-01

    Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions over a wide range of timescales. Several lines of evidence have suggested that the distribution of cAMP within cells is not uniform, and that cAMP compartmentalization is largely responsible for signaling specificity within the cAMP signaling pathway. However, to date, no studies have experimentally measured three dimensional (3D) cAMP distributions within cells. Here we use both 2D and 3D hyperspectral microscopy to visualize cAMP gradients in endothelial cells from the pulmonary microvasculature (PMVECs). cAMP levels were measured using a FRETbased cAMP sensor comprised of a cAMP binding domain from EPAC sandwiched between FRET donors and acceptors -- Turquoise and Venus fluorescent proteins. Data were acquired using either a Nikon A1R spectral confocal microscope or custom spectral microscopy system. Analysis of hyperspectral image stacks from a single confocal slice or from summed images of all slices (2D analysis) indicated little or no cAMP gradients were formed within PMVECs under basal conditions or following agonist treatment. However, analysis of hyperspectral image stacks from 3D cellular geometries (z stacks) demonstrate marked cAMP gradients from the apical to basolateral membrane of PMVECs. These results strongly suggest that 2D imaging studies of cAMP compartmentalization -- whether epifluorescence or confocal microscopy -- may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D studies are required to assess mechanisms of signaling specificity.

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

    SciTech Connect

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

    1983-01-01

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

  6. Genetics Home Reference: cyclic neutropenia

    MedlinePlus

    ... Understand Genetics Home Health Conditions cyclic neutropenia cyclic neutropenia Enable Javascript to view the expand/collapse boxes. Download PDF Open All Close All Description Cyclic neutropenia is a disorder that causes frequent infections and ...

  7. Shaping the Murine Macrophage Phenotype: IL-4 and cAMP Synergistically Activate the Arginase I Promoter

    PubMed Central

    Sheldon, Kathryn E.; Shandilya, Harish; Kepka-Lenhart, Diane; Poljakovic, Mirjana; Ghosh, Arundhati; Morris, Sidney M.

    2013-01-01

    Arginase I is a marker of murine M2 macrophages and is highly expressed in many inflammatory diseases. The basis for high arginase I expression in macrophages in vivo is incompletely understood but likely reflects integrated responses to combinations of stimuli. Our objective was to elucidate mechanisms involved in modulating arginase I induction by IL-4, the prototypical activator of M2 macrophages. IL-4 and 8-bromo-cAMP (8-Br-cAMP) individually induce arginase I, but together they rapidly and synergistically induce arginase I mRNA, protein, and promoter activity in murine macrophage cells. Arginase I induction by IL-4 requires binding of the transcription factors STAT6 and C/EBPβ to the IL-4 response element of the arginase I gene. Chromatin immunoprecipitation (ChIP) showed that the synergistic response involves binding of both transcription factors to the IL-4 response element at levels significantly greater than in response to IL-4 alone. The results suggest that C/EBPβ is a limiting factor for the level of STAT6 bound to the IL-4 response element. The enhanced binding in the synergistic response was not due to increased expression of either STAT6 or C/EBPβ but correlated primarily with increased nuclear abundance of C/EBPβ. Our findings also suggest that induction of arginase I expression is stochastic; i.e., differences in induction reflect differences in probability of transcriptional activation and not simply differences in rate of transcription. Results of the present study also may be useful for understanding mechanisms underlying regulated expression of other genes in macrophages and other myeloid-derived cells in health and disease. PMID:23913966

  8. Novel phosphorylation of aquaporin-5 at its threonine 259 through cAMP signaling in salivary gland cells.

    PubMed

    Hasegawa, Takahiro; Azlina, Ahmad; Javkhlan, Purevjav; Yao, Chenjuan; Akamatsu, Tetsuya; Hosoi, Kazuo

    2011-09-01

    Aquaporin-5 (AQP5), a water channel, plays key roles in salivary secretion. The novel phosphorylation of AQP5 was investigated by using human salivary gland (HSG) cells and mouse salivary glands. In the HSG cells stably transfected with a wild-type mouse AQP5 construct, a protein band immunoreactive with antibody against phosphorylated PKA substrate was detected in the AQP5 immunoprecipitated sample, and its intensity was enhanced by short-term treatment of the cells with 8-bromo-cAMP, forskolin, or phorbol 12-myristate 13-acetate, but not by that with A23187 calcium ionophore. Such enhancement was inhibited in the presence of H-89, a PKA inhibitor. An AQP5 mutant (AQP5-T259A) expressed by transfection of HSG cells was not recognized by anti-phosphorylated PKA substrate antibody, even when the cells were stimulated with the protein kinase activators. Immunoblotting and immunofluorescence studies using a specific antibody detecting AQP5 phosphorylated at its Thr259 demonstrated that AQP5 was rapidly and transiently phosphorylated at the apical membrane of acinar cells in the submandibular and parotid glands after administration of isoproterenol, but not pilocarpine. Furthermore, both AQP5 and AQP5-T259A were constitutively localized at the plasma membrane in HSG cells under the resting and forskolin-stimulated conditions. These results suggest that AQP5 is phosphorylated at its Thr259 by PKA through cAMP, but not Ca(2+), signaling pathways, and that this phosphorylation does not contribute to AQP5 trafficking in the salivary gland cells.

