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Sample records for adenosine monophosphate amp

  1. Adenosine Monophosphate (AMP)-Activated Protein Kinase: A New Target for Nutraceutical Compounds

    PubMed Central

    Marín-Aguilar, Fabiola; Pavillard, Luis E.; Giampieri, Francesca; Bullón, Pedro; Cordero, Mario D.

    2017-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) is an important energy sensor which is activated by increases in adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio and/or adenosine diphosphate (ADP)/ATP ratio, and increases different metabolic pathways such as fatty acid oxidation, glucose transport and mitochondrial biogenesis. In this sense, AMPK maintains cellular energy homeostasis by induction of catabolism and inhibition of ATP-consuming biosynthetic pathways to preserve ATP levels. Several studies indicate a reduction of AMPK sensitivity to cellular stress during aging and this could impair the downstream signaling and the maintenance of the cellular energy balance and the stress resistance. However, several diseases have been related with an AMPK dysfunction. Alterations in AMPK signaling decrease mitochondrial biogenesis, increase cellular stress and induce inflammation, which are typical events of the aging process and have been associated to several pathological processes. In this sense, in the last few years AMPK has been identified as a very interesting target and different nutraceutical compounds are being studied for an interesting potential effect on AMPK induction. In this review, we will evaluate the interaction of the different nutraceutical compounds to induce the AMPK phosphorylation and the applications in diseases such as cancer, type II diabetes, neurodegenerative diseases or cardiovascular diseases. PMID:28146060

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

  3. Cyclic adenosine monophosphate (cAMP)-induced histone hyperacetylation contributes to its antiproliferative and differentiation-inducing activities.

    PubMed

    Yoo, Seungwan; Lee, Yong Gyu; Kim, Ji Hye; Byeon, Se Eun; Rho, Ho Sik; Cho, Jae Youl; Hong, Sungyoul

    2012-01-01

    Histone acetylation is linked to the control of chromatin remodeling, which is involved in cell growth, proliferation, and differentiation. It is not fully understood whether cyclic adenosine monophosphate (cAMP), a representative differentiation-inducing molecule, is able to modulate histone acetylation as part of its anticancer activity. In the present study, we aimed to address this issue using cell-permeable cAMP, i.e. dibutyryl cAMP (dbcAMP) and C6 glioma cells. As reported previously, under the conditions of our studies, treatment with dbcAMP clearly arrested C6 cell proliferation and altered their morphology. Its antiproliferative and differentiation-inducing activity in C6 glioma cells involved upregulation of p219WAF/CIP), p27(kip1), glial fibrillary acidic protein (GFAP), and Cx43, as well as downregulation of vimentin. Furthermore, dbcAMP modulated the phosphorylation of ERK and Akt in a time-dependent manner and altered the colocalization pattern of phospho-Src and the actin cytoskeleton. Interestingly, dbcAMP upregulated the enzyme activity of histone acetyltransferase (HAT) and, in parallel, enhanced cellular acetyllysine levels. Finally, the hyperacetylation-inducing compound, sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor, displayed similar anticancer activity to dbcAMP. Therefore, our data suggest that antiproliferative and differentiation-inducing activities of dbcAMP may be generated by its enhanced hyperacetylation function.

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

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

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

  5. Effect of electromagnetic field on cyclic adenosine monophosphate (cAMP) in a human mu-opioid receptor cell model.

    PubMed

    Ross, Christina L; Teli, Thaleia; Harrison, Benjamin S

    2016-01-01

    During the cell communication process, endogenous and exogenous signaling affect normal as well as pathological developmental conditions. Exogenous influences such as extra-low-frequency electromagnetic field (EMF) have been shown to effect pain and inflammation by modulating G-protein receptors, down-regulating cyclooxygenase-2 activity, and affecting the calcium/calmodulin/nitric oxide pathway. Investigators have reported changes in opioid receptors and second messengers, such as cyclic adenosine monophosphate (cAMP), in opiate tolerance and dependence by showing how repeated exposure to morphine decreases adenylate cyclase activity causing cAMP to return to control levels in the tolerant state, and increase above control levels during withdrawal. Resonance responses to biological systems using exogenous EMF signals suggest that frequency response characteristics of the target can determine the EMF biological response. In our past research we found significant down regulation of inflammatory markers tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B (NFκB) using 5 Hz EMF frequency. In this study cAMP was stimulated in Chinese Hamster Ovary (CHO) cells transfected with human mu-opioid receptors, then exposed to 5 Hz EMF, and outcomes were compared with morphine treatment. Results showed a 23% greater inhibition of cAMP-treating cells with EMF than with morphine. In order to test our results for frequency specific effects, we ran identical experiments using 13 Hz EMF, which produced results similar to controls. This study suggests the use of EMF as a complementary or alternative treatment to morphine that could both reduce pain and enhance patient quality of life without the side-effects of opiates.

  6. “cAMP Sponge”: A Buffer for Cyclic Adenosine 3′, 5′-Monophosphate

    PubMed Central

    Lefkimmiatis, Konstantinos; Moyer, Mary Pat; Curci, Silvana; Hofer, Aldebaran M.

    2009-01-01

    Background While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP). Methods/Principal Findings Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIβ of protein kinase A (PKA). Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named “cAMP sponge”) was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets. Conclusions This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events. PMID:19888343

  7. Application and optimization of the tenderization of pig Longissimus dorsi muscle by adenosine 5'-monophosphate (AMP) using the response surface methodology.

    PubMed

    Deng, Shaoying; Wang, Daoying; Zhang, Muhan; Geng, Zhiming; Sun, Chong; Bian, Huan; Xu, Weimin; Zhu, Yongzhi; Liu, Fang; Wu, Haihong

    2016-03-01

    Based on single factor experiments, NaCl concentration, adenosine 5'-monophosphate (AMP) concentration and temperature were selected as independent variables for a three-level Box-Behnken experimental design, and the shear force and cooking loss were response values for regression analysis. According to the statistical models, it showed that all independent variables had significant effects on shear force and cooking loss, and optimal values were at the NaCl concentration of 4.15%, AMP concentration of 22.27 mmol/L and temperature of 16.70°C, which was determined with three-dimensional response surface diagrams and contour plots. Under this condition, the observed shear force and cooking loss were 0.625 kg and 8.07%, respectively, exhibiting a good agreement with their predicted values, showing the good applicability and feasibility of response surface methodology (RSM) for improving pork tenderness. Compared with control pig muscles, AMP combined with NaCl treatment demonstrated significant effects on improvement of meat tenderness and reduction of cooking loss. Therefore, AMP could be regarded as an effective tenderization agent for pork.

  8. Herkinorin dilates cerebral vessels via kappa opioid receptor and cyclic adenosine monophosphate (cAMP) in a piglet model.

    PubMed

    Ji, Fang; Wang, Zhenhong; Ma, Nan; Riley, John; Armstead, William M; Liu, Renyu

    2013-01-15

    Since herkinorin is the first non-opioid mu agonist derived from salvinorin A that has the ability to induce cerebral vascular dilatation, we hypothesized that herkinorin could have similar vascular dilatation effect via the mu and kappa opioid receptors and the cAMP pathway. The binding affinities of herkinorin to kappa and mu opioid receptors were determined by in-vitro competition binding assays. The cerebral arteries were monitored in piglets equipped with a closed cranial window and the artery responses were recorded before and every 30s after injection of artificial cerebrospinal fluid (CSF) in the presence or absence of the investigated drugs: herkinorion, norbinaltorphimine (NTP), a kappa opioid receptor antagonist, β-funaltrexamine (β-FNA), a mu opioid receptor antagonist, or Rp-8-Br-cAMPS (Rp-cAMPS), an inhibitor of protein kinase A (PKA). CSF samples were collected before and 10 min after herkinorin and NTP administration for the measurement of cAMP levels. Data were analyzed by repeated-measures analysis of variance. Our results show that herkinorin binds to both kappa and mu opioid receptors. Its vasodilation effect is totally abolished by NTP, but is not affected by β-FNA. The levels of cAMP in the CSF elevate after herkinorin administration, but are abolished with NTP administration. The cerebral vasodilative effect of herkinorin is also blunted by Rp-cAMPS. In conclusion, as a non-opioid kappa and mu opioid receptor agonist, herkinorin exhibits cerebral vascular dilatation effect. The dilatation is mediated though the kappa opioid receptor rather than the mu opioid receptor. cAMP signaling also plays an important role in this process.

  9. 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.

  10. Cyclic adenosine 3'-5'-monophosphate (cAMP) exerts proliferative and anti-proliferative effects in pituitary cells of different types by activating both cAMP-dependent protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac).

    PubMed

    Vitali, E; Peverelli, E; Giardino, E; Locatelli, M; Lasio, G B; Beck-Peccoz, P; Spada, A; Lania, A G; Mantovani, G

    2014-03-05

    In the pituitary the activation of cyclic adenosine 3'-5'-monophosphate (cAMP) dependent pathways generates proliferative signals in somatotrophs, whereas in pituitary cells of other lineages its effect remains uncertain. Moreover, the specific role of the two main cAMP effectors, protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac), has not been defined. Aim of this study was to investigate the effect of cAMP on pituitary adenomatous cells proliferation and to identify PKA and Epac differential involvement. We found that cAMP increased DNA synthesis and cyclin D1 expression in somatotropinomas, whereas it reduced both parameters in prolactinomas and nonfunctioning adenomas, these effects being replicated in corresponding cell lines. Moreover, the divergent cAMP effects were mimicked by Epac and PKA analogs, which activated Rap1 and CREB, respectively. In conclusion, we demonstrated that cAMP exerted opposite effects on different pituitary cell types proliferation, these effects being mediated by both Epac and PKA.

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

    PubMed

    Guo, Li; Breakspear, Andrew; Zhao, Guoyi; Gao, Lixin; Kistler, H Corby; Xu, Jin-Rong; Ma, Li-Jun

    2016-02-01

    The cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway is a central signalling cascade that transmits extracellular stimuli and governs cell responses through the second messenger cAMP. The importance of cAMP signalling in fungal biology has been well documented and the key conserved components, adenylate cyclase (AC) and the catalytic subunit of PKA (CPKA), have been functionally characterized. However, other genes involved in this signalling pathway and their regulation are not well understood in filamentous fungi. Here, we performed a comparative transcriptomics analysis of AC and CPKA mutants in two closely related fungi: Fusarium graminearum (Fg) and F. verticillioides (Fv). Combining available Fg transcriptomics and phenomics data, we reconstructed the Fg cAMP signalling pathway. We developed a computational program that combines sequence conservation and patterns of orthologous gene expression to facilitate global transcriptomics comparisons between different organisms. We observed highly correlated expression patterns for most orthologues (80%) between Fg and Fv. We also identified a subset of 482 (6%) diverged orthologues, whose expression under all conditions was at least 50% higher in one genome than in the other. This enabled us to dissect the conserved and unique portions of the cAMP-PKA pathway. Although the conserved portions controlled essential functions, such as metabolism, the cell cycle, chromatin remodelling and the oxidative stress response, the diverged portions had species-specific roles, such as the production and detoxification of secondary metabolites unique to each species. The evolution of the cAMP-PKA signalling pathway seems to have contributed directly to fungal divergence and niche adaptation.

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

    PubMed Central

    Bowman, Lisa; Zeden, Merve S.; Kaever, Volkhard

    2016-01-01

    Nucleotide signaling networks are key to facilitate alterations in gene expression, protein function, and enzyme activity in response to diverse stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced by the human pathogen Staphylococcus aureus and is involved in regulating a number of physiological processes including potassium transport. S. aureus must ensure tight control over its cellular levels as both high levels of the dinucleotide and its absence result in a number of detrimental phenotypes. Here we show that in addition to the membrane-bound Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterase (PDE) GdpP, S. aureus produces a second cytoplasmic DHH/DHHA1 PDE Pde2. Although capable of hydrolyzing c-di-AMP, Pde2 preferentially converts linear 5′-phosphadenylyl-adenosine (pApA) to AMP. Using a pde2 mutant strain, pApA was detected for the first time in S. aureus, leading us to speculate that this dinucleotide may have a regulatory role under certain conditions. Moreover, pApA is involved in a feedback inhibition loop that limits GdpP-dependent c-di-AMP hydrolysis. Another protein linked to the regulation of c-di-AMP levels in bacteria is the predicted regulator protein YbbR. Here, it is shown that a ybbR mutant S. aureus strain has increased acid sensitivity that can be bypassed by the acquisition of mutations in a number of genes, including the gene coding for the diadenylate cyclase DacA. We further show that c-di-AMP levels are slightly elevated in the ybbR suppressor strains tested as compared with the wild-type strain. With this, we not only identified a new role for YbbR in acid stress resistance in S. aureus but also provide further insight into how c-di-AMP levels impact acid tolerance in this organism. PMID:27834680

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

    PubMed

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

    2016-12-30

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

  14. Adenosine Monophosphate-Based Detection of Bacterial Spores

    NASA Technical Reports Server (NTRS)

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

    2009-01-01

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

  15. Cyclic 3′,5′-Adenosine Monophosphate Phosphodiesterase of Escherichia coli

    PubMed Central

    Nielsen, L. D.; Monard, D.; Rickenberg, H. V.

    1973-01-01

    The cyclic 3′,5′-adenosine monophosphate (c-AMP) phosphodiesterase from Escherichia coli has been partially purified. The enzyme has an apparent molecular weight of 30,000, a Michaelis constant of 0.5 mM c-AMP, and a pH optimum of 7. The partially purified enzyme requires for activity the presence of a reducing compound and of either iron or a protein which seemingly acts as iron carrier. PMID:4355491

  16. Selective adenosine-5'-monophosphate uptake by water-compatible molecularly imprinted polymer.

    PubMed

    Breton, Florent; Delépée, Raphaël; Jégourel, Damien; Deville-Bonne, Dominique; Agrofoglio, Luigi A

    2008-06-02

    Molecularly imprinted polymers (MIPs) were prepared for adenosine-5'-monophosphate (AMP), a substrate of AMP-activated protein kinase. The template molecule was formed by the vinylphenylboronate diester of adenosine on which 5'-free hydroxide was protected by tert-butyldimethylsilyl group in order to mimic the steric hindrance of the phosphate moiety of AMP. Molecular imprinting was performed by complexing acrylamide and the template in a highly cross-linked polymer. MIPs were tested in batch experiments with aqueous samples of nucleotides and a number of parameters were investigated. The use of tetrabutylammonium hydroxide (TBAH) was necessary to obtain a rebinding of nucleotides on MIP. The adsorption of AMP was optimal in 5 mM ammonium acetate buffer solution pH 9.5 for 30 min, with 30 mM of TBAH. The imprinted polymer was selective for AMP towards others nucleotides or deoxi analogues.

  17. Effect of ionizing irradiation on the physiological activity of cyclic adenosine monophosphate on smooth muscle preparations.

    PubMed

    Schachinger, L; Michailov, M; Owusa Daaku, S; Prechter, I; Klöter, H; Schippel, C

    1982-01-01

    The effect of ionizing irradiation on the physiological activity of cyclic adenosine monophosphate (cAMP) in smooth muscle preparations from frog lung was studied. cAMP, given as dibutyryl salt (dib-cAMP) inhibited the radiation induced contractions of the muscle in a manner similar to the action of theophylline. In vitro irradiation of dib-cAMP resulted in an alteration of the chemical structure of this substance, i.e., formation of monobutyryl-cAMP and further derivatives as well as a decomposition of the purine structure. There was also a loss of the relaxing activity of irradiated cAMP on the muscle tone of frog lung preparations. The physiologically measured inactivation of dib-cAMP was far more pronounced than the chemical alteration. An inhibitory effect of the reaction products is postulated.

  18. Genetics Home Reference: adenosine monophosphate deaminase deficiency

    MedlinePlus

    ... that can affect the muscles used for movement ( skeletal muscles ). In many affected individuals, AMP deaminase deficiency does ... called AMP deaminase. This enzyme is found in skeletal muscles , where it plays a role in producing energy. ...

  19. [Conformation study of cyclic adenosine-3',5'-monophosphate and some of its derivatives by means of circular dichroism].

    PubMed

    Tunitskaia, V L; Guliaev, N N; Poletaev, A I; Severin, E S

    1977-04-01

    Circular dichroism spectra of adenosine and cyclic adenosine-3',5'-monophosphate (cAMP) and their derivatives, having different substituents in 8-position of heterocycle, are studied, cAMP is suggested to have preferable anti-conformation in the solution, while its derivatives with substituents in 8-position of purine base are preferable in sin-conformation. An exception is 8-(beta aminoethylamine-)cAMP, which has an anti-conformation within pH range from 4.5 to 9.5. This is probably due to the formation of intra-molecular ionic bond between cyclophosphate group and aliphatic amino group of 8-position substituent.

  20. Cyclic adenosine 3',5'-monophosphate levels and activities of adenylate cyclase and cyclic adenosine 3',5'-monophosphate phosphodiesterase in Pseudomonas and Bacteroides.

    PubMed Central

    Siegel, L S; Hylemon, P B; Phibbs, P V

    1977-01-01

    A modified Gilman assay was used to determine the concentrations of cyclic adenosine 3',5'-monophosphate (cAMP) in rapidly filtered cells and in the culture filtrates of Pseudomonas aeruginosa, Escherichia coli K-12, and Bacteroides fragilis. In P. aeruginosa cultures, levels of cAMP in the filtrate increased with the culture absorbance (3.5 to 19.8 X 10(-9) M) but did not vary significantly with the carbon source used to support growth. Intracellular concentrations (0.8 to 3.2 X 10(-5) M) were substantially higher and did not vary appreciably during growth or with carbon source. Sodium cAMP (5 mM) failed to reverse the catabolite repression of inducible glucose-6-phosphate dehydrogenase (EC 1.1.1.49) synthesis caused by the addition of 10 mM succinate. Exogenous cAMP also had no discernible effect on the catabolite repression control of inducible mannitol dehydrogenase (EC 1.1.1.67). P. aeruginosa was found to contain both soluble cAMP phosphodiesterase (EC 3.1.4.17) and membrane-associated adenylate cyclase (EC 4.6.1.1) activity, and these were compared to the activities detected in crude extracts of E. coli. B. fragilis crude cell extracts contain neither of these enzyme activities, and little or no cAMP was detected in cells or culture filtrates of this anaerobic bacterium. PMID:187575

  1. Effect of adenosine and adenosine analogues on cyclic AMP accumulation in cultured mesangial cells and isolated glomeruli of the rat.

    PubMed Central

    Olivera, A.; Lopez-Novoa, J. M.

    1992-01-01

    1. Changes in intracellular levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) were studied in rat isolated glomeruli and cultured glomerular mesangial cells exposed to adenosine and to the preferential A1 receptor agonist N6-R-1-methyl-2-phenylethyl adenosine (R-PIA), or the potent A2 adenosine receptor agonist 5-(N-ethylcarboxamide)adenosine (NECA). 2. Whereas NECA and adenosine triggered a dose-dependent increase in cyclic AMP values with EC50 values of approximately 10(-6) M and 3 x 10(-5) M respectively, R-PIA lowered cyclic AMP levels at concentrations of 10(-6) M or less and increased them at higher concentrations. 3. The time-course of the increase induced by 10(-6) M NECA was slower than that induced by 10(-4) M adenosine. Adenosine produced a maximal stimulation within the first minute, whereas the effect of NECA in both glomeruli and mesangial cells was noticeable only from the second minute of incubation. 4. The effects of the agonists R-PIA and NECA on the cyclic AMP system were blocked respectively by the A1 adenosine receptor antagonist, 8-cyclopentyl-1, 3-dipropylxanthihe (DPCPX) at 10(-6) M and the A2 antagonist N-(2-dimethylaminoethyl)-N-methyl-4-(2, 3, 6, 7-tetrahydro-2,b-dioxo-1, 3-dipropyl-1H-purin-8-yl) benzene sulphonamide (PD115,199) at 10(-6) M. Theophylline, a known antagonist of adenosine receptors, inhibited the action of adenosine on cyclic AMP in mesangial cells. Dipyridamole, an inhibitor of the uptake of adenosine by the cells, enhanced the response to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1330173

  2. 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.

  3. Alteration of sodium, potassium-adenosine triphosphatase activity in rabbit ciliary processes by cyclic adenosine monophosphate-dependent protein kinase

    SciTech Connect

    Delamere, N.A.; Socci, R.R.; King, K.L. )

    1990-10-01

    The response of sodium, potassium-adenosine triphosphatase (Na,K-ATPase) to cyclic adenosine monophosphate (cAMP)-dependent protein kinase was examined in membranes obtained from rabbit iris-ciliary body. In the presence of the protein kinase together with 10(-5) M cAMP, Na,K-ATPase activity was reduced. No change in Na,K-ATPase activity was detected in response to the protein kinase without added cAMP. Likewise cAMP alone did not alter Na,K-ATPase activity. Reduction of Na,K-ATPase activity was also observed in the presence of the cAMP-dependent protein kinase catalytic subunit. The response of the enzyme to the kinase catalytic subunit was also examined in membranes obtained from rabbit ciliary processes. In the presence of 8 micrograms/ml of the catalytic subunit, ciliary process Na,K-ATPase activity was reduced by more than 50%. To examine whether other ATPases were suppressed by the protein kinase, calcium-stimulated ATPase activity was examined; its activity was stimulated by the catalytic subunit. To test whether the response of the ciliary process Na,K-ATPase is unique, experiments were also performed using membrane preparations from rabbit lens epithelium or rabbit kidney; the catalytic subunit significantly reduced the activity of Na,K-ATPase from the kidney but not the lens. These Na,K-ATPase studies suggest that in the iris-ciliary body, cAMP may alter sodium pump activity. In parallel 86Rb uptake studies, we observed that ouabain-inhibitable potassium uptake by intact pieces of iris-ciliary body was reduced by exogenous dibutryl cAMP or by forskolin.

  4. The effects of cyclic adenosine 3',5'-monophosphate and other adenine nucleotides on body temperature.

    PubMed Central

    Dascombe, M J; Milton, A S

    1975-01-01

    1. Adenosine 3',5'-monophosphate (cAMP), its dibutyryl derivative (Db-cAMP) and other adenine nucleotides have been micro-injected into the hypothalamic region of the unanaesthetized cat and the effects on body temperature, and on behavioural and autonomic thermoregulatory activities observed. 2. Db-cAMP and cAMP both produced hypothermia when applied to the pre-optic anterior hypothalamus. With Db-cAMP the hypothermia was shown to be dose dependent between 50 and 500 mug (0-096-0-96 mumole). 3. AMP, ADP and ATP also produced hypothermia when injected into the pre-optic anterior hypothalamus. 4. The order of relative potencies of the adenine nucleotides with respect both to the hypothermia produced and to the autonomic thermoregulatory effects observed were similar. Db-cAMP was most potent and cAMP least. 5. Micro-injection into the pre-optic anterior hypothalamus of many substances including saline produced in most cats a non-specific rise in body temperature apparently the result of tissue damage. Intraperitoneal injection of 4-acetamidophenol (paracetamol 50 mg/kg) reduced or abolished this febrile response. 6. The hypothermic effect of the adenine nucleotides has been compared with the effects produced in these same cats by micro-injections of noradrenaline, 5-hydroxytryptamine, a mixture of acetylcholine and physostigmine (1:1), EDTA and excess Ca2+ ions. 7. It is concluded that as Db-cAMP and cAMP both produce hypothermia, it is unlikely that endogenous cAMP in the pre-optic anterior hypothalamus mediates the hyperthermic responses to pyrogens and prostaglandins. PMID:170396

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  6. Cyclic adenosine monophosphate-dependent vascular responses to purinergic agonists adenosine triphosphate and uridine triphosphate in the anesthetized mouse.

    PubMed

    Shah, Mrugeshkumar K; Kadowitz, Philip J

    2002-01-01

    The mechanism by which purinergic agonist adenosine triphosphate (ATP) and uridine triphosphate (UTP) decrease systemic arterial pressure in the anesthetized mouse was investigated. Intravenous injections of adenosine triphosphate (ATP) and uridine triphosphate (UTP) produced dose-dependent decreases in systemic blood pressure in the mouse. The order of potency was ATP > UTP. Vasodilator responses to ATP and UTP were altered by the cyclic adenosine monophosphate (cAMP) phosphodiesterase inhibitor rolipram. The vascular responses to ATP and UTP were not altered by a nitric oxide synthase inhibitor, a cyclooxygenase inhibitor, a cGMP phosphodiesterase inhibitor, or a particular P2 receptor antagonist. These data suggest that ATP and UTP cause a decrease in systemic arterial pressure in the mouse via a cAMP-dependent pathway via a novel P2 receptor linked to adenylate cyclase and that nitric oxide release, prostaglandin synthesis, cGMP, and P2X1, P2Y1, and P2Y4 receptors play little or no role in the vascular effects of these purinergic agonists in the mouse.

  7. Metabolism of cyclic adenosine 3',5'-monophosphate and induction of tryptophanase in Escherichia coli.

    PubMed Central

    Botsford, J L

    1975-01-01

    The relationship between cyclic adenosine 3',5'-monophosphate (cyclic AMP) metabolism and the induction of tryptophanase and beta-galactosidase was studied in several strains of Escherichia coli grown with succinate, acetate, glycerol, or glucose as the carbon source. No consistent relationship between the intracellular concentration of cyclic AMP in the several strains cultured and the various carbon sources was discerned. In E. coli K-12-1 the induction of tryptophanase was found to vary in the order: succinate greater than acetate greater than glycerol greater than glucose, and that of beta-galactosidase was found in the order: glycerol greater than acetate greater than succinate greater than glucose. Rate of accumulation of cyclic AMP in the culture filtrate was in the order: succinate greater than acetate greater than glycerol greater than glucose. The addition of glycerol to E. coli K-12-1 grown in acetate caused inhibition of tryptophanase and slight inhibition of accumulation of extracellular cyclic AMP. These same conditions caused beta-galactosidase induction to be stimulated. The addition of exogenous cyclic AMP to cultures grown with four different carbon sources had an effect characteristic for each of the two enzymes studied as well as each individual carbon source. The results suggest that there are control elements distinct from cyclic AMP and its receptor protein which respond to the catabolic situation of the cell. PMID:170248

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

    SciTech Connect

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

    1989-08-08

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

  9. Adenosine 3',5'-monophosphate in relation to inhibition of cervical smooth muscle activity in early pregnant women.

    PubMed

    Norström, A; Bryman, I

    1991-08-01

    Contractile activity was registered in strips of cervical tissue obtained by needle biopsy from women in the first trimester of pregnancy. Dibutyryl cyclic adenosine-3',5'-monophosphate (5 x 10(-6) mol/l), isobutyryl methylxanthine (10(-4) mol/l), and forskolin (10(-5)-10(-4) mol/l), the latter two drugs known to increase the levels of endogenous cAMP, inhibited spontaneous muscle activity. The levels of tissue cAMP were determined in strips during relaxation induced by prostaglandin E2 or purified porcine relaxin and compared with cAMP levels in strips from the same women during contractile activity. Exposure to prostaglandin E2 but not to relaxin was followed by increased levels of cAMP. It is suggested that cAMP has a role as a second messenger in the prostaglandin E2-mediated relaxation of cervical smooth muscle.

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed

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

    1993-02-01

    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.

  12. Small molecule adenosine 5'-monophosphate activated protein kinase (AMPK) modulators and human diseases.

    PubMed

    Rana, Sandeep; Blowers, Elizabeth C; Natarajan, Amarnath

    2015-01-08

    Adenosine 5'-monophosphate activated protein kinase (AMPK) is a master sensor of cellular energy status that plays a key role in the regulation of whole-body energy homeostasis. AMPK is a serine/threonine kinase that is activated by upstream kinases LKB1, CaMKKβ, and Tak1, among others. AMPK exists as αβγ trimeric complexes that are allosterically regulated by AMP, ADP, and ATP. Dysregulation of AMPK has been implicated in a number of metabolic diseases including type 2 diabetes mellitus and obesity. Recent studies have associated roles of AMPK with the development of cancer and neurological disorders, making it a potential therapeutic target to treat human diseases. This review focuses on the structure and function of AMPK, its role in human diseases, and its direct substrates and provides a brief synopsis of key AMPK modulators and their relevance in human diseases.

  13. STUDIES ON THE MECHANISM OF ACTION OF CYCLIC 3’,5’-ADENOSINE MONOPHOSPHATE ON STEROID HYDROXYLATIONS IN ADRENAL HOMOGENATES,

    DTIC Science & Technology

    Cyclic 3’,5’-adenosine monophosphate (cyclic 3’,5’AMP) has recently been shown to stimulate selectively steroid C-11- beta hydroxylase activity in rat...to be mediated via stimulation of alpha- glucan phosphorylase, which in turn led to enhanced production of G-6-P from glycogen and a concomitant...increase in NADPH generation. However, if cyclic 3’,5’-AMP stimulated steroid 11- beta -hydroxylation in adrenal homogenates only by this mechanism, its

  14. Host layer buckling in the compounds formed by exfoliation and restacking of cadmium phosphorus trisulphide with adenosine monophosphate included

    SciTech Connect

    Westreich, Philippe . E-mail: pwestreich@alumni.sfu.ca; Yang Datong; Frindt, Robert F.

    2006-03-09

    Exfoliated single layer cadmium phosphorus trisulphide has been combined with the biological molecule adenosine monophosphate (AMP) to form the novel restacked compound Li{sub x}Cd{sub 0.8}PS{sub 3}(AMP){sub z}(H{sub 2}O){sub y}. Composition was determined using energy dispersive X-ray spectroscopy, and the structure of these compounds was studied using X-ray diffraction on oriented films. In the 0-80% relative humidity range, for (AMP){sub 0.5}, a host plane spacing near 19.6 A was found. Electron density calculations based on the X-ray diffraction pattern suggest a model for the arrangement of guest AMP molecules between the host layers, with an accompanying water molecule. The calculations also suggest that there is a buckling in the host layer of about {+-}0.6 A.

  15. Unhydrolyzable analogues of adenosine 3':5'-monophosphate demonstrating growth inhibition and differentiation in human cancer cells.

    PubMed

    Yokozaki, H; Tortora, G; Pepe, S; Maronde, E; Genieser, H G; Jastorff, B; Cho-Chung, Y S

    1992-05-01

    A set of adenosine 3':5'-monophosphate (cAMP) analogues that combine exocyclic sulfur substitutions in the equatorial (Rp) or the axial (Sp) position of the cyclophosphate ring with modifications in the adenine base of cAMP were tested for their effect on the growth of HL-60 human promyelocytic leukemia cells and LS-174T human colon carcinoma cells. Both diasteromeres of the phosphorothioate derivatives were growth inhibitory, exhibiting a concentration inhibiting 50% of cell proliferation of 3-100 microM. Among the analogues tested, Rp-8-Cl-cAMPS and Sp-8-Br-cAMPS were the two most potent. Rp-8-Cl-cAMPS was 5- to 10-fold less potent than 8-Cl-cAMP while Sp-8-Br-cAMPS was approximately 6-fold more potent than 8-Br-cAMP. The growth inhibition was not due to a block in a specific phase of the cell cycle or due to cytotoxicity. Rp-8-Cl-cAMPS enhanced its growth-inhibitory effect when added together with 8-Cl-cAMP and increased differentiation in combination with N6-benzyl-cAMP. The binding kinetics data showed that these Sp and Rp modifications brought about a greater decrease in affinity for Site B than for Site A of RI (the regulatory subunit of type I cAMP-dependent protein kinase) and a substantial decrease of affinity for Site A of RII (the regulatory subunit of type II protein kinase) but only a small decrease in affinity for Site B of RII, indicating the importance of the Site B binding of RII in the growth-inhibitory effect. These results show that the phosphorothioate analogues of cAMP are useful tools to investigate the mechanism of action of cAMP in growth control and differentiation and may have practical implication in the suppression of malignancy.

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

    SciTech Connect

    Francko, D.A.

    1980-01-01

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

  17. Aspirin-triggered resolvin D1 attenuates PDGF-induced vascular smooth muscle cell migration via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway

    PubMed Central

    Chatterjee, Anuran; Wu, Bian; Chen, Mian; Conte, Michael S.

    2017-01-01

    Background and objectives Resolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been previously shown to attenuate vascular smooth muscle cell (VSMC) migration, a key process in the development of intimal hyperplasia. We sought to investigate the role of the cAMP/PKA pathway in mediating the effects of the aspirin-triggered epimer 17R-RvD1 (AT-RvD1) on VSMC migration. Methods VSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity. Results Treatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor. Conclusions Our results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects

  18. Attempts to Detect Cyclic Adenosine 3′:5′-Monophosphate in Higher Plants by Three Assay Methods 12

    PubMed Central

    Bressan, Ray A.; Ross, Cleon W.; Vandepeute, Jozef

    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-8-14C 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

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

    SciTech Connect

    Francko, D.A.

    1980-01-01

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

  20. Ratiometric bioluminescence indicators for monitoring cyclic adenosine 3',5'-monophosphate in live cells based on luciferase-fragment complementation.

    PubMed

    Takeuchi, Masaki; Nagaoka, Yasutaka; Yamada, Toshimichi; Takakura, Hideo; Ozawa, Takeaki

    2010-11-15

    Bioluminescent indicators for cyclic 3',5'-monophosphate AMP (cAMP) are powerful tools for noninvasive detection with high sensitivity. However, the absolute photon counts are affected substantially by adenosine 5'-triphosphate (ATP) and d-luciferin concentrations, limiting temporal analysis in live cells. This report describes a genetically encoded bioluminescent indicator for detecting intracellular cAMP based on complementation of split fragments of two-color luciferase mutants originated from click beetles. A cAMP binding domain of protein kinase A was connected with an engineered carboxy-terminal fragment of luciferase, of which ends were connected with amino-terminal fragments of green luciferase and red luciferase. We demonstrated that the ratio of green to red bioluminescence intensities was less influenced by the changes of ATP and d-luciferin concentrations. We also showed an applicability of the bioluminescent indicator for time-course and quantitative assessments of intracellular cAMP in living cells and mice. The bioluminescent indicator will enable quantitative analysis and imaging of spatiotemporal dynamics of cAMP in opaque and autofluorescent living subjects.

  1. Cyclic adenosine monophosphate-dependent mechanisms induce von Willebrand factor expression in the Dami megakaryoblastic cell line.

    PubMed

    Deguine, V; Kerbiriou-Nabias, D; Lecoq, D; Greenberg, S M; Meyer, D; Dosne, A M

    1995-02-01

    It has been proposed that cyclic adenosine monophosphate (cAMP) is involved in the differentiation of several cell types and this study analysed whether von Willebrand factor (vWf) synthesis, which is a marker of the megakaryocyte maturation of these cells, would be enhanced by agents acting on cAMP formation. Different compounds known to stimulate cAMP accumulation in cells were used: dibutyryl cAMP (db-cAMP), isobutyl-methylxanthine (IBMX) or pentoxifylline (PTX) and forskolin. Treatments with db-cAMP or IBMX (10-1,000 microM) induced a dose-dependent increase in vWf synthesis. Associations of IBMX with forskolin produced a synergistic enhancement in vWf synthesis. PTX alone did not enhance vWf synthesis but a latent effect was revealed in the presence of forskolin or db-cAMP. The increase in vWf mRNA shown by Northern blot analysis demonstrates that the protein synthesis correlates with the transcript expression after db-cAMP or IBMX treatments. vWf synthesis paralleled the accumulation of cAMP in the cells. Moreover vWf expression induced by combination of IBMX with forskolin was associated with a moderate increase in the percentage of GPIIb/IIIa positive cells and in the ploidy level related to an important inhibition of cell growth. These data provide evidence that agents acting on cAMP metabolism induce vWf synthesis in the Dami megakaryoblastic cells.

  2. Hypothermia induced by adenosine 5'-monophosphate attenuates early stage injury in an acute gouty arthritis rat model.

    PubMed

    Miao, Zhimin; Guo, Weiting; Lu, Shulai; Lv, Wenshan; Li, Changgui; Wang, Yangang; Zhao, Shihua; Yan, Shengli; Tao, Zhenyin; Wang, Yunlong

    2013-08-01

    To investigate whether the hypothermia induced by Adenosine 5'-Monophosphate (5'-AMP) could attenuate early stage injury in a rat acute gouty arthritis model. Ankle joint injection with monosodium urate monohydrate crystals (MSU crystals) in hypothermia rat model which was induced by 5'-AMP and then observe whether hypothermia induced by 5'-AMP could be effectively inhibit the inflammation on acute gouty arthritis in rats. AMP-induced hypothermia has protective effects on our acute gouty arthritis, which was demonstrated by the following criteria: (1) a significant reduction in the ankle swelling (p < 0.001); (2) a significant decrease in the occurrence of leukocyte infiltration and mild hemorrhage; (3) a significant reduction in the presence of serum Interleukin-1β (IL-1β, p < 0.001) and metalloproteinase-9 (MMP-9, p < 0.001); and (4) a significant inhibition in the Nuclear Factor -κappaB (NF-κB) activity (p < 0.001). AMP-induced hypothermia could inhibit acute inflammation reaction and protect the synovial tissue against acute injury in a rat acute gouty arthritis model.

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

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

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

  4. Crystallization and preliminary X-ray crystallographic analysis of adenosine 5′-monophosphate deaminase (AMPD) from Arabidopsis thaliana in complex with coformycin 5′-phosphate

    SciTech Connect

    Han, Byung Woo; Bingman, Craig A.; Mahnke, Donna K.; Sabina, Richard L.; Phillips, George N. Jr

    2005-08-01

    Adenosine 5′-monophosphate deaminase from A. thaliana has been crystallized in complex with coformycin 5′-phosphate. Diffraction data have been collected to 3.34 Å resolution. Adenosine 5′-monophosphate deaminase (AMPD) is a eukaryotic enzyme that converts adenosine 5′-monophosphate (AMP) to inosine 5′-monophosphate (IMP) and ammonia. AMPD from Arabidopsis thaliana (AtAMPD) was cloned into the baculoviral transfer vector p2Bac and co-transfected along with a modified baculoviral genome into Spodoptera frugiperda (Sf9) cells. The resulting recombinant baculovirus were plaque-purified, amplified and used to overexpress recombinant AtAMPD. Crystals of purified AtAMPD have been obtained to which coformycin 5′-phosphate, a transition-state inhibitor, is bound. Crystals belong to space group P6{sub 2}22, with unit-cell parameters a = b = 131.325, c = 208.254 Å, α = β = 90, γ = 120°. Diffraction data were collected to 3.34 Å resolution from a crystal in complex with coformycin 5′-phosphate and to 4.05 Å resolution from a crystal of a mercury derivative.

  5. Intraventricular injection of agents that enhance cyclic adenosine monophosphate formation leads to inhibition of proestrous luteinizing hormone surge in rats.