  9. Effect of Thyrocalcitonin on Adenosine 3′:5′-Cyclic Phosphate Formation by Rat Kidney and Bone

    PubMed Central

    Murad, Ferid; Brewer, H. Bryan; Vaughan, Martha

    1970-01-01

    Thyrocalcitonin (TCT) increased the rate of accumulation of adenosine 3′:5′-cyclic phosphate (cyclic AMP) when added to incubations containing washed particles from whole rat kidney, adenosine triphosphate (ATP), MgSO4, and caffeine. The maximum stimulatory effect of TCT, 44 ± 6.7 per cent, was always less than the 150 to 250 per cent increase produced by parathyroid hormone (PTH). The effect of both hormones together was no greater than that of PTH alone when each was present at a maximally effective concentration. Since neither TCT nor PTH altered the rate of degradation of cyclic AMP by the kidney preparation, it may be inferred that their effects on cyclic AMP accumulation are the result of increased formation of cyclic AMP. Adenyl cyclase activity in homogenates of renal cortex was stimulated to a greater extent by TCT and PTH than was that of medulla, whereas, as reported earlier, the effect of vasopressin was much larger with homogenates of medulla. The accumulation of cyclic AMP in incubations of rat kidney cortex slices was increased 20 to 60 per cent by TCT and 50 to 140 per cent by PTH. The accumulation of cyclic AMP in incubations of rat calvaria was increased about threefold with TCT and nine to tenfold with PTH, while reduced and alkylated TCT had less than 10 per cent of the activity of TCT. These observations are consistent with the view that the physiological effects of TCT and PTH in kidney and bone are secondary to the enhanced formation of cyclic AMP. PMID:4313199

  10. Functional Analysis of a c-di-AMP-specific Phosphodiesterase MsPDE from Mycobacterium smegmatis

    PubMed Central

    Tang, Qing; Luo, Yunchao; Zheng, Cao; Yin, Kang; Ali, Maria Kanwal; Li, Xinfeng; He, Jin

    2015-01-01

    Cyclic di‑AMP (c-di-AMP) is a second signaling molecule involved in the regulation of bacterial physiological processes and interaction between pathogen and host. However, the regulatory network mediated by c-di-AMP in Mycobacterium remains obscure. In M. smegmatis, a diadenylate cyclase (DAC) was reported recently, but there is still no investigation on c-di-AMP phosphodiesterase (PDE). Here, we provide a systematic study on signaling mechanism of c-di-AMP PDE in M. smegmatis. Based on our enzymatic analysis, MsPDE (MSMEG_2630), which contained a DHH-DHHA1 domain, displayed a 200-fold higher hydrolytic efficiency (kcat/Km) to c-di-AMP than to c-di-GMP. MsPDE was capable of converting c-di-AMP to pApA and AMP, and hydrolyzing pApA to AMP. Site-directed mutations in DHH and DHHA1 revealed that DHH domain was critical for the phosphodiesterase activity. To explore the regulatory role of c-di-AMP in vivo, we constructed the mspde mutant (Δmspde) and found that deficiency of MsPDE significantly enhanced intracellular C12-C20 fatty acid accumulation. Deficiency of DAC in many bacteria results in cell death. However, we acquired the M. smegmatis strain with DAC gene disrupted (ΔmsdisA) by homologous recombination approach. Deletion of msdisA reduced bacterial C12-C20 fatty acids production but scarcely affected bacterial survival. We also provided evidences that superfluous c-di-AMP in M. smegmatis could lead to abnormal colonial morphology. Collectively, our results indicate that MsPDE is a functional c-di-AMP-specific phosphodiesterase both in vitro and in vivo. Our study also expands the regulatory network mediated by c-di-AMP in M. smegmatis. PMID:26078723

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

    PubMed

    van Ool, P J; Buck, H M

    1982-01-01

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

  12. Cyclic control stick

    DOEpatents

    Whitaker, Charles N.; Zimmermann, Richard E.