    PubMed

    Taleisnik, S; Haymal, B; Caligaris, L

    1993-09-01

    The effect of increasing hypothalamic levels of 3',5'-cyclic adenosine monophosphate (cAMP) on the preovulatory surge of luteinizing hormone (LH) and ovulation was studied in cycling rats. Animals hearing chronically implanted guiding cannulae into the third ventricle were injected with agents known to enhance the cellular levels of cAMP. Hourly blood samples from the unanesthetized, unrestrained rats were obtained between 11.00 and 17.00 h through a plastic cannula inserted into the jugular vein. Intraventricular injections of serotonin (7.5 mg/ml; 2 microliters) in the morning of proestrous blocked the preovulatory surge of LH and ovulation. This effect was assigned to an increased neuronal level of cAMP because it was prevented by a serum anti-cAMP. Third-ventricle injections of 2 microliters of forskolin (0.5 mmol/l), guanosine 5'-O-(3-thiotriphosphate)(2 mmol/l) or dibutyryl-cAMP (1 mmol/l) at 11.00 h on the day of proestrus mimicked the inhibitory effect of serotonin on the proestrous release of LH. It is suggested that serotonin inhibits LH surge by acting directly on LH-releasing hormone neurons and/or on neurons that provide inputs to these neurons involving cAMP as a second messenger. Neurons releasing gamma-aminobutyric acid (GABA) may serve as interneurons sensitive to serotonin, as well as to cAMP, inasmuch as the inhibitory effect of forskolin on the release of LH was partially blocked by the GABA antagonists, picrotoxin and bicuculline.

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

    PubMed

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

    2016-09-01

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

  7. Effects of Catecholamines and their Interaction with Other Hormones on Cyclic 3′,5′-Adenosine Monophosphate of the Kidney

    PubMed Central

    Beck, Nama P.; Reed, Sarah W.; Murdaugh, H. V.; Davis, Bernard B.

    1972-01-01

    Catecholamines have several physiological effects on the kidney. These include: (a) stimulation of renin synthesis in the cortex: (b) antidiuresis by beta adrenergic agents; and (c) diuresis by alpha adrenergic stimulation. The role of cyclic 3′,5′-adenosine monophosphate (cyclic AMP) in the renal actions of catecholamines was evaluated by measuring the effects of several adrenergic agents on cyclic AMP concentration in the dog kidney. Beta adrenergic activity increased cyclic AMP concentration in the renal cortex, a finding consistent with the hypothesis that beta-adrenergic stimulation augments renin synthesis by increasing cyclic AMP generation. Beta adrenergic stimulation, like vasopressin, increased cyclic AMP concentration in the renal medulla. This suggests that beta adrenergic stimulation causes antidiuresis by augmenting cyclic AMP generation in the renal medulla. Alpha adrenergic activity inhibited the effect of vasopressin to stimulate cyclic AMP generation. These results support the hypothesis that the diuretic effect of alpha adrenergic stimulation is mediated by inhibition of the effect of vasopressin to increase cyclic AMP generation. PMID:4335447

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

    PubMed

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

    2015-10-01

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

  9. Phenotype, virulence and immunogenicity of Edwardsiella ictaluri cyclic adenosine 3',5'-monophosphate receptor protein (Crp) mutants in catfish host.

    PubMed

    Santander, Javier; Mitra, Arindam; Curtiss, Roy

    2011-12-01

    Edwardsiella ictaluri is an Enterobacteriaceae that causes lethal enteric septicemia in catfish. Being a mucosal facultative intracellular pathogen, this bacterium is an excellent candidate to develop immersion-oral live attenuated vaccines for the catfish aquaculture industry. Deletion of the cyclic 3',5'-adenosine monophosphate (cAMP) receptor protein (crp) gene in several Enterobacteriaceae has been utilized in live attenuated vaccines for mammals and birds. Here we characterize the crp gene and report the effect of a crp deletion in E. ictaluri. The E. ictaluri crp gene and encoded protein are similar to other Enterobacteriaceae family members, complementing Salmonella enterica Δcrp mutants in a cAMP-dependent fashion. The E. ictaluri Δcrp-10 in-frame deletion mutant demonstrated growth defects, loss of maltose utilization, and lack of flagella synthesis. We found that the E. ictaluri Δcrp-10 mutant was attenuated, colonized lymphoid tissues, and conferred immune protection against E. ictaluri infection to zebrafish (Danio rerio) and catfish (Ictalurus punctatus). Evaluation of the IgM titers indicated that bath immunization with the E. ictaluri Δcrp-10 mutant triggered systemic and skin immune responses in catfish. We propose that deletion of the crp gene in E. ictaluri is an effective strategy to develop immersion live attenuated antibiotic-sensitive vaccines for the catfish aquaculture industry.

  10. Application of graphene-ionic liquid-chitosan composite-modified carbon molecular wire electrode for the sensitive determination of adenosine-5'-monophosphate.

    PubMed

    Shi, Fan; Gong, Shixing; Xu, Li; Zhu, Huanhuan; Sun, Zhenfan; Sun, Wei

    2013-12-01

    In this paper, a graphene (GR) ionic liquid (IL) 1-octyl-3-methylimidazolium hexafluorophosphate and chitosan composite-modified carbon molecular wire electrode (CMWE) was fabricated by a drop-casting method and further applied to the sensitive electrochemical detection of adenosine-5'-monophosphate (AMP). CMWE was prepared with diphenylacetylene (DPA) as the modifier and the binder. The properties of modified electrode were examined by scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. Electrochemical behaviors of AMP was carefully investigated with enhanced responses appeared, which was due to the presence of GR-IL composite on the electrode surface with excellent electrocatalytic ability. A well-defined oxidation peak of AMP appeared at 1.314 V and the electrochemical parameters were calculated by electrochemical methods. Under the selected conditions, the oxidation peak current of AMP was proportional to its concentration in the range from 0.01 μM to 80.0 μM with the detection limit as 3.42 nM (3σ) by differential pulse voltammetry. The proposed method exhibited good selectivity and was applied to the detection of vidarabine monophosphate injection samples with satisfactory results.

  11. Separate 5-hydroxytryptamine receptors on the salivary gland of the blowfly are linked to the generation of either cyclic adenosine 3',5'-monophosphate or calcium signals.

    PubMed Central

    Berridge, M. J.; Heslop, J. P.

    1981-01-01

    1 5'-Hydroxytryptamine (5-HT) stimulates the formation of two separate second messengers in the salivary gland of the blowfly. Activation of adenylate cyclase raises adenosine 3',5'-monophosphate (cyclic AMP) whereas the hydrolysis of phosphatidylinositol (PI) is associated with an increase in calcium permeability. The possibility that these two signal pathways might be controlled by separate 5-HT receptors was studied by testing the specificity of 5-HT analogues and antagonists. 2 The parent compound 5-HT was found to stimulate both cyclic AMP formation and the related parameters of PI hydrolysis and calcium transport with similar dose-response relationships. 3 Certain analogues such as 4- and 5-fluoro-alpha-methyltryptamine were capable of raising cyclic AMP levels and stimulating fluid secretion but did not stimulate the hydrolysis of PI or the entry of calcium. 4 Other analogues, which had chloro or methyl substituents at the 5-position, were found to stimulate the hydrolysis of PI and the transport of calcium at much lower doses than those required to stimulate the formation of cyclic AMP. 5 Antagonists were also found to exert selective effects. Methysergide was a potent inhibitor of PI hydrolysis whereas cinanserin was far more selective in blocking the stimulatory effect of 5-HT on cyclic AMP formation. 6 It is concluded that 5-HT acts on two separate receptors, a 5-HT1 receptor acting through calcium and a 5-HT2 receptor which mediates its effects through cyclic AMP. PMID:6265018

  12. IMPDH2 genetic polymorphism: a promoter single-nucleotide polymorphism disrupts a cyclic adenosine monophosphate responsive element.

    PubMed

    Garat, Anne; Cauffiez, Christelle; Hamdan-Khalil, Rima; Glowacki, François; Devos, Aurore; Leclerc, Julie; Lionet, Arnaud; Allorge, Delphine; Lo-Guidice, Jean-Marc; Broly, Franck

    2009-12-01

    Inosine 5'-monophosphate dehydrogenase (IMPDH), which catalyzes a key step in the de novo biosynthesis of guanine nucleotide, is mediated by two highly conserved isoforms, IMPDH1 and IMPDH2. In this study, IMPDH2 genetic polymorphism was investigated in 96 individuals of Caucasian origin. Four single-nucleotide polymorphisms were identified, comprising one previously described single base-pair substitution in the close vicinity of the consensus donor splice site of intron 7 (IVS7+10T>C), and three novel polymorphisms, one silent substitution in exon 9 (c.915C>G), one single base-pair insertion (g.6971_6972insT) within the 3'-untranslated region of the gene, and one substitution located in the promoter region (c.-95T>G) in a transcription factor binding site CRE(A) (cyclic adenosine monophosphate [cAMP] response element). Considering the nature and location of this latter polymorphism, its functional relevance was examined by transfecting HEK293 and Jurkat cell lines with constructs of the related region of IMPDH2/luciferase reporter gene. The c.-95T>G mutation leads to a significant decrease of luciferase activity (HEK293: 55% decrease, p < 0.05; Jurkat: 65% decrease, p < 0.05) compared with the wild-type promoter sequence and, therefore, is likely to determine interindividual differences in IMPDH2 transcriptional regulation. These results might contribute to a better understanding of the variability in clinical outcome and dose adjustments of certain immunosuppressors that are metabolized through the IMPDH pathway or that are IMPDH inhibitors.

  13. Adenosine monophosphate-activated protein kinase activation and suppression of inflammatory response by cell stretching in rabbit synovial fibroblasts.

    PubMed

    Kunanusornchai, Wanlop; Muanprasat, Chatchai; Chatsudthipong, Varanuj

    2016-12-01

    Joint mobilization is known to be beneficial in osteoarthritis (OA) patients. This study aimed to investigate the effect of stretching on adenosine monophosphate-activated protein kinase (AMPK) activity and its role in modulating inflammation in rabbit synovial fibroblasts. Uniaxial stretching of isolated rabbit synovial fibroblasts for ten min was performed. Stretching-induced AMPK activation, its underlying mechanism, and its anti-inflammatory effect were investigated using Western blot. Static stretching at 20 % of initial length resulted in AMPK activation characterized by expression of phosphorylated AMPK and phosphorylated acetyl-Co A carboxylase. AMP-activated protein kinase phosphorylation peaked 1 h after stretching and declined toward resting activity. Using cell viability assays, static stretching did not appear to cause cellular damage. Activation of AMPK involves Ca(2+) influx via a mechanosensitive L-type Ca(2+) channel, which subsequently raises intracellular Ca(2+) and activates AMPK via Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ). Interestingly, stretching suppressed TNFα-induced expression of COX-2, iNOS, and phosphorylated NF-κB. These effects were prevented by pretreatment with compound C, an AMPK inhibitor. These results suggest that mechanical stretching suppressed inflammatory responses in synovial fibroblasts via a L-type Ca(2+)-channel-CaMKKβ-AMPK-dependent pathway which may underlie joint mobilization's ability to alleviate OA symptoms.

  14. GROWTH AND DEVELOPMENT SYMPOSIUM: Adenosine monophosphate-activated protein kinase and mitochondria in Rendement Napole pig growth.

    PubMed

    Scheffler, T L; Gerrard, D E

    2016-09-01

    The Rendement Napole mutation (RN-), which is well known to influence pork quality, also has a profound impact on metabolic characteristics of muscle. Pigs with RN- possess a SNP in the γ3 subunit of adenosine monophosphate (AMP)-activated protein kinase (AMPK); AMPK, a key energy sensor in skeletal muscle, modulates energy producing and energy consuming pathways to maintain cellular homeostasis. Importantly, AMPK regulates not only acute response to energy stress but also facilitates long-term adaptation via changes in gene and protein expression. The RN- allele increases AMPK activity, which alters the metabolic phenotype of skeletal muscle by increasing mitochondrial content and oxidative capacity. Fibers with greater oxidative capacity typically exhibit increased protein turnover and smaller fiber size, which indicates that RN- pigs may exhibit decreased efficiency and growth potential. However, whole body and muscle growth of RN- pigs appear similar to that of wild-type pigs and despite increased oxidative capacity, fibers maintain the capacity for hypertrophic growth. This indicates that compensatory mechanisms may allow RN- pigs to achieve rates of muscle growth similar to those of wild-type pigs. Intriguingly, lipid oxidation and mitochondria function are enhanced in RN- pig muscle. Thus far, characteristics of RN- muscle are largely based on animals near market weight. To better understand interaction between energy signaling and protein accretion in muscle, further work is needed to define age-dependent relationships between AMPK signaling, metabolism, and muscle growth.

  15. 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

  16. Mitogen-stimulated glucose transport in thymocytes. Possible role of Ca++ and antagonism by adenosine 3':5'-monophosphate

    PubMed Central

    1977-01-01

    The plant lectin, concanavalin A (Con-A), and the ionophore, A-23187 (specific for divalent cations), stimulated glucose transport in rat thymocytes. Con-A stimulation developed more slowly and was somewhat less extensive than that of stimulation developed more slowly and was somewhat less extensive than that of A-23187. Both responses showed saturation dose dependencies. The two responses were poorly additive, suggesting that A-23187 may saturate regulatory processes shared by the two stimulatory mechanisms. Doses of methylisobutylxanthine (MIX) and prostaglandin E2 which raised adenosine 3':5'-monophosphate (cAMP) levels in these cells also antagonized the Con-A stimulation of glucose transport but did not inhibit basal glucose transport or the A-23187 stimulation. Dibutyryl-cAMP and 8-bromo-cAMP also natagonized Con-A stimulation without inhibiting basal glucose transport. MIX antagonized high Con-A doses about as strongly as it did low Con-A doses, suggesting that MIX did not compete in the Con-A binding step or other process saturable by Con-A. [3H-A1Con-A binding was not affected by MIX. The stimulatory effects of Con-A and A-23187 were reduced by reduction of Ca++ in the medium. Both Con-A and A-23187 enhanced 45Ca++ influx and cellular Ca++ content. The A-23187 dose, which was saturating for glucose transport stimulation, enhanced Ca++ influx and cellular Ca++ content more than did the Con-A dose which was saturating for glucose transport stimulation. The dose fo MIX which specifically antagonized Con-A stimulation of glucose transport proved also to reduce Ca++ influx and cellular Ca++ in the presence of Con-A but not in the presence of A-23187. Thus, glucose transport correlates rather well with cellular Ca++. These results are compatible with the view that Ca++ in a cellular compartment can promote glucose transport, the Con-A's enhancement of Ca++ entry contributes to its stimulation of glucose transport, and the MIX antagonized Con-A action at least

  17. Metabolic syndrome: adenosine monophosphate-activated protein kinase and malonyl coenzyme A.

    PubMed

    Ruderman, Neil B; Saha, Asish K

    2006-02-01

    The metabolic syndrome can be defined as a state of metabolic dysregulation characterized by insulin resistance, central obesity, and a predisposition to type 2 diabetes, dyslipidemia, premature atherosclerosis, and other diseases. An increasing body of evidence has linked the metabolic syndrome to abnormalities in lipid metabolism that ultimately lead to cellular dysfunction. We review here the hypothesis that, in many instances, the cause of these lipid abnormalities could be a dysregulation of the adenosine monophosphate-activated protein kinase (AMPK)/malonyl coenzyme A (CoA) fuel-sensing and signaling mechanism. Such dysregulation could be reflected by isolated increases in malonyl CoA or by concurrent changes in malonyl CoA and AMPK, both of which would alter intracellular fatty acid partitioning. The possibility is also raised that pharmacological agents and other factors that activate AMPK and/or decrease malonyl CoA could be therapeutic targets.

  18. Adenosine 5′-monophosphate blocks acetaminophen toxicity by increasing ubiquitination-mediated ASK1 degradation

    PubMed Central

    Sun, Qi; Xu, Xi; Kong, Yi; Zhang, Jianfa

    2017-01-01

    Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver failure in the world. Hepatic c-jun NH2-terminal protein kinase (JNK) activation is thought to be a consequence of oxidative stress produced during APAP metabolism. Activation of JNK signals causes hepatocellular damage with necrotic and apoptotic cell death. Here we found that APAP caused a feedback increase in plasma adenosine 5′-monophsphate (5′-AMP). We demonstrated that co-administration of APAP and 5′-AMP significantly ameliorated APAP-induced hepatotoxicity in mice, without influences on APAP metabolism and its analgesic function. The mechanism of protection by 5′-AMP was through inhibiting APAP-induced activation of JNK, and attenuating downstream c-jun and c-fos gene expression. This was triggered by attenuating apoptosis signal-regulated kinase 1(ASK1) methylation and increasing ubiquitination-mediated ASK1 protein degradation. Our findings indicate that replacing the current APAP with a safe and functional APAP/5′-AMP formulation could prevent APAP-induced hepatotoxicity. PMID:28031524

  19. Release of the β-Galactosidase-Synthesizing System from Ultraviolet Catabolite Repression by Cyclic 3′,5′-Adenosine Monophosphate, Dark Repair, Photoreactivation, and Cold Treatment

    PubMed Central

    Swenson, P. A.

    1972-01-01

    Recovery from the inhibitory effect of ultraviolet irradiation on the induced synthesis of β-galactosidase was studied in Escherichia coli B/r. When irradiated cells (520 ergs/mm2 at 254 nm) were induced and incubated in minimal medium supplemented with Casamino Acids (conditions of catabolite repression), the ability to form enzyme was greatly reduced for about 100 min and then recovery began. The inhibition observed immediately after ultraviolet irradiation was partially reversed by cyclic 3′,5′-adenosine monophosphate (cyclic AMP) or by photoreactivation treatment. Inhibition was reduced if the cells were given cold treatment (5 C) before or during irradiation; the kinetics of induced enzyme formation in each case were similar to those of irradiated cells receiving cyclic AMP. These kinetics suggest that the cold treatments, like cyclic AMP, cause the release of the β-galactosidase-synthesizing system from catabolite repression. When irradiated cells were incubated for various times before cyclic AMP or photoreactivation treatment, some reversal of the inhibition of induced enzyme formation was obtained, but by 100 min the treatments were ineffective. Because 100 min was also the time at which dark recovery of enzyme formation began, the recovery process was interpreted to be the result of completion of DNA repair, which, in turn, released the β-galactosidase-synthesizing system from catabolite repression. PMID:4333380

  20. Probing the Interaction between a DNA Nucleotide (Adenosine-5'-Monophosphate Disodium) and Surface Active Ionic Liquids by Rotational Relaxation Measurement and Fluorescence Correlation Spectroscopy.

    PubMed

    Roy, Arpita; Banerjee, Pavel; Dutta, Rupam; Kundu, Sangita; Sarkar, Nilmoni

    2016-10-02

    This article demonstrates the interaction of a deoxyribonucleic acid (DNA) nucleotide, adenosine-5'-monophosphate disodium (AMP) with a cationic surface active ionic liquid (SAIL) 1-dodecyl-3-methylimidazoium chloride (C12mimCl) and an anionic SAIL, 1-butyl-3-methylimidazolium n-octylsulfate ([C4mim][C8SO4]). Dynamic light scattering (DLS) measurements and 1H NMR (nuclear magnetic resonance) studies indicate that substantial interaction is taking place among the DNA nucleotide, AMP and the SAILs. Moreover, cryogenic transmission electron microscopy (cryo-TEM) suggests that SAILs containing micellar assemblies are transformed into larger micellar assemblies in presence of DNA nucleotide. Additionally, the rotational motion of two oppositely charged molecules, Rhodamine 6G perchlorate (R6G) and Fluorescein sodium salt (Fl-Na) have been monitored in these aggregates. The rotational motion of R6G and Fl-Na differs significantly between SAILs micelles, and SAILs-AMP containing larger micellar aggregates. The effect of negatively charged DNA nucleotide (AMP) addition into the cationic and anionic SAILs is more prominent for the cationic charged molecule R6G than that of anionic probe Fl-Na due to the favourable electrostatic interaction between the AMP and cationic R6G. Moreover, the influence of the anionic DNA nucleotide on the cationic and anionic SAIL micelles is monitored through the variation of the lateral diffusion motion of oppositely charged probe molecules (R6G and Fl-Na) inside these aggregates. This variation in diffusion coefficient values also suggests that interaction pattern of these oppositely charged probes are different within the SAILs-nucleotide containing aggregates. Therefore, both rotational and translational diffusion measurements confirm that the DNA nucleotide (AMP) renders more rigid microenvironment within the micellar solution of SAILs.

  1. The antilipolytic agent 3,5-dimethylpyrazole inhibits insulin release in response to both nutrient secretagogues and cyclic adenosine monophosphate agonists in isolated rat islets.

    PubMed

    Masiello, P; Novelli, M; Bombara, M; Fierabracci, V; Vittorini, S; Prentki, M; Bergamini, E

    2002-01-01

    This study intended to test the hypothesis that intracellular lipolysis in the pancreatic beta cells is implicated in the regulation of insulin secretion stimulated by nutrient secretagogues or cyclic adenosine monophosphate (cAMP) agonists. Indeed, although lipid signaling molecules were repeatedly reported to influence beta-cell function, the contribution of intracellular triglycerides to the generation of these molecules has remained elusive. Thus, we have studied insulin secretion of isolated rat pancreatic islets in response to various secretagogues in the presence or absence of 3,5-dimethylpyrazole (DMP), a water-soluble and highly effective antilipolytic agent, as previously shown in vivo. In vitro exposure of islets to DMP resulted in an inhibition (by approximately 50%) of the insulin release stimulated not only by high glucose, but also by another nutrient secretagogue, 2-ketoisocaproate, as well as the cAMP agonists 3-isobutyl-1-methylxanthine and glucagon. The inhibitory effect of DMP, which was not due to alteration of islet glucose oxidation, could be reversed upon addition of sn-1,2-dioctanoylglycerol, a synthetic diglyceride, which activates protein kinase C. The results provide direct pharmacologic evidence supporting the concept that endogenous beta-cell lipolysis plays an important role in the generation of lipid signaling molecules involved in the control of insulin secretion in response to both fuel stimuli and cAMP agonists.

  2. Adenosine monophosphate-activated protein kinase-based classification of diabetes pharmacotherapy.

    PubMed

    Dutta, D; Kalra, S; Sharma, M

    2016-09-21

    The current classification of both diabetes and antidiabetes medication is complex, preventing a treating physician from choosing the most appropriate treatment for an individual patient, sometimes resulting in patient-drug mismatch. We propose a novel, simple systematic classification of drugs, based on their effect on adenosine monophosphate-activated protein kinase (AMPK). AMPK is the master regular of energy metabolism, an energy sensor, activated when cellular energy levels are low, resulting in activation of catabolic process, and inactivation of anabolic process, having a beneficial effect on glycemia in diabetes. This listing of drugs makes it easier for students and practitioners to analyze drug profiles and match them with patient requirements. It also facilitates choice of rational combinations, with complementary modes of action. Drugs are classified as stimulators, inhibitors, mixed action, possible action, and no action on AMPK activity. Metformin and glitazones are pure stimulators of AMPK. Incretin-based therapies have a mixed action on AMPK. Sulfonylureas either inhibit AMPK or have no effect on AMPK. Glycemic efficacy of alpha-glucosidase inhibitors, sodium glucose co-transporter-2 inhibitor, colesevelam, and bromocriptine may also involve AMPK activation, which warrants further evaluation. Berberine, salicylates, and resveratrol are newer promising agents in the management of diabetes, having well-documented evidence of AMPK stimulation medicated glycemic efficacy. Hence, AMPK-based classification of antidiabetes medications provides a holistic unifying understanding of pharmacotherapy in diabetes. This classification is flexible with a scope for inclusion of promising agents of future.

  3. 5'-adenosine monophosphate-activated protein kinase and the metabolic syndrome.

    PubMed

    Mor, Vijay; Unnikrishnan, M K

    2011-09-01

    Lifestyle changes such as physical inactivity combined with calorie-rich, low-fibre diets have triggered an explosive surge in metabolic syndrome, outlined as a cluster of heart attack risk factors such as insulin resistance, raised fasting plasma glucose, abdominal obesity, high cholesterol and high blood pressure. By acting as a master-switch of energy homeostasis and associated pathophysiological phenomena, 5'-adenosine monophosphate-activated protein kinase (AMPK) appears to orchestrate the adaptive physiology of energy deficit, suggesting that the sedentary modern human could be suffering from chronic suboptimal AMPK activation. Addressing individual targets with potent ligands with high specificity may be inappropriate (it has not yielded any molecule superior to the sixty year old metformin) because this strategy cannot address a cluster of interrelated pathologies. However, spices, dietary supplements and nutraceuticals attenuate the multiple symptoms of metabolic syndrome in a collective and perhaps more holistic fashion with fewer adverse events. Natural selection could have favoured races that developed a taste for spices and dietary supplements, most of which are not only antioxidants but also activators of AMPK. The review will outline the various biochemical mechanisms and pathophysiological consequences of AMPK activation involving the cluster of symptoms that embrace metabolic syndrome and beyond. Recent advances that integrate energy homeostasis with a number of overarching metabolic pathways and physiological phenomena, including inflammatory conditions, cell growth and development, malignancy, life span, and even extending into environmental millieu, as in obesity mediated by gut microflora and others will also be outlined.

  4. Cardiac myocyte–secreted cAMP exerts paracrine action via adenosine receptor activation

    PubMed Central

    Sassi, Yassine; Ahles, Andrea; Truong, Dong-Jiunn Jeffery; Baqi, Younis; Lee, Sang-Yong; Husse, Britta; Hulot, Jean-Sébastien; Foinquinos, Ariana; Thum, Thomas; Müller, Christa E.; Dendorfer, Andreas; Laggerbauer, Bernhard; Engelhardt, Stefan

    2014-01-01

    Acute stimulation of cardiac β-adrenoceptors is crucial to increasing cardiac function under stress; however, sustained β-adrenergic stimulation has been implicated in pathological myocardial remodeling and heart failure. Here, we have demonstrated that export of cAMP from cardiac myocytes is an intrinsic cardioprotective mechanism in response to cardiac stress. We report that infusion of cAMP into mice averted myocardial hypertrophy and fibrosis in a disease model of cardiac pressure overload. The protective effect of exogenous cAMP required adenosine receptor signaling. This observation led to the identification of a potent paracrine mechanism that is dependent on secreted cAMP. Specifically, FRET-based imaging of cAMP formation in primary cells and in myocardial tissue from murine hearts revealed that cardiomyocytes depend on the transporter ABCC4 to export cAMP as an extracellular signal. Extracellular cAMP, through its metabolite adenosine, reduced cardiomyocyte cAMP formation and hypertrophy by activating A1 adenosine receptors while delivering an antifibrotic signal to cardiac fibroblasts by A2 adenosine receptor activation. Together, our data reveal a paracrine role for secreted cAMP in intercellular signaling in the myocardium, and we postulate that secreted cAMP may also constitute an important signal in other tissues. PMID:25401477

  5. Effect of bucladesine, Pentoxifylline, and H-89 as cyclic adenosine monophosphate analog, phosphodiesterase and protein kinase A inhibitor on acute pain.

    PubMed

    Salehi, Forouz; Hosseini-Zare, Mahshid Sadat; Aghajani, Haleh; Seyedi, Yalda; Hosseini-Zare, Maryam Sadat; Sharifzadeh, Mohammad

    2017-03-07

    The aim of the present study was to determine the effects of Cyclic adenosine monophosphate (cAMP) and its dependent pathway on thermal nociception in a mouse model of acute pain. Here we studied the effect of H-89 (protein kinase A inhibitor), Bucladesine (Db-cAMP) (membrane permeable analog of cAMP) and pentoxifylline (PTX) (non-specific phosphodiesterase (PDE) inhibitor) on pain sensation. Different doses of H-89 (0.05, 0.1 and 0.5 mg/100g), PTX (5, 10 and 20 mg/100g), and Db-cAMP (50, 100 and 300 nM/mouse) were administered intraperitoneally (I.p.) 15 minutes before a tail-flick test. In combination groups, we injected the first and the second compound 30 and 15 minutes before the tail-flick test, respectively. I.p. administration of H-89 and PTX significantly decreased the thermal-induced pain sensation in their low applied doses. Bucladesine, however, decreased the pain sensation in a dose-dependent manner. The highest applied dose of H-89 (0.5 mg/100g) attenuated the anti-nociceptive effect of Db-cAMP in doses of 50 and 100 nM/mouse. Surprisingly, Db-cAMP decreased the anti-nociceptive effect of the lowest dose of H-89 (0.05mg/100g). All applied doses of PTX reduced the effect of 0.05mg/100g H-89 on pain sensation; however, the highest dose of H-89 compromised the anti-nociceptive effect of 20 mg/100g dose of PTX. Co-administration of Db-cAMP and PTX increased the anti-nociceptive effect of each compound on thermal-induced pain. In conclusion, PTX, H-89, and Db-cAMP affect the thermal-induced pain by probably interacting with intracellular cAMP and cGMP signaling pathways and cyclic nucleotide-dependent protein kinases. This article is protected by copyright. All rights reserved.

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

    PubMed Central

    Newby, A C

    1980-01-01

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

  7. 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.

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

    PubMed Central

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

    2016-01-01

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

  9. Evidence for a substrate cycle between AMP and adenosine in isolated hepatocytes.

    PubMed Central

    Bontemps, F; Van den Berghe, G; Hers, H G

    1983-01-01

    The effect of adenosine on the metabolism of prelabeled adenine nucleotides was investigated in isolated hepatocytes. Adenosine caused an approximately equal to 2-fold increase in the ATP content of the cells. This effect was in part counteracted by an increased rate of adenine nucleotide catabolism that could be explained by a stimulation of both AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) and the cytoplasmic 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) because of the increased concentration of ATP. The unexpected finding that labeled adenosine was formed immediately after the addition of the unlabeled nucleoside could be explained by the trapping effect of adenosine. An accumulation of labeled adenosine was observed also in the presence of 5-iodotubercidin, a potent inhibitor of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20). Under these conditions, there was a decrease in the concentration of ATP in the cell and a 2- to 3-fold increase in the rate of formation of allantoin. This formation of adenosine was only slightly decreased by inhibition of the membranous 5'-nucleotidase; it led to the accumulation of S-adenosylhomocysteine in the presence of coformycin and an excess of L-homocysteine. It was concluded that, under basal conditions, the cytoplasmic 5'-nucleotidase present in the liver cell continuously produces adenosine, which is immediately reconverted into AMP by adenosine kinase, without giving rise to allantoin. This futile cycle between AMP and adenosine amounts to at least 20 nmol/min per g of liver and, thus, exceeds the basic rate of allantoin formation. PMID:6304684

  10. Compartmentalized Cyclic Adenosine 3′,5′-Monophosphate at the Plasma Membrane Clusters PDE3A and Cystic Fibrosis Transmembrane Conductance Regulator into Microdomains

    PubMed Central

    Penmatsa, Himabindu; Zhang, Weiqiang; Yarlagadda, Sunitha; Li, Chunying; Conoley, Veronica G.; Yue, Junming; Bahouth, Suleiman W.; Buddington, Randal K.; Zhang, Guangping; Nelson, Deborah J.; Sonecha, Monal D.; Manganiello, Vincent; Wine, Jeffrey J.

    2010-01-01

    Formation of multiple-protein macromolecular complexes at specialized subcellular microdomains increases the specificity and efficiency of signaling in cells. In this study, we demonstrate that phosphodiesterase type 3A (PDE3A) physically and functionally interacts with cystic fibrosis transmembrane conductance regulator (CFTR) channel. PDE3A inhibition generates compartmentalized cyclic adenosine 3′,5′-monophosphate (cAMP), which further clusters PDE3A and CFTR into microdomains at the plasma membrane and potentiates CFTR channel function. Actin skeleton disruption reduces PDE3A–CFTR interaction and segregates PDE3A from its interacting partners, thus compromising the integrity of the CFTR-PDE3A–containing macromolecular complex. Consequently, compartmentalized cAMP signaling is lost. PDE3A inhibition no longer activates CFTR channel function in a compartmentalized manner. The physiological relevance of PDE3A–CFTR interaction was investigated using pig trachea submucosal gland secretion model. Our data show that PDE3A inhibition augments CFTR-dependent submucosal gland secretion and actin skeleton disruption decreases secretion. PMID:20089840

  11. [Cloning of the gene controlling catabolite repression with the participation of cyclic adenosine monophosphate in Escherichia coli K-12].

    PubMed

    Lisenkov, A F; Smirnov, Iu V; Sukhodolets, V V

    1983-05-01

    The crp gene coding for cyclic adenosine monophosphate receptor protein has been cloned on the vehicle pBR325 using restriction endonuclease PstI and the recipient strain C600 crp. The pCAP2 hybrid plasmid obtained has a molecular weight 7.0 MD and in the pBR325 with the insertion into a PstI site. Bacterial clones carrying pCAP2 restore Crp+ phenotype, as judged by the capacity of bacteria for utilization of various carbohydrates and by the activity of catabolite sensitive enzymes.

  12. A model for the chemical interactions of adenosine 3':5'-monophosphate with the R subunit of protein kinase type I. Refinement of the cyclic phosphate binding moiety of protein kinase type I.

    PubMed

    Jastorff, B; Hoppe, J; Morr, M

    1979-11-01

    The cAMP receptor site in the regulatory subunit of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase type I was mapped using analogues of cAMP in which the ribose phosphate moiety was systematically modified. Electronical alteration of the cyclophosphate ring at the 3' and 5' positions by sulfur and nitrogen decreased the affinity of these analogues towards the kinase. Substituents at these positions are not tolerated. Testing the separated diastereomers of derivatives in which one of the exocyclic oxygens at the phosphorus has been substituted by sulfur, it was found that one diastereoisomer is preferentially recognized. Based on these results it is proposed that the hydrophylic cyclic phosphate-ribose moiety of cAMP is bound to the kinase via its 3' and 5'-oxygens, the 2'-hydroxy group and the negative charge in a fixed position. Based on our and other published results it is further proposed, that the adenine moiety is bound in a hydrophobic cleft without any hydrogen bond interactions. The chemical interactions between cAMP and the R subunit of protein kinase type I differ from those found for the binding of cAMP to the chemoreceptor of Dictyostelium discoideum [18].

  13. Metabolite gene regulation: imidazole and imidazole derivatives which circumvent cyclic adenosine 3',5'-monophosphate in induction of the Escherichia coli L-arabinose operon.

    PubMed

    Kline, E L; Bankaitis, V A; Brown, C S; Montefiori, D C

    1980-02-01

    Imidazole, histidine, histamine, histidinol phosphate, urocanic acid, or imidazolepropionic acid were shown to induce the L-arabinose operon in the absence of cyclic adenosine 3',5'-monophosphate. Induction was quantitated by measuring the increased differential rate of synthesis of L-arabinose isomerase in Escherichia coli strains which carried a deletion of the adenyl cyclase gene. The crp gene product (cyclic adenosine 3',5'-monophosphate receptor protein) and the araC gene product (P2) were essential for induction of the L-arabinose operon by imidazole and its derivatives. These compounds were unable to circumvent the cyclic adenosine 3',5'-monophosphate in the induction of the lactose or the maltose operons. The L-arabinose regulon was catabolite repressed upon the addition of glucose to a strain carrying an adenyl cyclase deletion growing in the presence of L-arabinose with imidazole. These results demonstrated that several imidazole derivatives may be involved in metabolite gene regulation (23).

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

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

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

  15. Adenosine A2B-receptor-mediated cyclic AMP accumulation in primary rat astrocytes.

    PubMed Central

    Peakman, M. C.; Hill, S. J.

    1994-01-01

    1. The effects of adenosine receptor agonists and antagonists on the accumulation of cyclic AMP have been investigated in primary cultures of rat astrocytes. 2. Adenosine A2-receptor stimulation caused a concentration-dependent increase in the accumulation of [3H]-cyclic AMP in cells prelabelled with [3H]-adenine. The rank order of agonist potencies was 5'-N-ethylcarboxamidoadenosine (NECA; EC50 = 1 microM) > adenosine (EC50 = 5 microM) > 2-chloroadenosine (EC50 = 20 microM) >> CGS 21680 (EC50 > 10 microM). The presence of 0.5 microM dipyridamole, an adenosine uptake blocker, had no effect on the potency of adenosine. 3. The response to 10 microM NECA was antagonized in a concentration-dependent manner by the non-selective adenosine receptor antagonists, xanthine amine congener (apparent KD = 12 nM), PD 115,199 (apparent KD = 134 nM) and 8-phenyltheophylline (apparent KD = 126 nM). However, the A1-receptor-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine, had no significant effect on the responses to NECA or 2-chloroadenosine at concentrations up to 1 microM. 4. Stimulation of A1-receptors with the selective agonist, N6-cyclopentyladenosine, did not alter the basal accumulation of [3H]-cyclic AMP but inhibited a forskolin-mediated elevation of [3H]-cyclic AMP accumulation by a maximal value of 42%. This inhibition was fully reversed in the presence of 0.1 microM, 8-cyclopentyl-1,3-dipropylxanthine. 5. The time course for NECA-mediated [3H]-cyclic AMP accumulation was investigated. The results suggest that there is a substantial efflux of cyclic AMP from the cells in addition to the rapid and sustained elevation of intracellular cyclic AMP (5 fold over basal) which was also observed. 6. These data indicate that rat astrocytes in primary culture express an A2B-adenosine receptor coupled positively to adenylyl cyclase. Furthermore, the presence of A1-receptors negatively coupled to adenylyl cyclase appears to have no significant effect on the A2B

  16. Cyclic adenosine monophosphate metabolism in synaptic growth, strength, and precision: neural and behavioral phenotype-specific counterbalancing effects between dnc phosphodiesterase and rut adenylyl cyclase mutations.

    PubMed

    Ueda, Atsushi; Wu, Chun-Fang

    2012-03-01

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

  17. Cyclic adenosine monophosphate acutely inhibits and chronically stimulates Na/H antiporter in OKP cells.

    PubMed Central

    Cano, A; Preisig, P; Alpern, R J

    1993-01-01

    Parathyroid hormone, dopamine, alpha-adrenergic catecholamines, and angiotensin II regulate renal Na excretion, at least in part through modulation of acute cyclic (c)AMP-induced proximal tubule Na/H antiporter inhibition. The present studies examined the effect of chronic increases in cell cAMP on Na/H antiporter activity in OKP cells. Whereas 8-bromo cAMP acutely inhibited Na/H antiporter activity, chronic application for 6 h led to a 24% increase in Na/H antiporter activity measured 16-20 h after cAMP removal. This chronic persistent activation of the Na/H antiporter required > 2 h exposure. This effect was not a nonspecific effect of 8-bromo cAMP, in that addition of forskolin or forskolin + 3-isobutyl-1-methylxanthine for 6 h also led to a chronic persistent increase in Na/H antiporter activity. Inhibition of protein synthesis with cycloheximide prevented 8-bromo cAMP-induced Na/H antiporter stimulation. Although 8-bromo cAMP addition decreased cell pH by 0.15-0.20 pH U, Na/H antiporter stimulation could be dissociated from cell acidification. In summary, while cAMP acutely inhibits Na/H antiporter activity, it chronically increases antiporter activity. This chronic activation occurs with exogenous addition or endogenous generation of cAMP. These results imply that for hormones that modulate renal Na excretion and proximal tubule Na/H antiporter activity via cAMP and protein kinase A, acute effects may not predict chronic effects. PMID:7691881

  18. Viscothionin isolated from Korean mistletoe improves nonalcoholic fatty liver disease via the activation of adenosine monophosphate-activated protein kinase.