    1989-01-01

    A cyclic control stick of the type used in helicopters for reducing the safety hazards associated with such a mechanism in the event of a crewman being thrown violently into contact with the cyclic control stick resulting from a crash or the like. The cyclic control stick is configured to break away upon the exertion of an impact force which exceeds a predetermined value and/or is exerted for more than a momentary time duration. The cyclic control stick is also configured to be adjustable so as to locate the grip thereof as far away from the crewman as possible for safety reasons without comprising the comfort of the crewman or the use of the control stick, and a crushable pad is provided on the top of the grip for impact energy absorbing purposes.

  13. Cyclic steps on ice

    NASA Astrophysics Data System (ADS)

    Yokokawa, M.; Izumi, N.; Naito, K.; Parker, G.; Yamada, T.; Greve, R.

    2016-05-01

    Boundary waves often form at the interface between ice and fluid flowing adjacent to it, such as ripples under river ice covers, and steps on the bed of supraglacial meltwater channels. They may also be formed by wind, such as the megadunes on the Antarctic ice sheet. Spiral troughs on the polar ice caps of Mars have been interpreted to be cyclic steps formed by katabatic wind blowing over ice. Cyclic steps are relatives of upstream-migrating antidunes. Cyclic step formation on ice is not only a mechanical but also a thermodynamic process. There have been very few studies on the formation of either cyclic steps or upstream-migrating antidunes on ice. In this study, we performed flume experiments to reproduce cyclic steps on ice by flowing water, and found that trains of steps form when the Froude number is larger than unity. The features of those steps allow them to be identified as ice-bed analogs of cyclic steps in alluvial and bedrock rivers. We performed a linear stability analysis and obtained a physical explanation of the formation of upstream-migrating antidunes, i.e., precursors of cyclic steps. We compared the results of experiments with the predictions of the analysis and found the observed steps fall in the range where the analysis predicts interfacial instability. We also found that short antidune-like undulations formed as a precursor to the appearance of well-defined steps. This fact suggests that such antidune-like undulations correspond to the instability predicted by the analysis and are precursors of cyclic steps.

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

    SciTech Connect

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

    2009-04-03

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

  15. cAMP signaling microdomains and their observation by optical methods

    PubMed Central

    Calebiro, Davide; Maiellaro, Isabella

    2014-01-01

    The second messenger cyclic AMP (cAMP) is a major intracellular mediator of many hormones and neurotransmitters and regulates a myriad of cell functions, including synaptic plasticity in neurons. Whereas cAMP can freely diffuse in the cytosol, a growing body of evidence suggests the formation of cAMP gradients and microdomains near the sites of cAMP production, where cAMP signals remain apparently confined. The mechanisms responsible for the formation of such microdomains are subject of intensive investigation. The development of optical methods based on fluorescence resonance energy transfer (FRET), which allow a direct observation of cAMP signaling with high temporal and spatial resolution, is playing a fundamental role in elucidating the nature of such microdomains. Here, we will review the optical methods used for monitoring cAMP and protein kinase A (PKA) signaling in living cells, providing some examples of their application in neurons, and will discuss the major hypotheses on the formation of cAMP/PKA microdomains. PMID:25389388

  16. Cyclic polymers from alkynes

    NASA Astrophysics Data System (ADS)

    Roland, Christopher D.; Li, Hong; Abboud, Khalil A.; Wagener, Kenneth B.; Veige, Adam S.

    2016-08-01

    Cyclic polymers have dramatically different physical properties compared with those of their equivalent linear counterparts. However, the exploration of cyclic polymers is limited because of the inherent challenges associated with their synthesis. Conjugated linear polyacetylenes are important materials for electrical conductivity, paramagnetic susceptibility, optical nonlinearity, photoconductivity, gas permeability, liquid crystallinity and chain helicity. However, their cyclic analogues are unknown, and therefore the ability to examine how a cyclic topology influences their properties is currently not possible. We have solved this challenge and now report a tungsten catalyst supported by a tetraanionic pincer ligand that can rapidly polymerize alkynes to form conjugated macrocycles in high yield. The catalyst works by tethering the ends of the polymer to the metal centre to overcome the inherent entropic penalty of cyclization. Gel-permeation chromatography, dynamic and static light scattering, viscometry and chemical tests are all consistent with theoretical predictions and provide unambiguous confirmation of a cyclic topology. Access to a wide variety of new cyclic polymers is now possible by simply choosing the appropriate alkyne monomer.