    PubMed

    Kim, Sokho; Lee, Dongho; Kim, Jae-Kyung; Kim, Jae-Hun; Park, Jong-Heum; Lee, Ju-Woon; Kwon, Jungkee

    2014-12-10

    The present study investigated the effects of viscothionin, a compound isolated from Korean mistletoe (Viscum album coloratum), on nonalcoholic fatty liver disease (NAFLD) in both in vitro and in vivo models. A connection was discovered between viscothionin and the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, which is involved in lipid metabolism. Viscothionin was shown to significantly attenuate lipid accumulation in HepG2 cells treated with oleic acid, which induces lipid accumulation. Moreover, the phosphorylation of AMPK and acetyl-coenzyme A carboxylase in HepG2 cells was increased by viscothionin treatment. Viscothionin was orally administered to high fat diet-induced obese mice and subsequently histopathological analysis associated with AMPK signaling pathways was evaluated. A significant reduction in the extent of hepatic steatosis was revealed in viscothionin-treated obese mice. Thus, viscothionin mediates its beneficial effects on NAFLD via AMPK signaling pathways, suggesting that it may be a potential target for novel NAFLD treatments.

  19. Electroacupuncture preconditioning attenuates ischemic brain injury by activation of the adenosine monophosphate-activated protein kinase signaling pathway

    PubMed Central

    Ran, Qiang-qiang; Chen, Huai-long; Liu, Yan-li; Yu, Hai-xia; Shi, Fei; Wang, Ming-shan

    2015-01-01

    Electroacupuncture has therapeutic effects on ischemic brain injury, but its mechanism is still poorly understood. In this study, mice were stimulated by electroacupuncture at the Baihui (GV20) acupoint for 30 minutes at 1 mA and 2/15 Hz for 5 consecutive days. A cerebral ischemia model was established by ligating the bilateral common carotid artery for 15 minutes. At 72 hours after injury, neuronal injury in the mouse hippocampus had lessened, and the number of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-positive cells reduced after electroacupuncture treatment. Moreover, expression of adenosine monophosphate-activated protein kinase α (AMPKα) and phosphorylated AMPKα was up-regulated. Intraperitoneal injection of the AMPK antagonist, compound C, suppressed this phenomenon. Our findings suggest that electroacupuncture preconditioning alleviates ischemic brain injury via AMPK activation. PMID:26330828

  20. Adenosine monophosphate-activated protein kinase: a central regulator of metabolism with roles in diabetes, cancer, and viral infection.

    PubMed

    Hardie, D G

    2011-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy sensor activated by metabolic stresses that inhibit catabolic ATP production or accelerate ATP consumption. Once activated, AMPK switches on catabolic pathways, generating ATP, while inhibiting cell growth and proliferation, thus promoting energy homeostasis. AMPK is activated by the antidiabetic drug metformin, and by many natural products including "nutraceuticals" and compounds used in traditional medicines. Most of these xenobiotics activate AMPK by inhibiting mitochondrial ATP production. AMPK activation by metabolic stress requires the upstream kinase, LKB1, whose tumor suppressor effects may be largely mediated by AMPK. However, many tumor cells appear to have developed mechanisms to reduce AMPK activation and thus escape its growth-restraining effects. A similar phenomenon occurs during viral infection. If we can establish how down-regulation occurs in tumors and virus-infected cells, there may be therapeutic avenues to reverse these effects.

  1. Sarcoplasmic reticulum-associated cyclic adenosine 5'-monophosphate phosphodiesterase activity in normal and failing human hearts.

    PubMed Central

    Movsesian, M A; Smith, C J; Krall, J; Bristow, M R; Manganiello, V C

    1991-01-01

    Sarcoplasmic reticulum-associated cAMP phosphodiesterase activity was examined in microsomes prepared from the left ventricular myocardium of eight heart transplant recipients with end-stage idiopathic dilated cardiomyopathy and six unmatched organ donors with normal cardiac function. At cAMP concentrations less than or equal to 1.0 microM, sarcoplasmic reticulum-associated cAMP phosphodiesterase activity was functionally homogeneous. cAMP phosphodiesterase activity was inhibited competitively by cGMP (Ki = 0.031 +/- 0.008 microM) and the cilostamide derivative OPC 3911 (Ki = 0.018 +/- 0.004 microM), but was essentially insensitive to rolipram. Vmax and Km were 781.7 +/- 109.2 nmol/mg per min and 0.188 +/- 0.031 microM, respectively, in microsomes prepared from nonfailing hearts and 793.9 +/- 68.9 nmol/mg per min and 0.150 +/- 0.027 microM in microsomes prepared from failing hearts. Microsomes prepared from nonfailing and failing hearts did not differ with respect to either the ratio of cAMP phosphodiesterase activity to ATP-dependent Ca2+ accumulation activity or the sensitivity of cAMP phosphodiesterase activity to inhibition by OPC 3911. These data suggest that the diminished inotropic efficacy of phosphodiesterase inhibitors in failing human hearts does not result from changes in the level, kinetic properties, or pharmacologic sensitivity of sarcoplasmic reticulum-associated cAMP phosphodiesterase activity. PMID:1647414

  2. Relaxin-induced changes in adenosine 3',5'-monophosphate levels in the human cervix.

    PubMed

    Norström, A; Wiqvist, I

    1985-05-01

    The effects of porcine relaxin on the levels of cAMP in human cervical tissue were studied in vitro. The specimens were obtained by needle biopsy from women undergoing hysterectomy, legal abortion in the first trimester or elective Caesarean section at term, and were incubated in Krebs-Ringer buffer for 15 min in the presence of porcine relaxin (5 micrograms/ml, 3000 GPU/mg). cAMP was determined using a modified protein binding assay. The concentration of cAMP was higher in pregnant than in non-pregnant women. Relaxin stimulated the production of cAMP in the 7th-8th week of gestation and at term but did not significantly alter the cervical cAMP levels in neither non-pregnant women nor in women in the 10th-12th week of pregnancy. Previous studies have shown that porcine relaxin reduces collagen synthesis in tissue from the human cervix and lower uterine segment. The present observations indicate that these effects can be mediated by cAMP.

  3. Differential regulation of kit ligand A (kitlga) expression in the zebrafish ovarian follicle cells--evidence for the existence of a cyclic adenosine 3', 5' monophosphate-mediated binary regulatory system during folliculogenesis.

    PubMed

    Yao, Kai; Ge, Wei

    2015-02-15

    Kit ligand (Kitl) is an important paracrine factor involved in the activation of primordial follicles from the quiescent pool and in the maintenance of meiotic arrest before germinal vesicle breakdown (GVBD). It has been reported that follicle-stimulating hormone (FSH) stimulates but luteinizing hormone (LH) suppresses the expression of Kitl in the granulosa cells in mammals. Considering that both gonadotropins signal in the follicle cells mainly by activating cyclic adenosine 3', 5'-monophosphate (cAMP) pathway, we are intrigued by how cAMP differentially regulates Kitl expression. In the present study, we demonstrated that both human chorionic gonadotropin (hCG) and pituitary adenylate cyclase activating polypeptide (PACAP) inhibited insulin-like growth factor I (IGF-I)-induced Akt phosphorylation and kitlga expression in the zebrafish follicle cells. Further experiments showed that cAMP was involved in regulating the expression of kitlga. However, two cAMP-activated effectors, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), had converse effects. PKA promoted whereas Epac inhibited the expression of kitlga, as demonstrated by the respective activators. Interestingly, cAMP also appeared to exert differential effects on kitlga expression at different stages of follicle development during folliculogenesis, significantly stimulating kitlga expression at the early growth stage but suppressing it at the full-grown stage before final oocyte maturation, implying a potential mechanism for differential effects of the same pathway at different stages. The inhibitory effect of forskolin (activator of adenylate cyclase) and H89 (inhibitor of PKA) on IGF-I-induced expression of kitlga suggested cross-talk between the cAMP and IGF-I-activated PI3K-Akt pathways. This study, together with our previous findings on IGF-I regulation of kitlga expression, provides important clues to the underlying mechanism that regulates Kit ligand expression during

  4. Comparison of three inhaled non-steroidal anti-inflammatory drugs on the airway response to sodium metabisulphite and adenosine 5'-monophosphate challenge in asthma.

    PubMed Central

    Wang, M.; Wisniewski, A.; Pavord, I.; Knox, A.; Tattersfield, A.

    1996-01-01

    BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) are used to assess the role of prostaglandins in asthma but their effects on bronchoconstrictor challenges have been inconsistent. The effects of three nebulised nonsteroidal anti-inflammatory drugs on the airway response to inhaled sodium metabisulphite (MBS) and adenosine 5'-monophosphate (AMP) were compared in the same asthmatic subjects to see whether contractile prostaglandins were involved in MBS or AMP induced bronchoconstriction. A possible protective effect of the osmolarity or pH of the inhaled solutions was also assessed. METHODS: Two double blind placebo controlled studies were carried out. In study 1, 15 non-aspirin sensitive patients with mild asthma attended on four occasions and inhaled 5 ml of lysine aspirin (L-aspirin) 900 mg, indomethacin 50 mg, sodium salicylate 800 mg, or saline 20 minutes before an inhaled MBS challenge. On four further occasions 14 of the patients inhaled the same solutions followed by an inhaled AMP challenge. In study 2, 10 of the patients attended on four additional occasions and inhaled 5 ml of 0.9%, 3%, 10%, or 9.5% saline with indomethacin 50 mg 20 minutes before an inhaled MBS challenge. RESULTS: In study 1 inhaled lysine aspirin had a similar effect on MBS and AMP induced bronchoconstriction, increasing the provocative dose causing a 20% fall in FEV1 (PD20) by 1.29 (95% CI 0.54 to 2.03) and 1.23 (95% CI 0.53 to 1.93) doubling doses, respectively. Indomethacin increased the MBS PD20 and AMP PD20 by 0.64 (95% CI -0.1 to 1.38) and 0.99 (95% CI 0.29 to 1.69) doubling doses, respectively. Sodium salicylate had no significant effect on either challenge. The two solutions causing most inhibition were the most acidic and the most alkaline. In study 2 inhaled 9.5% saline with indomethacin (osmolarity 3005 mOsm/kg) increased the MBS PD20 by 1.1 doubling doses (95% CI 0.2 to 2.0) compared with only 0.09 (95% CI -0.83 to 1.0) and 0.04 (95% CI -0.88 to 0.95) doubling doses

  5. Magnesium sulphate increases lymphocyte adenosine 3':5'-cyclic monophosphate in humans.

    PubMed Central

    Von Mandach, U; Bürgi, M; Huch, R; Huch, A

    1993-01-01

    We determined the effect of i.v. magnesium sulphate, which is often combined with beta 2-adrenoceptor agonists for tocolytic therapy, on lymphocyte cyclic AMP production, extracellular magnesium and blood calcium concentrations. Sixteen healthy volunteers received i.v. magnesium sulphate 1 g h-1 over 8 h; seven volunteers also had infusion of NaCl 18 mg h-1 as control. Venous blood was taken pre- and post-infusion to determine basal lymphocyte cyclic AMP and the increase evoked by 0.1 mM isoprenaline, as well as serum and plasma concentrations of total and non-protein-bound magnesium and calcium. Following magnesium sulphate there was a significant rise in the isoprenaline-evoked increase in cyclic AMP (P < 0.05) and in the magnesium concentrations (P < 0.01) and a decrease in the free calcium concentration (P < 0.01). PMID:8385975

  6. Magnesium sulphate increases lymphocyte adenosine 3':5'-cyclic monophosphate in humans.

    PubMed

    Von Mandach, U; Bürgi, M; Huch, R; Huch, A

    1993-03-01

    We determined the effect of i.v. magnesium sulphate, which is often combined with beta 2-adrenoceptor agonists for tocolytic therapy, on lymphocyte cyclic AMP production, extracellular magnesium and blood calcium concentrations. Sixteen healthy volunteers received i.v. magnesium sulphate 1 g h-1 over 8 h; seven volunteers also had infusion of NaCl 18 mg h-1 as control. Venous blood was taken pre- and post-infusion to determine basal lymphocyte cyclic AMP and the increase evoked by 0.1 mM isoprenaline, as well as serum and plasma concentrations of total and non-protein-bound magnesium and calcium. Following magnesium sulphate there was a significant rise in the isoprenaline-evoked increase in cyclic AMP (P < 0.05) and in the magnesium concentrations (P < 0.01) and a decrease in the free calcium concentration (P < 0.01).

  7. Isolation of a glycogen synthase I kinase that is independent of adenosine 3':5'-monophosphate.

    PubMed Central

    Schlender, K K; Reimann, E M

    1975-01-01

    Three protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) were detected when the soluble fraction of rabbit kidney medulla was chromatographed on DEAE-cellulose with a linear NaC1 gradient. The first two kinases eluted (Peak 1 and Peak II) were cyclic-AMP-dependent, wheras Peak III was cyclic-AMP-independent. A procedure was developed to separate the catalytic subunit of Peak II cyclic-AMP-dependent protein kinase (representing the bulk of the histone kinase activity) from Peak III protein kinase. In contrast to the catalytic subunit, Peak III protein kinase phosphorylated casein more rapidly than histone. Peak III was insensitive to the heat-stable protein inhibitor of cyclic-AMP-dependent protein kinases and appeared to have a higher requirement for ATP than did the catalytic subunit. Peak III catalyzed the conversion of glycogen synthase (UDPglucose:glycogen alpha-4-glucosyltransferase, EC 2.4.1.11) from the I (glucose-6-phosphate-independent) to the D (glucose-6-phosphate-dependent) form. This conversion was dependent on Mg-2+ and ATP and was unaffected by cyclic AMP, cyclic GMP, or the protein inhibitor. Glycogen synthase I in the soluble fraction of kidney medulla could be converted to the D form by endogenous glycogen synthase I kinase if Mg-2+ and ATP were added. Most of this glycogen synthase I kinase activity was unaffected by cyclic AMP or by the protein inhibitor, suggesting that Peak III may be of major importance in the regulation of glycogen synthase in vivo. PMID:166380

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-08-01

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

  10. Regulation of 5'-adenosine monophosphate deaminase in the freeze tolerant wood frog, Rana sylvatica

    PubMed Central

    Dieni, Christopher A; Storey, Kenneth B

    2008-01-01

    Background The wood frog, Rana sylvatica, is one of a few vertebrate species that have developed natural freeze tolerance, surviving days or weeks with 65–70% of its total body water frozen in extracellular ice masses. Frozen frogs exhibit no vital signs and their organs must endure multiple stresses, particularly long term anoxia and ischemia. Maintenance of cellular energy supply is critical to viability in the frozen state and in skeletal muscle, AMP deaminase (AMPD) plays a key role in stabilizing cellular energetics. The present study investigated AMPD control in wood frog muscle. Results Wood frog AMPD was subject to multiple regulatory controls: binding to subcellular structures, protein phosphorylation, and effects of allosteric effectors, cryoprotectants and temperature. The percentage of bound AMPD activity increased from 20 to 35% with the transition to the frozen state. Bound AMPD showed altered kinetic parameters compared with the free enzyme (S0.5 AMP was reduced, Hill coefficient fell to ~1.0) and the transition to the frozen state led to a 3-fold increase in S0.5 AMP of the bound enzyme. AMPD was a target of protein phosphorylation. Bound AMPD from control frogs proved to be a low phosphate form with a low S0.5 AMP and was phosphorylated in incubations that stimulated PKA, PKC, CaMK, or AMPK. Bound AMPD from frozen frogs was a high phosphate form with a high S0.5 AMP that was reduced under incubation conditions that stimulated protein phosphatases. Frog muscle AMPD was activated by Mg·ATP and Mg·ADP and inhibited by Mg·GTP, KCl, NaCl and NH4Cl. The enzyme product, IMP, uniquely inhibited only the bound (phosphorylated) enzyme from muscle of frozen frogs. Activators and inhibitors differentially affected the free versus bound enzyme. S0.5 AMP of bound AMPD was also differentially affected by high versus low assay temperature (25 vs 5°C) and by the presence/absence of the natural cryoprotectant (250 mM glucose) that accumulates during freezing

  11. Coordinatively Unsaturated Lanthanide(III) Helicates: Luminescence Sensors for Adenosine Monophosphate in Aqueous Media.

    PubMed

    Sahoo, Jashobanta; Arunachalam, Rajendran; Subramanian, Palani S; Suresh, Eringathodi; Valkonen, Arto; Rissanen, Kari; Albrecht, Markus

    2016-08-08

    Coordinatively unsaturated double-stranded helicates [(H2 L)2 Eu2 (NO3 )2 (H2 O)4 ](NO3 )4 , [(H2 L)2 Tb2 (H2 O)6 ](NO3 )6 , and [(H2 L)2 Tb2 (H2 O)6 ]Cl6 (H2 L=butanedioicacid-1,4-bis[2-(2-pyridinylmethylene)hydrazide]) are easily obtained by self-assembly from the ligand and the corresponding lanthanide(III) salts. The complexes are characterized by X-ray crystallography showing the helical arrangement of the ligands. Co-ligands at the metal ions can be easily substituted by appropriate anions. A specific luminescence response of AMP in presence of ADP, ATP, and other anions is observed. Specificity is assigned to the perfect size match of AMP to bridge the two metal centers and to replace quenching co-ligands in the coordination sphere.

  12. Chemoattraction and chemotaxis in Dictyostelium discoideum: myxamoeba cannot read spatial gradients of cyclic adenosine monophosphate.

    PubMed

    Vicker, M G; Schill, W; Drescher, K

    1984-06-01

    Myxamoebae of the morphogenetic cellular slime mold Dictyostelium discoideum are thought to be able to accurately read and respond to directional information in spatial gradients of cyclic AMP. We examined the spatial and temporal mechanisms proposed for chemotaxis by comparing the behavior of spreading or evenly distributed cell populations after exposure to well-defined spatial gradients. The effects of gradient generation on cells were avoided by using predeveloped gradients. Qualitatively different responses were obtained using (a) isotropic, (b) static spatial, or (c) temporal (impulse) gradients in a simple chamber of penetrable micropore filters. We simulated models of chemotaxis and chemokinesis to aid our interpretations. The attractive and locomotory responses of populations were maximally stimulated by 0.05 microM cyclic AMP, provided that cellular phosphodiesterase was inhibited. But a single impulse of cyclic AMP during gradient development caused a greater and qualitatively different attraction. Attraction in spatial gradients was only transient, in that populations eventually developed a random distribution when confined to a narrow territory. Populations never accumulated nor lost their random distribution even in extremely steep spatial gradients. Attraction in spatial gradients was inducible only in spreading populations, not randomly distributed ones. Thus, spatial gradients effect biased-random locomotion: i.e., chemokinesis without adaptation. Cells cannot read gradients; the reaction of the cells is stochastic. Spatial gradients do not cause chemotaxis, which probably requires a sharp stimulant concentration increase (a temporal gradient) as a pulse or impulse. The results also bear on concepts of how embryonic cells might be able to decipher the positional information in a morphogen spatial gradient during development.