  17. Cyclic polymers from alkynes.

    PubMed

    Roland, Christopher D; Li, Hong; Abboud, Khalil A; Wagener, Kenneth B; Veige, Adam S

    2016-08-01

    Cyclic polymers have dramatically different physical properties compared with those of their equivalent linear counterparts. However, the exploration of cyclic polymers is limited because of the inherent challenges associated with their synthesis. Conjugated linear polyacetylenes are important materials for electrical conductivity, paramagnetic susceptibility, optical nonlinearity, photoconductivity, gas permeability, liquid crystallinity and chain helicity. However, their cyclic analogues are unknown, and therefore the ability to examine how a cyclic topology influences their properties is currently not possible. We have solved this challenge and now report a tungsten catalyst supported by a tetraanionic pincer ligand that can rapidly polymerize alkynes to form conjugated macrocycles in high yield. The catalyst works by tethering the ends of the polymer to the metal centre to overcome the inherent entropic penalty of cyclization. Gel-permeation chromatography, dynamic and static light scattering, viscometry and chemical tests are all consistent with theoretical predictions and provide unambiguous confirmation of a cyclic topology. Access to a wide variety of new cyclic polymers is now possible by simply choosing the appropriate alkyne monomer. PMID:27442285

  18. Profound Asymmetry in the Structure of the cAMP-free cAMP Receptor Protein (CRP) from Mycobacterium tuberculosis

    SciTech Connect

    Gallagher, D.; Smith, N; Kim, S; Robinson, H; Reddy, P

    2009-01-01

    The cyclic AMP receptor protein (CRP, also called catabolite gene activator protein or CAP) plays a key role in metabolic regulation in bacteria and has become a widely studied model allosteric transcription factor. On binding its effector cAMP in the N-terminal domain, CRP undergoes a structural transition to a conformation capable of specific DNA binding in the C-terminal domain and transcription initiation. The crystal structures of Escherichia coli CRP (EcCRP) in the cAMP-bound state, both with and without DNA, are known, although its structure in the off state (cAMP-free, apoCRP) remains unknown. We describe the crystal structure at 2.0A resolution of the cAMP-free CRP homodimer from Mycobacterium tuberculosis H37Rv (MtbCRP), whose sequence is 30% identical with EcCRP, as the first reported structure of an off-state CRP. The overall structure is similar to that seen for the cAMP-bound EcCRP, but the apo MtbCRP homodimer displays a unique level of asymmetry, with a root mean square deviation of 3.5A between all C? positions in the two subunits. Unlike structures of on-state EcCRP and other homologs in which the C-domains are asymmetrically positioned but possess the same internal conformation, the two C-domains of apo MtbCRP differ both in hinge structure and in internal arrangement, with numerous residues that have completely different local environments and hydrogen bond interactions, especially in the hinge and DNA-binding regions. Comparison of the structures of apo MtbCRP and DNA-bound EcCRP shows how DNA binding would be inhibited in the absence of cAMP and supports a mechanism involving functional asymmetry in apoCRP.

  19. 3',5'-cyclic adenosine monophosphate and adenylate cyclase in phototransduction by limulus ventral photoreceptors.

    PubMed Central

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

    1984-01-01

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

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

    PubMed

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

    1977-04-01

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

  1. Cyclic GMP reduces ventricular myocyte stunning after simulated ischemia-reperfusion.

    PubMed

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

    1999-12-01

    We tested the hypothesis that the second messenger activated by nitric oxide, cyclic GMP, would reduce the effects of myocyte stunning following simulated ischemia-reperfusion and that this was related to cyclic GMP protein kinase. Ventricular cardiac myocytes were isolated from New Zealand White rabbits (n = 8). Cell shortening was measured by a video edge detector and protein phosphorylation was determined autoradiographically after SDS gel electrophoresis. Cell shortening data were acquired at: (i) baseline followed by 8-Bromo-cGMP 10(-6) M (8-Br-cGMP) and then KT 5823 10(-6) M (cyclic GMP protein kinase inhibitor) and (ii) simulated ischemia (20 min of 95% N(2)-5% CO(2) at 37 degrees C) followed by simulated reperfusion (reoxygenation) with addition of 8-Br-cGMP 10(-6) M followed by KT 5823 10(-6) M, (iii) addition of 8-Br-cGMP prior to ischemia followed by the addition of KT 5823 10(-6) M after 30 min of reoxygenation. In the control group, 8-Br-cGMP 10(-6) M decreased percentage shortening (%short) (5.0 +/- 0.6 vs 3.8 +/- 0. 4) and the maximum velocity (V(max), microm/s) (48.6 +/- 6.9 vs 40.2 +/- 6.4). KT 5823 10(-6) M added after 8-Br-cGMP partially restored %short (4.6 +/- 0.5) and V(max) (46.6 +/- 8.0). After stunning, baseline myocytes had decreased %short (3.4 +/- 0.2) and V(max) (36. 0 +/- 4.2). After the addition of 8-Br-cGMP, the %short (2.7 +/- 0. 2) and V(max) (27.6 +/- 2.5) decreased further. The addition of KT 5823 did not change either the %short or the V(max). The myocytes with 8-Br-cGMP during ischemia had increased %short (4.2 +/- 0.2) and V(max) (37.2 +/- 3.4) when compared to the stunned group. The addition of KT 5823 did not significantly alter %short (3.3 +/- 0.4) or V(max) (29.2 +/- 5.0) in the myocytes pretreated with 8-Br-cGMP. Protein phosphorylation was increased by 8-Br-cGMP in control and stunned myocytes. KT 5823 blocked this effect in control but not stunned myocytes, suggesting some change in the cyclic GMP protein kinase

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

    PubMed Central

    Amrhein, Nikolaus; Filner, Philip

    1973-01-01

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

  3. Cyclic nucleotide phosphodiesterases in heart and vessels: A therapeutic perspective.

    PubMed

    Bobin, Pierre; Belacel-Ouari, Milia; Bedioune, Ibrahim; Zhang, Liang; Leroy, Jérôme; Leblais, Véronique; Fischmeister, Rodolphe; Vandecasteele, Grégoire

    2016-01-01

    Cyclic nucleotide phosphodiesterases (PDEs) degrade the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), thereby regulating multiple aspects of cardiac and vascular muscle functions. This highly diverse class of enzymes encoded by 21 genes encompasses 11 families that are not only responsible for the termination of cyclic nucleotide signalling, but are also involved in the generation of dynamic microdomains of cAMP and cGMP, controlling specific cell functions in response to various neurohormonal stimuli. In the myocardium and vascular smooth muscle, the PDE3 and PDE4 families predominate, degrading cAMP and thereby regulating cardiac excitation-contraction coupling and smooth muscle contractile tone. PDE3 inhibitors are positive inotropes and vasodilators in humans, but their use is limited to acute heart failure and intermittent claudication. PDE5 is particularly important for the degradation of cGMP in vascular smooth muscle, and PDE5 inhibitors are used to treat erectile dysfunction and pulmonary hypertension. There is experimental evidence that these PDEs, as well as other PDE families, including PDE1, PDE2 and PDE9, may play important roles in cardiac diseases, such as hypertrophy and heart failure, as well as several vascular diseases. After a brief presentation of the cyclic nucleotide pathways in cardiac and vascular cells, and the major characteristics of the PDE superfamily, this review will focus on the current use of PDE inhibitors in cardiovascular diseases, and the recent research developments that could lead to better exploitation of the therapeutic potential of these enzymes in the future. PMID:27184830

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed

    Bressan, R A; Ross, C W

    1976-01-01

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

  6. Activation of f-channels by cAMP analogues in macropatches from rabbit sino-atrial node myocytes.

    PubMed Central

    Bois, P; Renaudon, B; Baruscotti, M; Lenfant, J; DiFrancesco, D

    1997-01-01

    1. The action of the two diastereometric phosphorothioate derivatives of cAMP, Rp-cAMPs and Sp-cAMPs, was investigated on hyperpolarization-activated 'pacemaker' current (i(f)) recorded in inside-out macropatches from rabbit sino-atrial (SA) node myocytes. 2. When superfused on the intracellular side of f-channels at the concentration of 10 microM, both cAMP derivatives accelerated i(f) activation; their action was moderately less pronounced than that due to the same concentration of cAMP. 3. The measurement of the i(f) conductance-voltage relation by voltage ramp protocols indicated that both cAMP analogues shift the activation curve of i(f) to more positive voltages with no change in maximal (fully activated) conductance. 4. Dose-response relationships of the shift of the i(f) activation curve showed that both Rp-cAMPs and Sp-cAMPs act as agonists in the cAMP-dependent direct f-channel activation. Fitting data to the Hill equation resulted in maximal shifts of 9.6 and 9.5 mV, apparent dissociation constants of 0.82 and 5.4 microM, and Hill coefficients of 0.82 and 1.12 for Sp-cAMPs and Rp-cAMPs, respectively. 5. The activating action of Rp-cAMPs, a known antagonist of cAMP in the activation of cAMP-dependent protein kinase, confirms previously establi