  13. Chemoattraction and chemotaxis in Dictyostelium discoideum: myxamoeba cannot read spatial gradients of cyclic adenosine monophosphate

    PubMed Central

    1984-01-01

    Myxamoebae of the morphogenetic cellular slime mold Dictyostelium discoideum are thought to be able to accurately read and respond to directional information in spatial gradients of cyclic AMP. We examined the spatial and temporal mechanisms proposed for chemotaxis by comparing the behavior of spreading or evenly distributed cell populations after exposure to well-defined spatial gradients. The effects of gradient generation on cells were avoided by using predeveloped gradients. Qualitatively different responses were obtained using (a) isotropic, (b) static spatial, or (c) temporal (impulse) gradients in a simple chamber of penetrable micropore filters. We simulated models of chemotaxis and chemokinesis to aid our interpretations. The attractive and locomotory responses of populations were maximally stimulated by 0.05 microM cyclic AMP, provided that cellular phosphodiesterase was inhibited. But a single impulse of cyclic AMP during gradient development caused a greater and qualitatively different attraction. Attraction in spatial gradients was only transient, in that populations eventually developed a random distribution when confined to a narrow territory. Populations never accumulated nor lost their random distribution even in extremely steep spatial gradients. Attraction in spatial gradients was inducible only in spreading populations, not randomly distributed ones. Thus, spatial gradients effect biased-random locomotion: i.e., chemokinesis without adaptation. Cells cannot read gradients; the reaction of the cells is stochastic. Spatial gradients do not cause chemotaxis, which probably requires a sharp stimulant concentration increase (a temporal gradient) as a pulse or impulse. The results also bear on concepts of how embryonic cells might be able to decipher the positional information in a morphogen spatial gradient during development. PMID:6327727

  14. Methylene blue induces macroautophagy through 5' adenosine monophosphate-activated protein kinase pathway to protect neurons from serum deprivation.

    PubMed

    Xie, Luokun; Li, Wenjun; Winters, Ali; Yuan, Fang; Jin, Kunlin; Yang, Shaohua

    2013-01-01

    Methylene blue has been shown to be neuroprotective in multiple experimental neurodegenerative disease models. However, the mechanisms underlying the neuroprotective effects have not been fully elucidated. Previous studies have shown that macroautophagy has multiple beneficial roles for maintaining normal cellular homeostasis and that induction of macroautophagy after myocardial ischemia is protective. In the present study we demonstrated that methylene blue could protect HT22 hippocampal cell death induced by serum deprivation, companied by induction of macroautophagy. We also found that methylene blue-mediated neuroprotection was abolished by macroautophagy inhibition. Interestingly, 5' adenosine monophosphate-activated protein kinase (AMPK) signaling, but not inhibition of mammalian target of rapamycin signaling, was activated at 12 and 24 h after methylene blue treatment in a dose-dependent manner. Methylene blue-induced macroautophagy was blocked by AMPK inhibitor. Consistent with in vitro data, macroautophagy was induced in the cortex and hippocampus of mouse brains treated with methylene blue. Our findings suggest that methylene blue-induced neuroprotection is mediated, at least in part, by macroautophagy though activation of AMPK signaling.

  15. Adenosine monophosphate-activated kinase and its key role in catabolism: structure, regulation, biological activity, and pharmacological activation.

    PubMed

    Krishan, Sukriti; Richardson, Des R; Sahni, Sumit

    2015-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy sensor, which once activated, plays a role in several processes within the cell to restore energy homeostasis. The protein enhances catabolic pathways, such as β-oxidation and autophagy, to generate ATP, and inhibits anabolic processes that require energy, including fatty acid, cholesterol, and protein synthesis. Due to its key role in the regulation of critical cellular pathways, deregulation of AMPK is associated with the pathology of many diseases, including cancer, Wolff-Parkinson-White syndrome, neurodegenerative disorders, diabetes, and metabolic syndrome. In fact, AMPK is a target of some pharmacological agents implemented in the treatment of diabetes (metformin and thiazolidinediones) as well as other naturally derived products, such as berberine, which is used in traditional medicine. Due to its critical role in the cell and the pathology of several disorders, research into developing AMPK as a therapeutic target is becoming a burgeoning and exciting field of pharmacological research. A profound understanding of the regulation and activity of AMPK would enhance its development as a promising therapeutic target.

  16. Adenosine monophosphate-activated protein kinase attenuates cardiomyocyte hypertrophy through regulation of FOXO3a/MAFbx signaling pathway.

    PubMed

    Chen, Baolin; Wu, Qiang; Xiong, Zhaojun; Ma, Yuedong; Yu, Sha; Chen, Dandan; Huang, Shengwen; Dong, Yugang

    2016-09-01

    Control of cardiac muscle mass is thought to be determined by a dynamic balance of protein synthesis and degradation. Recent studies have demonstrated that atrophy-related forkhead box O 3a (FOXO3a)/muscle atrophy F-box (MAFbx) signaling pathway plays a central role in the modulation of proteolysis and exert inhibitory effect on cardiomyocyte hypertrophy. In this study, we tested the hypothesis that adenosine monophosphate-activated protein kinase (AMPK) activation attenuates cardiomyocyte hypertrophy by regulating FOXO3a/MAFbx signaling pathway and its downstream protein degradation. The results showed that activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) attenuated cardiomyocyte hypertrophy induced by angiotensin II (Ang II). The antihypertrophic effects of AICAR were blunted by AMPK inhibitor Compound C. In addition, AMPK dramatically increased the activity of transcription factor FOXO3a, up-regulated the expression of its downstream ubiquitin ligase MAFbx, and enhanced cardiomyocyte proteolysis. Meanwhile, the effects of AMPK on protein degradation and cardiomyocyte hypertrophy were blocked after MAFbx was silenced by transfection of cardiomyocytes with MAFbx-siRNA. These results indicate that AMPK plays an important role in the inhibition of cardiomyocyte hypertrophy by activating protein degradation via FOXO3a/MAFbx signaling pathway.

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

    PubMed Central

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

    2016-01-01

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

  18. Glicentin and oxyntomodulin modulate both the phosphoinositide and cyclic adenosine monophosphate signaling pathways in gastric myocytes.

    PubMed

    Rodier, G; Magous, R; Mochizuki, T; Le Nguyen, D; Martinez, J; Bali, J P; Bataille, D; Jarrousse, C; Geneviève, R

    1999-01-01

    We have investigated the transduction pathways mediating the contractile effect of two glucagon-containing peptides, glicentin (GLIC) and oxyntomodulin (OXM), on smooth muscle cells isolated from rabbit antrum. Low concentrations of GLIC induced a biphasic and rapid (first phase at 5-8 sec) Ins(1,4,5)P3 production. By comparison, higher concentrations of OXM or OXM(19-37) were required to obtain biphasic time-courses of Ins(1,4,5)P3 production. In a Ca2+ free medium, the first phase of Ins(1,4,5)P3 production induced by GLIC or OXM was maintained, while the second phase disappeared. In saponin-permeabilized cells, all three peptides induced cell contraction with similar efficacies and potencies. Exogenous Ins(1,4,5)P3 mimicked the contractile effect of the peptides and heparin, which inhibits the Ins(1,4,5)P3 binding to its receptor, prevented contraction stimulated by each effector. We conclude that a Ca2+ mobilization from the intracellular stores is essential in the contractile effects of GLIC and OXM. Using the fluo-3 probe, a [Ca2+]i increase was observed in the presence of GLIC, OXM, or OXM(19-37). The three peptides reduced by 30-40% the cAMP content of cells stimulated by forskolin. This effect was pertussis toxin sensitive as demonstrated with OXM(19-37). Our data constitute important clues for the existence in smooth muscle cells of receptor(s) specific for the GLIC/OXM hormones, coupled via G protein(s) to both Ca2+ and cAMP pathways.

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

    SciTech Connect

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

    1987-03-01

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

  20. Regulation of cyclic AMP formation in cultures of human foetal astrocytes by beta 2-adrenergic and adenosine receptors.

    PubMed

    Woods, M D; Freshney, R I; Ball, S G; Vaughan, P F

    1989-09-01

    Two cell cultures, NEP2 and NEM2, isolated from human foetal brain have been maintained through several passages and found to express some properties of astrocytes. Both cell cultures contain adenylate cyclase stimulated by catecholamines with a potency order of isoprenaline greater than adrenaline greater than salbutamol much greater than noradrenaline, which is consistent with the presence of beta 2-adrenergic receptors. This study reports that the beta 2-adrenergic-selective antagonist ICI 118,551 is approximately 1,000 times more potent at inhibiting isoprenaline stimulation of cyclic AMP (cAMP) formation in both NEP2 and NEM2 than the beta 1-adrenergic-selective antagonist practolol. This observation confirms the presence of beta 2-adrenergic receptors in these cell cultures. The formation of cAMP in NEP2 is also stimulated by 5'-(N-ethylcarboxamido)adenosine (NECA) more potently than by either adenosine or N6-(L-phenylisopropyl)adenosine (L-PIA), which suggests that this foetal astrocyte expresses adenosine A2 receptors. Furthermore, L-PIA and NECA inhibit isoprenaline stimulation of cAMP formation, a result suggesting the presence of adenosine A1 receptors on NEP2. The presence of A1 receptors is confirmed by the observation that the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine reverses the inhibition of isoprenaline stimulation of cAMP formation by L-PIA and NECA. Additional evidence that NEP2 expresses adenosine receptors linked to the adenylate cyclase-inhibitory GTP-binding protein is provided by the finding that pretreatment of these cells with pertussis toxin reverses the adenosine inhibition of cAMP formation stimulated by either isoprenaline or forskolin.

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

    PubMed

    Tamrakar, Pratistha; Briski, Karen P

    2015-01-01

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

  2. Airway administration of dexamethasone, 3'-5'-cyclic adenosine monophosphate, and isobutylmethylxanthine facilitates compensatory lung growth in adult mice.

    PubMed

    Takahashi, Yusuke; Izumi, Yotaro; Kohno, Mitsutomo; Kawamura, Masafumi; Ikeda, Eiji; Nomori, Hiroaki

    2011-03-01

    The combination of dexamethasone, 8-bromo-3'-5'-cyclic adenosine monophosphate, and isobutylmethylxanthine, referred to as DCI, has been reported to optimally induce cell differentiation in fetal lung explants and type II epithelial cells. DCI administration is also known to modulate the expression levels of many genes known to be involved in the facilitation of lung growth. Recently, we found that RNA silencing of thyroid transcription factor 1 (TTF-1) delayed compensatory lung growth. DCI is also known to induce TTF-1 expression in pulmonary epithelial cells. From these findings, we hypothesized that DCI administration may facilitate compensatory lung growth. In the present study, using a postpneumonectomy lung growth model in 9-wk-old male mice, we found that compensatory lung growth was significantly facilitated by airway administration of DCI immediately following left pneumonectomy, as indicated by the increase in the residual right lung dry weight index. TTF-1 expression was significantly elevated by DCI administration, and transient knockdown of TTF-1 attenuated the facilitation of compensatory lung growth by DCI. These results suggested that DCI facilitated compensatory lung growth, at least in part, through the induction of TTF-1. Morphological analyses suggested that DCI administration increased the number of alveoli, made each of them smaller, and produced a net increase in the calculated surface area of the alveoli per volume of lung. The effect of a single administration was maintained during the observation period, which was 28 days. DCI with further modifications may provide the material to potentially augment residual lung function after resection.

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

    PubMed Central

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

    2015-01-01

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

  4. Biospecific affinity chromatography of an adenosine 3′:5′-cyclic monophosphate-stimulated protein kinase (protamine kinase from trout testis) by using immobilized adenine nucleotides

    PubMed Central

    Jergil, Bengt; Guilford, Hugh; Mosbach, Klaus

    1974-01-01

    1. Two adenine nucleotides, 8-(6-aminohexyl)aminoadenosine 3′:5′-cyclic monophosphate and 8-(6-aminohexyl)amino-AMP, were synthesized. Their structures were established in particular by using mass spectroscopy. 2. Free cyclic AMP and 8-(6-aminohexyl)amino cyclic AMP both stimulate protamine kinase activity at low concentrations, but are inhibitory at concentrations above 0.1mm. AMP is an inhibitor of enzymic activity, whereas neither 8-(6-aminohexyl)amino-AMP nor the earlier synthesized N6-(6-aminohexyl)-AMP is inhibitory. 3. The nucleotides were coupled to Sepharose 4B and used for biospecific chromatography of partially purified protamine kinase. Enzyme applied at high buffer concentrations to the cyclic AMP–Sepharose material was retarded and thereby purified tenfold. At low buffer concentrations the enzyme was adsorbed to the affinity material, and was subsequently released by a pulse of the inhibitor AMP, yielding a 50–100-fold purification. Enzyme applied to immobilized 8-(6-aminohexyl)amino-AMP or N6-(6-aminohexyl)-AMP was eluted together with the main protein peak in the void volume. 4. Protamine kinase eluted from 8-(6-aminohexyl)amino cyclic AMP–Sepharose was no longer activated by cyclic AMP. Results from sucrose gradient centrifugation suggest that a dissociation of the enzyme took place on the immobilized nucleotide. 5. Further information on the mass spectroscopy has been deposited as Supplementary Publication SUP 50026 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5. PMID:4374933

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

    PubMed

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

    2005-11-09

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

  6. The P2Y12 antagonists, 2-methylthioadenosine 5'-monophosphate triethylammonium salt and cangrelor (ARC69931MX), can inhibit human platelet aggregation through a Gi-independent increase in cAMP levels.

    PubMed

    Srinivasan, Subhashini; Mir, Fozia; Huang, Jin-Sheng; Khasawneh, Fadi T; Lam, Stephen C-T; Le Breton, Guy C

    2009-06-12

    ADP plays an integral role in the process of hemostasis by signaling through two platelet G-protein-coupled receptors, P2Y1 and P2Y12. The recent use of antagonists against these two receptors has contributed a substantial body of data characterizing the ADP signaling pathways in human platelets. Specifically, the results have indicated that although P2Y1 receptors are involved in the initiation of platelet aggregation, P2Y12 receptor activation appears to account for the bulk of the ADP-mediated effects. Based on this consideration, emphasis has been placed on the development of a new class of P2Y12 antagonists (separate from clopidogrel and ticlopidine) as an approach to the treatment of thromboembolic disorders. The present work examined the molecular mechanisms by which two of these widely used adenosine-based P2Y12 antagonists (2-methylthioadenosine 5'-monophosphate triethylammonium salt (2MeSAMP) and ARC69931MX), inhibit human platelet activation. It was found that both of these compounds raise platelet cAMP to levels that substantially inhibit platelet aggregation. Furthermore, the results demonstrated that this elevation of cAMP did not require Gi signaling or functional P2Y12 receptors but was mediated through activation of a separate G protein-coupled pathway, presumably involving Gs. However, additional experiments revealed that neither 2MeSAMP nor ARC69931MX (cangrelor) increased cAMP through activation of A2a, IP, DP, or EP2 receptors, which are known to couple to Gs. Collectively, these findings indicate that 2MeSAMP and ARC69931MX interact with an unidentified platelet G protein-coupled receptor that stimulates cAMP-mediated inhibition of platelet function. This inhibition is in addition to that derived from antagonism of P2Y12 receptors.

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

    PubMed

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

    2008-01-01

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

  8. A1 adenosine receptors inhibit chloride transport in the shark rectal gland. Dissociation of inhibition and cyclic AMP.

    PubMed Central

    Kelley, G G; Poeschla, E M; Barron, H V; Forrest, J N

    1990-01-01

    In the in vitro perfused rectal gland of the dogfish shark (Squalus acanthias), the adenosine analogue 2-chloroadenosine (2Clado) completely and reversibly inhibited forskolin-stimulated chloride secretion with an IC50 of 5 nM. Other A1 receptor agonists including cyclohexyladenosine (CHA), N-ethylcarboxamideadenosine (NECA) and R-phenylisopropyl-adenosine (R-PIA) also completely inhibited forskolin stimulated chloride secretion. The "S" stereoisomer of PIA (S-PIA) was a less potent inhibitor of forskolin stimulated chloride secretion, consistent with the affinity profile of PIA stereoisomers for an A1 receptor. The adenosine receptor antagonists 8-phenyltheophylline and 8-cyclopentyltheophylline completely blocked the effect of 2Clado to inhibit forskolin-stimulated chloride secretion. When chloride secretion and tissue cyclic (c)AMP content were determined simultaneously in perfused glands, 2Clado completely inhibited secretion but only inhibited forskolin stimulated cAMP accumulation by 34-40%, indicating that the mechanism of inhibition of secretion by 2Clado is at least partially cAMP independent. Consistent with these results, A1 receptor agonists only modestly inhibited (9-15%) forskolin stimulated adenylate cyclase activity and 2Clado markedly inhibited chloride secretion stimulated by a permeant cAMP analogue, 8-chlorophenylthio cAMP (8CPT cAMP). These findings provide the first evidence for a high affinity A1 adenosine receptor that inhibits hormone stimulated ion transport in a model epithelia. A major portion of this inhibition occurs by a mechanism that is independent of the cAMP messenger system. PMID:1970583

  9. Gonadotropin stimulation of cyclic adenosine monophosphate and testosterone production without detectable high-affinity binding sites in purified Leydig cells from rat testis

    SciTech Connect

    Browne, E.S.; Bhalla, V.K. )

    1991-02-01

    Rat testicular interstitial cells were separated by three different gradient-density procedures and, with each, two biochemically and morphologically distinct cell fractions were isolated. The lighter density cells in fraction-I bound iodine 125-labeled human chorionic gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, Kd, approximately 10{sup {minus} 10} M) without producing either cyclic adenosine monophosphate or testosterone in response to hormone action. The heavier-density cells displayed morphologic features typical of Leydig cells and produced cyclic adenosine monophosphate and testosterone in the presence of hCG without detectable {sup 125}I-labeled hCG high-affinity binding. These cell fractions were further characterized by studies using deglycosylated hCG, a known antagonist to hCG action. Cell concentration-dependent studies with purified Leydig cells revealed that maximal testosterone production was achieved when lower cell concentrations (0.5 x 10(6) cells/250 microliters) were used for in vitro hCG stimulation assays. Under these conditions, the {sup 125}I-labeled hCG binding was barely detectable (2.24 fmol; 2,698 sites/cell). Furthermore, these studies revealed that the hCG-specific binding in Leydig cells is overestimated by the classic method for nonspecific binding correction using excess unlabeled hormone. An alternate method is presented.

  10. Molecular characterization of adenosine 5'-monophosphate deaminase--the key enzyme responsible for the umami taste of nori (Porphyra yezoensis Ueda, Rhodophyta).

    PubMed

    Minami, Seiko; Sato, Minoru; Shiraiwa, Yoshihiro; Iwamoto, Koji

    2011-12-01

    The enzyme adenosine 5'-monophosphate deaminase (AMPD, EC 3.5.4.6) catalyzes the conversion of adenosine 5'-monophosphate to inosine 5'-mononucleotide (IMP). IMP is generally known as the compound responsible for the umami taste of the edible red alga Porphyra yezoensis Ueda that is known in Japan as nori. Therefore, we suspect that AMPD plays a key role in providing a favorable nori taste. In this study, we undertake the molecular characterization of nori-derived AMPD. The nori AMPD protein has a molecular mass of 55 kDa as estimated from both gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The calculated molecular mass from the amino acid sequence deduced from cDNA is 57.1 kDa. The isoelectric point is 5.71. The coding region of AMPD consists of 1,566 bp encoding 522 amino acids and possesses a transmembrane domain and two N-glycosylation sites. The sequence identity of nori AMPD in human and yeast AMPDs was found to be less than 50% and 20% in DNA and amino acid sequences, respectively. Proline in the conserved motif of [SA]-[LIVM]-[NGS]-[STA]-D-D-P was found to be converted to glutamate. These results indicate that nori AMPD is a novel type of AMPD.

  11. [The ratio of hormones of system "hypophysis - thyroid" with level of dopamine and cyclic adenosine mono-phosphate of males in European north].

    PubMed

    Tipisova, E V; Molodovskaia, I N

    2014-03-01

    The study sampling consisted of 96 males from Arkhangelsk and 52 males from village of Nes. The examination was carried out to find out predominant regulative effect of dopamine on the system "hypophysis - thyroid" depending on territory of residence. In males of Zapolyarye, against the background of higher levels of T4, fT3 and TSH and cyclic adenosine mono-phosphate in blood occurs decreasing of levels of thyroglobulin and dopamine in comparison with males of circumpolar territories in case of registration of positive correlation between levels of dopamine and fT3. In males from circumpolar territories age-related decreasing of range of variations of level of dopamine and fT4 under increase of concentration of TSH was registered. At that, negative correlation between content of dopamine and T4 was registered. The age-related dynamics of alteration of level of cyclic adenosine monophosphate with tendency to increase in males of Zapolyarye at the age of 36-60 years in comparison with age group of 22-35 years.

  12. 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…

  13. Effects of electric stress on glucose metabolism, glucose-stimulated cyclic adenosine 3',5'-monophosphate accumulation and 45 Ca++ efflux in isolated pancreatic islets from rats fed with a high fat diet.

    PubMed

    Yamaguchi, K; Goko, H; Matsuoka, A

    1979-10-01

    The effects of the electric stress on glucose oxidation, cyclic adenosine 3', 5'-monophosphate (AMP) accumulation and 45Ca++ efflux in response to glucose were studied in pancreatic islets isolated from rats fed on a control (C) or a high fat diet (F) for 12 weeks. The half of rats on each diet were subjected to electrical shocks in the random time schedule for 1 hr per day for the last 3 weeks of the feeding period (group C-S and F-S). The remaining rats were not given any shocks (group C-NS and F-NS). The rats in F-S group had the high levels of plasma epinephrine, dopamine and blood glucose. The basal content of cyclic AMP after 20 min of incubation with 2.8 mM glucose was decreased in islets from F-S group without affecting insulin release. After 20 min of incubation with 25 mM glucose, the cyclic AMP content in islets from F-S group, which was identical with that in F-NS group, was only 50% of that in C-S group. Insulin release in response to high glucose was significantly inhibited in islets from F-S group. In spite of a remarkable increase of cyclic AMP content in islets from C-S group, insulin release did not differ from that in C-NS group. Glucose (16.7 mM)-stimulated 45Ca++ efflux from the perfused islets was greatly inhibited by the high fat diet rather than by stress. The rate of glucose oxidation with 16.7 mM glucose was decreased in islets from F-S group. It is suggested that the decreased insulin release in response to glucose provoked by the combined effects of the feeding of a high fat diet and electric stress may be mediated by changes of the adenylate cyclase-cyclic AMP system on the plasma membrane of the B-cell or be related to changes in glucose metabolism in islets.

  14. Post-Translational Regulation of the Glucose-6-Phosphatase Complex by Cyclic Adenosine Monophosphate Is a Crucial Determinant of Endogenous Glucose Production and Is Controlled by the Glucose-6-Phosphate Transporter.

    PubMed

    Soty, Maud; Chilloux, Julien; Delalande, François; Zitoun, Carine; Bertile, Fabrice; Mithieux, Gilles; Gautier-Stein, Amandine

    2016-04-01

    The excessive endogenous glucose production (EGP) induced by glucagon participates in the development of type 2 diabetes. To further understand this hormonal control, we studied the short-term regulation by cyclic adenosine monophosphate (cAMP) of the glucose-6-phosphatase (G6Pase) enzyme, which catalyzes the last reaction of EGP. In gluconeogenic cell models, a 1-h treatment by the adenylate cyclase activator forskolin increased G6Pase activity and glucose production independently of any change in enzyme protein amount or G6P content. Using specific inhibitors or protein overexpression, we showed that the stimulation of G6Pase activity involved the protein kinase A (PKA). Results of site-directed mutagenesis, mass spectrometry analyses, and in vitro phosphorylation experiments suggested that the PKA stimulation of G6Pase activity did not depend on a direct phosphorylation of the enzyme. However, the temperature-dependent induction of both G6Pase activity and glucose release suggested a membrane-based mechanism. G6Pase is composed of a G6P transporter (G6PT) and a catalytic unit (G6PC). Surprisingly, we demonstrated that the increase in G6PT activity was required for the stimulation of G6Pase activity by forskolin. Our data demonstrate the existence of a post-translational mechanism that regulates G6Pase activity and reveal the key role of G6PT in the hormonal regulation of G6Pase activity and of EGP.

  15. Rapid transcriptional down-regulation of c-myc expression during cyclic adenosine monophosphate-promoted differentiation of leukemic cells.

    PubMed

    Slungaard, A; Confer, D L; Schubach, W H

    1987-05-01

    Pharmacologic elevation of cyclic AMP (cAMP) promotes growth arrest and differentiation in a variety of transformed mammalian cells, including the HL-60 human promyelocytic leukemia cell line. However, mechanisms underlying this phenomenon are poorly understood. Because cellular oncogenes play a pivotal role in regulating proliferation and differentiation, we examined whether cAMP-promoted differentiation of HL-60 was preceded by a decrease in the expression of c-myc, a cellular oncogene both amplified and constitutively expressed in HL-60. We find that cyclic AMP elevation in HL-60 caused by three different pharmacologic regimens is followed by an abrupt, greater than 90% decrease in steady state c-myc mRNA levels within 3 h, well before detectable changes in proliferation and differentiation. This decrease, which occurs despite protein synthetic blockade, is attributable to transcriptional down-regulation of c-myc and is accompanied by changes in chromatin structure near c-myc promoter sites. Our findings establish that cAMP, a ubiquitous intracellular regulatory messenger previously known only to enhance gene transcriptional activity in higher eukaryotic cells, can also suppress transcription of a cellular oncogene, thereby suggesting a potential mechanism for cAMP-promoted differentiation.

  16. Rapid transcriptional down-regulation of c-myc expression during cyclic adenosine monophosphate-promoted differentiation of leukemic cells.

    PubMed Central

    Slungaard, A; Confer, D L; Schubach, W H

    1987-01-01

    Pharmacologic elevation of cyclic AMP (cAMP) promotes growth arrest and differentiation in a variety of transformed mammalian cells, including the HL-60 human promyelocytic leukemia cell line. However, mechanisms underlying this phenomenon are poorly understood. Because cellular oncogenes play a pivotal role in regulating proliferation and differentiation, we examined whether cAMP-promoted differentiation of HL-60 was preceded by a decrease in the expression of c-myc, a cellular oncogene both amplified and constitutively expressed in HL-60. We find that cyclic AMP elevation in HL-60 caused by three different pharmacologic regimens is followed by an abrupt, greater than 90% decrease in steady state c-myc mRNA levels within 3 h, well before detectable changes in proliferation and differentiation. This decrease, which occurs despite protein synthetic blockade, is attributable to transcriptional down-regulation of c-myc and is accompanied by changes in chromatin structure near c-myc promoter sites. Our findings establish that cAMP, a ubiquitous intracellular regulatory messenger previously known only to enhance gene transcriptional activity in higher eukaryotic cells, can also suppress transcription of a cellular oncogene, thereby suggesting a potential mechanism for cAMP-promoted differentiation. Images PMID:2437157

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

    PubMed

    Weaver, J L; Williams, R W

    1988-12-13

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

  18. Crystal Structures of the Adenylate Sensor from Fission Yeast AMP-Activated Protein Kinase

    SciTech Connect

    Townley,R.; Shapiro, L.

    2007-01-01

    The 5'-AMP (adenosine monophosphate)-activated protein kinase (AMPK) coordinates metabolic function with energy availability by responding to changes in intracellular adenosine triphosphate (ATP) and AMP levels. Here we report crystal structures at 2.6 and 2.9 Angstrom resolution for ATP- and AMP-bound forms of a core {alpha}{beta}{gamma} adenylate-binding domain from the fission yeast AMPK homologue. ATP and AMP bind competitively to a single site in the {gamma} subunit, with their respective phosphate groups positioned near function-impairing mutants. Surprisingly, ATP binds without counter ions, amplifying its electrostatic effects on a critical regulatory region where all three subunits converge.

  19. Modulation of calcium channels of twitch skeletal muscle fibres of the frog by adrenaline and cyclic adenosine monophosphate.

    PubMed Central

    Arreola, J; Calvo, J; García, M C; Sánchez, J A

    1987-01-01

    1. Modulation of fast and slow Ca2+ channels of frog skeletal muscle by adrenaline (10(-6) to 10(-5) M) and cyclic AMP was investigated using intracellular voltage recordings in intact fibres and a voltage-clamp technique in cut fibres. 2. In tetraethylammonium (TEA), Cl(-)-free Ringer solution, adrenaline increased the maximum rate of rise of Ca2+ spikes by 85% and in a similar solution, peak slow Ca2+ current (ICa,s) by 51%. 3. Application of cyclic AMP to the cut ends of fibres, produced a relative increase of ICa,s of ca. 24%. The effect was maintained for ca. 2 h. 4. Changes in the time course of ICa,s were produced by adrenaline and cyclic AMP: the limiting values of time-to-peak current measured as a function of membrane potential were lower (ca. 41% in adrenaline and ca. 34% in cyclic AMP) than those found in control experiments. Also, ICa,s decayed faster in the presence of adrenaline or cyclic AMP. These changes can be explained by exhaustion of Ca2+ in the lumen of transverse tubular system and do not require the assumption of kinetic variations. 5. Fast Ca2+ currents (ICa,f) which could not be blocked by nifedipine were also recorded. Cyclic AMP greatly increased the amplitude of ICa,f but had no obvious effects on ICa,f kinetics. 6. Application of catalytic subunit of cyclic AMP-dependent protein kinase by diffusion or by pressure injection also increased the amplitude of ICa,s and ICa,f. Pressure injection brought about modifications in the time course of ICa,s that cannot be explained by depletion of Ca2+. 7. Mechanical experiments were performed on single fibres. Nominally Ca2+-free solutions prevented the development and the maintenance of positive inotropic effect of adrenaline on twitch tension. Development of twitch potentiation was dependent upon the frequency of stimulation. Adrenaline was practically ineffective if no stimulation was applied. 8. It is concluded that both populations of Ca2+ channels are modulated by adrenergic stimulation

  20. Adenosine 3',5'-cyclic monophosphate involvement in hepatic triacylglyceride lipase release from prazosin-stimulated primary cultured rat hepatocytes.

    PubMed

    Nakamura, Tetsuya; Morita, Tetsuo

    2014-01-01

    We recently found that hepatic triglyceride lipase (HTGL) was released from primary cultured rat hepatocytes after treatment with prazosin, an antagonist of alpha-1 adrenoceptors. However, the details of prazosin-induced HTGL release remain uncertain. Here we investigated whether changes in cAMP levels in hepatocytes were related to HTGL release from prazosin-stimulated hepatocytes. When hepatocytes were treated with prazosin, cAMP levels during stimulated release of HTGL increased in a time- and dose-dependent manner. Stimulated release of HTGL was suppressed by the adenylate cyclase inhibitors MDL-12,330A and 2',5'-dideoxyadenosine. Further, cAMP-dependent protein kinase A (PKA) activity in prazosin-stimulated hepatocytes also increased in a time- and dose-dependent manner. Moreover, prazosin-stimulated HTGL release was suppressed by the PKA inhibitors H-89 and KT5720. These results suggest that prazosin-stimulated HTGL release from hepatocytes was due to cAMP production and partly due to subsequent PKA activation in hepatocytes.

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

    PubMed

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

    2014-09-01

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

  2. 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

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

    PubMed

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

    2015-02-01

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

  4. Phosphorylation of Cytokinin by Adenosine Kinase from Wheat Germ 1

    PubMed Central

    Chen, Chong-Maw; Eckert, Richard L.

    1977-01-01

    Adenosine kinase was partially purified from wheat germ. This enzyme preparation, which was devoid of adenine phosphoribosyltransferase and nearly free of adenosine deaminase but contained adenylate kinase, rapidly phosphorylated adenosine and a cytokinin, N6-(δ2-isopentenyl)adenosine. Electrophoretic analysis indicated that only N6-(δ2-isopentenyl)adenosine-monophosphate was formed from the cytokinin while about 55% AMP, 45% ADP, and a trace of ATP were formed from adenosine. The biosynthesized nucleoside monophosphates were quantitatively hydrolyzed to the corresponding nucleosides by 5′-nucleotidase and the isopentenyl side chain of the phosphorylated cytokinin was not cleaved. The enzyme did not catalyze phosphorylation of inosine. The phosphorylation of the cytokinin and adenosine required ATP and Mg2+. The pH optimum was from 6.8 to 7.2 for both the cytokinin and adenosine. At pH 7 and 37 C the Km and Vmax for the cytokinin were 31 μm and 8.3 nmoles per mg protein per minute, and the values for adenosine were 8.7 μm and 46 nmoles per mg protein per minute. Crude enzyme preparations from tobacco callus tissue and wheat germ phosphorylated N6-(δ2-isopentenyl)adenosine. These preparations also phosphorylated N6-(δ2-isopentenyl)adenine when 5-phosphorylribose-1-pyrophosphate was present. PMID:16659870

  5. Estradiol reduces nonclassical transcription at cyclic adenosine 3',5'-monophosphate response elements in glioma cells expressing estrogen receptor alpha.

    PubMed

    Mhyre, Andrew J; Shapiro, Robert A; Dorsa, Daniel M

    2006-04-01

    Estradiol can protect the brain from a variety of insults by activating membrane-initiated signaling pathways, and thereby modulate gene expression and lead to functional changes in neurons. These direct neuronal effects of the hormone have been well documented; however, it is less understood what effects estradiol may have on nonneuronal cells of the central nervous system. There is evidence that estradiol levels can induce the release of glial-derived growth factors and other cytokines, suggesting that estradiol may both directly and indirectly protect neurons. To determine whether 17beta-estradiol (E2) can activate rapid signaling and modulate nonclassical transcription in astrocytes, we stably transfected the C6 rat glioblastoma cell line with human estrogen receptor (ER) alpha (C6ERalpha) or rat ERbeta (C6ERbeta). Introduction of a cAMP response element-luciferase reporter gene into C6, C6ERalpha, and C6ERbeta cells leads to the observation that E2 treatment reduced isoproterenol-stimulated luciferase activity by 35% in C6ERalpha but had no effect on reporter gene expression in C6ERbeta or untransfected C6 cells. A similar effect was seen with a membrane-impermeable estrogen (E2-BSA), suggesting the modulation of nonclassical transcription by estradiol treatment is mediated by the activation of a membrane-initiated signaling pathway. Furthermore, pretreatment with wortmannin (phosphatidylinsositol 3-kinase) or U73122 (phospholipase C) attenuated the E2-induced reduction in nonclassical transcription. We conclude that E2 treatment reduces cAMP response element-mediated transcription in glioma cells expressing ERalpha and that this reduction is dependent on the activation of membrane-initiated signaling. These findings suggest a novel model of estrogen rapid signaling in astrocytes that leads to modulation of nonclassical transcription.

  6. Atorvastatin and pitavastatin enhance lipoprotein lipase production in L6 skeletal muscle cells through activation of adenosine monophosphate-activated protein kinase.

    PubMed

    Ohira, Masahiro; Endo, Kei; Saiki, Atsuhito; Miyashita, Yoh; Terai, Kensuke; Murano, Takeyoshi; Watanabe, Fusako; Tatsuno, Ichiro; Shirai, Kohji

    2012-10-01

    Pravastatin and atorvastatin increase the serum level of lipoprotein lipase (LPL) mass in vivo but do not increase LPL activity in 3T3-L1 preadipocytes in vitro. LPL is mainly produced by adipose tissue and skeletal muscle cells. Metformin enhances LPL in skeletal muscle through adenosine monophosphate-activated protein kinase (AMPK) activation but not in adipocytes. This study aimed to examine the effect of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) on LPL production and to investigate the mechanism by which statins enhance skeletal muscle cell LPL production. L6 skeletal muscle cells were incubated with pravastatin, simvastatin, atorvastatin or pitavastatin. LPL activity, protein levels and mRNA expression were measured. Atorvastatin and pitavastatin significantly increased LPL activity, protein levels and mRNA expression in L6 skeletal muscle cells at 1 μmol/L, but neither statin had an effect at 10 μmol/L. We measured AMPK to clarify the mechanism by which statins increase LPL production in skeletal muscle cells. At 1 μmol/L, both atorvastatin and pitavastatin enhanced AMPK activity, but this enhancement was abolished when AMPK signaling was blocked by compound C. The increased expressions of LPL protein and mRNA by atorvastatin and pitavastatin were reduced by compound C. In addition, mevalonic acid abolished atorvastatin- and pitavastatin-induced AMPK activation and LPL expression. These results suggest that atorvastatin and pitavastatin increase LPL activity, protein levels and LPL mRNA expression by activating AMPK in skeletal muscle cells.

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

    PubMed

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

    2015-09-01

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

  8. 5-Aminoimidazole-4-carboxamide ribonucleoside-mediated adenosine monophosphate-activated protein kinase activation induces protective innate responses in bacterial endophthalmitis.

    PubMed

    Kumar, Ajay; Giri, Shailendra; Kumar, Ashok

    2016-12-01

    The retina is considered to be the most metabolically active tissue in the body. However, the link between energy metabolism and retinal inflammation, as incited by microbial infection such as endophthalmitis, remains unexplored. In this study, using a mouse model of Staphylococcus aureus (SA) endophthalmitis, we demonstrate that the activity (phosphorylation) of 5' adenosine monophosphate-activated protein kinase alpha (AMPKα), a cellular energy sensor and its endogenous substrate; acetyl-CoA carboxylase is down-regulated in the SA-infected retina. Intravitreal administration of an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), restored AMPKα and acetyl-CoA carboxylase phosphorylation. AICAR treatment reduced both the bacterial burden and intraocular inflammation in SA-infected eyes by inhibiting NF-kB and MAP kinases (p38 and JNK) signalling. The anti-inflammatory effects of AICAR were diminished in eyes pretreated with AMPK inhibitor, Compound C. The bioenergetics (Seahorse) analysis of SA-infected microglia and bone marrow-derived macrophages revealed an increase in glycolysis, which was reinstated by AICAR treatment. AICAR also reduced the expression of SA-induced glycolytic genes, including hexokinase 2 and glucose transporter 1 in microglia, bone marrow-derived macrophages and the mouse retina. Interestingly, AICAR treatment enhanced the bacterial phagocytic and intracellular killing activities of cultured microglia, macrophages and neutrophils. Furthermore, AMPKα1 global knockout mice exhibited increased susceptibility towards SA endophthalmitis, as evidenced by increased inflammatory mediators and bacterial burden and reduced retinal function. Together, these findings provide the first evidence that AMPK activation promotes retinal innate defence in endophthalmitis by modulating energy metabolism and that it can be targeted therapeutically to treat ocular infections.

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

    PubMed Central

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

    2016-01-01

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

  10. Adenosine Monophosphate-Activated Protein Kinase (AMPK) as a New Target for Antidiabetic Drugs: A Review on Metabolic, Pharmacological and Chemical Considerations

    PubMed Central

    Gruzman, Arie; Babai, Gali; Sasson, Shlomo

    2009-01-01

    In view of the epidemic nature of type 2 diabetes and the substantial rate of failure of current oral antidiabetic drugs the quest for new therapeutics is intensive. The adenosine monophosphate-activated protein kinase (AMPK) is an important regulatory protein for cellular energy balance and is considered a master switch of glucose and lipid metabolism in various organs, especially in skeletal muscle and liver. In skeletal muscles, AMPK stimulates glucose transport and fatty acid oxidation. In the liver, it augments fatty acid oxidation and decreases glucose output, cholesterol and triglyceride synthesis. These metabolic effects induced by AMPK are associated with lowering blood glucose levels in hyperglycemic individuals. Two classes of oral antihyperglycemic drugs (biguanidines and thiazolidinediones) have been shown to exert some of their therapeutic effects by directly or indirectly activating AMPK. However, side effects and an acquired resistance to these drugs emphasize the need for the development of novel and efficacious AMPK activators. We have recently discovered a new class of hydrophobic D-xylose derivatives that activates AMPK in skeletal muscles in a non insulin-dependent manner. One of these derivatives (2,4;3,5-dibenzylidene-D-xylose-diethyl-dithioacetal) stimulates the rate of hexose transport in skeletal muscle cells by increasing the abundance of glucose transporter-4 (GLUT-4) in the plasma membrane through activation of AMPK. This compound reduces blood glucose levels in diabetic mice and therefore offers a novel strategy of therapeutic intervention strategy in type 2 diabetes. The present review describes various classes of chemically-related compounds that activate AMPK by direct or indirect interactions and discusses their potential for candidate antihyperglycemic drug development. PMID:19557293

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

    PubMed

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

    2014-04-01

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

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

    PubMed

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

    2014-08-01

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

  13. Adenosine Monophosphate-Activated Protein Kinase Abates Hyperglycaemia-Induced Neuronal Injury in Experimental Models of Diabetic Neuropathy: Effects on Mitochondrial Biogenesis, Autophagy and Neuroinflammation.

    PubMed

    Yerra, Veera Ganesh; Kumar, Ashutosh

    2017-04-01

    Impaired adenosine monophosphate kinase (AMPK) signalling under hyperglycaemic conditions is known to cause mitochondrial dysfunction in diabetic sensory neurons. Facilitation of AMPK signalling is previously reported to ameliorate inflammation and induce autophagic response in various complications related to diabetes. The present study assesses the role of AMPK activation on mitochondrial biogenesis, autophagy and neuroinflammation in experimental diabetic neuropathy (DN) using an AMPK activator (A769662). A769662 (15 and 30 mg/kg, i.p) was administered to Sprague-Dawley rats (250-270 g) for 2 weeks after 6 weeks of streptozotocin (STZ) injection (55 mg/kg, i.p.). Behavioural parameters (mechanical/thermal hyperalgesia) and functional characteristics (motor/sensory nerve conduction velocities (MNCV and SNCV) and sciatic nerve blood flow (NBF)) were assessed. For in vitro studies, Neuro2a (N2A) cells were incubated with 25 mM glucose to simulate high glucose condition and then studied for mitochondrial dysfunction and protein expression changes. STZ administration resulted in significant hyperglycaemia (>250 mg/dl) in rats. A769662 treatment significantly improved mechanical/thermal hyperalgesia threshold and enhanced MNCV, SNCV and NBF in diabetic animals. A769662 exposure normalised the mitochondrial superoxide production, membrane depolarisation and markedly increased neurite outgrowth of N2A cells. Further, AMPK activation also abolished the NF-κB-mediated neuroinflammation. A769662 treatment increased Thr-172 phosphorylation of AMPK results in stimulated PGC-1α-directed mitochondrial biogenesis and autophagy induction. Our study supports that compromised AMPK signalling in hyperglycaemic conditions causes defective mitochondrial biogenesis ultimately leading to neuronal dysfunction and associated deficits in DN and activation of AMPK can be developed as an attractive therapeutic strategy for the management of DN.

  14. Association study of three single-nucleotide polymorphisms in the cyclic adenosine monophosphate response element binding 1 gene and major depressive disorder.

    PubMed

    Wei, Yange; Bu, Shufang; Liu, Xican; Li, Hengfen

    2015-06-01

    Major depressive disorder is a common chronic emotional disorder, and cyclic adenosine monophosphate response element binding protein 1 (CREB1) is hypothesized to play a role in its pathogenesis. The aim of the present study was to investigate the associations between major depressive disorder and relevant single nucleotide polymorphisms (SNPs) in the CREB1 gene. A total of 1,038 subjects of Han Chinese descent were recruited, including 456 patients with major depressive disorder (case group) and 582 healthy volunteers (control group). The frequency distributions of the genotypes and alleles were estimated in the case and control groups, and analyzed for any correlation with major depressive disorder. Three relevant SNP sites in CREB1 were analyzed using quantitative polymerase chain reaction, and statistical analyses were performed to estimate their use as risk factors for major depressive disorder. The analyses revealed that rs2254137 and rs16839883 in CREB1 showed polymorphisms in the sample population, and the genotype and allele frequencies of rs16839883 differed significantly when comparing the patients and healthy controls (P<0.05). No statistically significant differences were detected in the two SNP sites between the male and female patients (P>0.05). Furthermore, no statistically significant differences were detected in rs2254137 genotype and allele distribution when comparing the male and female patients with their corresponding control groups (P>0.05). However, statistically significant differences were observed in the genotype and allele frequencies of rs16839883 when the male and female patients were compared with their respective controls (P<0.05). Therefore, the results demonstrated that there is a close correlation between the rs16839883 polymorphism in CREB1 and major depressive disorder, which suggests that this SNP site should be further studied as a potential biomarker for major depressive disorder.

  15. Effect of the growth stage and cultivar on policosanol profiles of barley sprouts and their adenosine 5'-monophosphate-activated protein kinase activation.

    PubMed

    Seo, Woo Duck; Yuk, Heung Joo; Curtis-Long, Marcus J; Jang, Ki Chang; Lee, Jin Hwan; Han, Sang-Ik; Kang, Hang Won; Nam, Min Hee; Lee, Sung-Joon; Lee, Ji Hae; Park, Ki Hun

    2013-02-06

    Adenosine 5'-monophosphate-activated protein kinase (AMPK) is an intracellular sensor that can regulate glucose levels within the cell. For this reason, it is well-known to be a target for drugs against diabetes and obesity. AMPK was activated significantly by the hexane extract of barley sprouts. This AMPK activation emerges across the growth stages of the sprout, becoming most significant (3 times above the initial stages) 10 days after sprouting. After this time, the activation decreased between 13 and 20 days post-sprouting. Analysis of the hexane extracts by gas chromatography-mass spectrometry showed that the amounts of policosanols (PCs, which are linear, primary aliphatic alcohols with 20-30 carbons) in the plant dramatically increased between 5 days (109.7 mg/100 g) and 10 days (343.7 mg/100 g) post-sprouting and then levels fell back down, reaching 76.4 mg/100 g at 20 days post-sprouting. This trend is consistent with PCs being the active ingredient in the barley plants. We validate this by showing that hexacosanol is an activator of AMPK. The richest cultivar for PCs was found to be the Daejin cultivar. Cultivars had a significant effect on the total PC content (113.2-183.5 mg/100 g) within the plant up to 5 days post-sprouting. However this dependence upon the cultivar was not so apparent at peak stages of PC production (10 days post-sprouting). The most abundant PC in barley sprout, hexacosanol, contributed 62-80% of the total PC content at every stage. These results are valuable to determine the optimal times of harvest to obtain the highest yield of PCs.

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

    PubMed

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

    2012-11-13

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

  17. Mdivi-1 Protects Against Ischemic Brain Injury via Elevating Extracellular Adenosine in a cAMP/CREB-CD39-Dependent Manner.

    PubMed

    Cui, Mei; Ding, Hongyan; Chen, Fangzhe; Zhao, Yanxin; Yang, Qi; Dong, Qiang

    2016-01-01

    This study aimed to examine whether the neuroprotective effects of Mdivi-1 are attributable to extracellular ATP and adenosine. Mdivi-1 was administered prior to or post middle cerebral artery occlusion (MCAO). The extracellular adenosine was measured by in vivo microdialysis and high-pressure liquid chromatography (HPLC) in MCAO mouse model. Western blot was done to determine the influence of Mdivi-1 on the expression of CD39 and CREB phosphorylation both in vivo and in the cultured astrocytes. Intracellular cAMP and protein kinase A (PKA) activity were detected in primary astrocytes. Results showed that Mdivi-1 significantly reduced infarct volume and neurological scores when administered either prior to or post MCAO. Interestingly, pretreatment with Mdivi-1 resulted in marked increase of extracellular adenosine and concomitant decrease in ATP. The expression of CD39, but not CD73, was upregulated by Mdivi-1, which was associated with the elevated phosphorylated cAMP response element-binding protein (CREB), a transcription factor potentially regulating CD39 expression. In primary astrocytes, Mdivi-1 treatment induced increases in intracellular cAMP, PKA activity and CREB phosphorylation, and PKA-specific inhibitor completely reversed Mdivi-1-induced CD39 expression. Our results demonstrate that Mdivi-1 protects against ischemic brain injury through increasing extracellular adenosine, a process involving elevated CD39 expression that is likely modulated by cAMP/PKA/CREB cascade. Figure Potential mechanisms by which Mdivi-1 mediates the neuroprotection on cerebral ischemic stroke. Results from the present study indicate that Mdivi-1 protects against ischemic brain injury through increasing extracellular adenosine, a process involving elevated CD39 expression that is likely modulated by the cAMP/PKA/CREB cascades.

  18. The Role of Adenosine Signaling in Headache: A Review

    PubMed Central

    Fried, Nathan T.; Elliott, Melanie B.; Oshinsky, Michael L.

    2017-01-01

    Migraine is the third most prevalent disease on the planet, yet our understanding of its mechanisms and pathophysiology is surprisingly incomplete. Recent studies have built upon decades of evidence that adenosine, a purine nucleoside that can act as a neuromodulator, is involved in pain transmission and sensitization. Clinical evidence and rodent studies have suggested that adenosine signaling also plays a critical role in migraine headache. This is further supported by the widespread use of caffeine, an adenosine receptor antagonist, in several headache treatments. In this review, we highlight evidence that supports the involvement of adenosine signaling in different forms of headache, headache triggers, and basic headache physiology. This evidence supports adenosine A2A receptors as a critical adenosine receptor subtype involved in headache pain. Adenosine A2A receptor signaling may contribute to headache via the modulation of intracellular Cyclic adenosine monophosphate (cAMP) production or 5' AMP-activated protein kinase (AMPK) activity in neurons and glia to affect glutamatergic synaptic transmission within the brainstem. This evidence supports the further study of adenosine signaling in headache and potentially illuminates it as a novel therapeutic target for migraine. PMID:28335379

  19. 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...

  20. Molecular imprinting of AMP by an ionic-noncovalent dual approach.

    PubMed

    Breton, Florent; Delépée, Raphaël; Agrofoglio, Luigi A

    2009-10-01

    In order to mimic recognition properties of adenylate kinase, molecularly imprinted polymers (MIPs) were prepared for adenosine 5'-monophosphate (AMP), a substrate of the enzyme. Different functional monomers interacting with the phosphate moiety were tested, and the MIP giving the best specific binding of AMP was composed with one equivalent of 2-(dimethylamino)ethyl methacrylate and ten equivalents of acrylamide compared to AMP. Packed into solid phase cartridge, this polymer showed similar characteristics than the enzyme, since it was specific for AMP toward other nucleotides.

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

    PubMed Central

    Davis, William M.; White, David C.

    1980-01-01

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

  2. Cocaine-amphetamine-regulated transcript expression in the rat nucleus accumbens is regulated by adenylyl cyclase and the cyclic adenosine 5'-monophosphate/protein kinase a second messenger system.

    PubMed

    Jones, Douglas C; Kuhar, Michael J

    2006-04-01

    Cocaine-amphetamine-regulated transcript (CART), a neuropeptide involved in the brain's reward/reinforcement circuit, modulates the effects of psychostimulants, including cocaine. The CART gene has been characterized, and binding sites for multiple transcription factors have been identified within the promoter region, including the cAMP-response element, which serves as a binding site for cAMP-response element-binding protein (CREB). CART expression appears to be regulated via cAMP/protein kinase A (PKA)/CREB-mediated signaling in cell culture. Therefore, the goal of these studies was to examine the involvement of cAMP/PKA/CREB-mediated signaling in CART mRNA and peptide expression in vivo in the rat nucleus accumbens. Intra-accumbal injections of forskolin, an adenylyl cyclase activator, stimulated the phosphorylation of CREB and increased both CART mRNA and peptide levels, an effect attenuated by inhibition of PKA with H89 [N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinoline-sulfonamide hydrochloride] and adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In addition, Rp-cAMPS alone decreased CART mRNA compared with saline-injected controls, suggesting that CART expression may be tonically regulated by PKA. Under certain conditions, cocaine increases CART mRNA levels; thus, we examined the effects of cocaine on forskolin-induced CART mRNA expression in the rat nucleus accumbens. Cocaine plus forskolin significantly increased CART mRNA over either of the drugs administered independently, suggesting that under conditions of heightened cAMP signaling, cocaine may impact CART gene expression. These results suggest that CART expression in vivo in the rat nucleus accumbens is regulated by adenylyl cyclase and cAMP/PKA-mediating signaling and, likely, through the activation of CREB.

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

    PubMed

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

    1975-02-13

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

  4. Properties of enzyme fraction A from Chlorella and copurification of 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase, adenosine 5'phosphosulfate sulfohydrolase and adenosine-5'-phosphosulfate cyclase activities.

    PubMed

    Lik-Shing Tsang, M; Schiff, J A

    1976-05-17

    Enzyme fraction A from Chlorella which catalyzes the formation of adenosine 5'-phosphosulfate from adenosine 3'-phosphate 5'-phosphosulfate is further characterized. Fraction A is found to contain an Mg2+ -activated and Ca2+ -inhibited 3' (2')-nucleotidase specific for 3' (2'), 5'-biphosphonucleosides. This activity has been named 3' (2), 5'-biphosphonucleoside 3' (2')-phosphohydrolase. The A fraction is also found to contain an activity which catalyzes the formation of adenosine 3':5'-monophosphate (cyclic AMP) from adenosine 5'-phosphosulfate (adenosine 5'-phosphosulfate cyclase). Under the same conditions of assay, 5'-ATP and 5'-ADP are not substrated for cyclic AMP formation. Unlike the 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase activity, the adenosine 5'-phosphosulfate cyclase activity does not require Mg2+, requires NH+4 or Na+, and is not inhibited by Ca2+. The A fraction also contains an adenosine 5'-phospho sulfate sulfohydrolase activity which forms 5'-AMP and sulfate. The three activities remain together during purification and acrylamide gel electrophoresis of the purified preparation yields a pattern where only one protein band has all three activities. The phosphohydrolase can be separated from the other two activities by affinity chromatography on agarose-hexyl-adenosine 3'n5'-bisphosphate yielding a phosphohydrolase preparation showing a single band on gel electrophoresis. The adenosine 5'-phosphosulfate cyclase may provide an alternate route of cyclic AMP formation from sulfate via ATP sulfurylase, but its regulatory significance in Chlorella, if any, remains to be demonstrated. In sulfate reduction, the phosphohydrolase may serve to provide a readily utilized pool of adenosine 5'-phosphosulfate as needed by the adenosine 5'-phosphosulfate sulfotransferase. The cyclase and sulfohydrolase activities would be regarded as side reactions incidental to this pathway, but may be of importance in other metabolic and regulatory reactions.

  5. Extracellular adenosine triphosphate affects the response of human macrophages infected with Mycobacterium tuberculosis.

    PubMed

    Dubois-Colas, Nicolas; Petit-Jentreau, Laetitia; Barreiro, Luis B; Durand, Sylvère; Soubigou, Guillaume; Lecointe, Cécile; Klibi, Jihène; Rezaï, Keyvan; Lokiec, François; Coppée, Jean-Yves; Gicquel, Brigitte; Tailleux, Ludovic

    2014-09-01

    Granulomas are the hallmark of Mycobacterium tuberculosis infection. As the host fails to control the bacteria, the center of the granuloma exhibits necrosis resulting from the dying of infected macrophages. The release of the intracellular pool of nucleotides into the surrounding medium may modulate the response of newly infected macrophages, although this has never been investigated. Here, we show that extracellular adenosine triphosphate (ATP) indirectly modulates the expression of 272 genes in human macrophages infected with M. tuberculosis and that it induces their alternative activation. ATP is rapidly hydrolyzed by the ecto-ATPase CD39 into adenosine monophosphate (AMP), and it is AMP that regulates the macrophage response through the adenosine A2A receptor. Our findings reveal a previously unrecognized role for the purinergic pathway in the host response to M. tuberculosis. Dampening inflammation through signaling via the adenosine A2A receptor may limit tissue damage but may also favor bacterial immune escape.

  6. Adrenocorticotropin and adenosine 3',5'-monophosphate stimulate de novo synthesis of adrenal phosphatidic acid by a cycloheximide-sensitive, CA++-dependent mechanism

    SciTech Connect

    Farese, R.V.; Sabir, M.A.; Larson, R.E.

    1981-12-01

    We tested further our postulate that enhanced de novo synthesis of phosphatidic acid is responsible for ACTH- and cAMP-induced increases in adrenal phospholipids in the phosphatidate polyphosphoinositide pathway. During incubation of adrenal sections or cells in vitro, ACTH and cAMP increased the concentrations of and incorporation of (3H)glycerol and (14C)palmitate into phosphatidylcholine and phosphatidylethanolamine, two major phospholipids which are derived from phosphatidic acid, but are extrinsic to the inositide pathway. Thus, it is unlikely that ACTH and cAMP increase inositide phospholipids at the expense of other phospholipids. Similar to previously reported effects on phosphatidic acid and inositide phospholipids, cycloheximide blocked the effects of ACTH and cAMP on phosphatidylcholine and phosphatidylethanolamine. In addition, Ca++ was required for these effects, as well as for cAMP-induced increases in phosphatidic acid, inositide phospholipids, and steroidogenesis. Our findings strongly suggest that ACTH, via cAMP, stimulates de novo phosphatidate synthesis by a cycloheximide-sensitive, Ca++-dependent process, and this stimulation causes a rapid generalized increase in adrenal phospholipids. Moreover, the increased incorporation of labeled glycerol and palmitate into phospholipids suggests that ACTH and cAMP may stimulate the glycerol-3'-PO4 acyltransferase reaction. This stimulatory effect may play a central role in the steroidogenic and trophic actions of ACTH and cAMP.

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

    PubMed Central

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

    2010-01-01

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

  8. Glial restricted precursor cell transplant with cyclic adenosine monophosphate improved some autonomic functions but resulted in a reduced graft size after spinal cord contusion injury in rats.

    PubMed

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

    2011-01-01

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

  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. Adenosine 3',5-cyclic monophosphate phosphodiesterase activity in granulosa cells from Booroola x Romney ewes with and without the F gene.

    PubMed

    McNatty, K P; Heath, D A; Lun, S; Hudson, N L

    1989-02-01

    Granulosa cells from ovarian follicles (greater than or equal to 1 mm diameter) in Booroola ewes which are homozygous (FF) or heterozygous (F+) for the F gene have previously been shown to produce significantly more cAMP in response to FSH or LH than those from similar sized follicles in ewes without the F gene (++). The aim of these studies was to test whether these F gene-specific differences arose because of differences in cAMP-phosphodiesterase (cAMP-PDE) activity. In the first study using 1 mumol cAMP/l as substrate, no F gene-specific effects were noted in cAMP-PDE activity in granulosa cells from small (1-2.5 mm diameter, n = 4 per genotype) or large (greater than or equal to 3 mm diameter, n = 4 per genotype) follicles from FF, F+ or ++ ewes, despite F gene-specific effects in FSH (1 microgram/ml)- and LH (0.1 microgram/ml)-induced cAMP accumulation in these same cell preparations. The overall mean levels of cAMP-PDE across all genotypes in cells from small and large follicles were 0.47 +/- 0.04 (S.E.M., n = 12) and 0.28 +/- 0.03 pmol cAMP/10(6) cells per min respectively; the mean PDE activity in cells from small follicles was significantly (P less than 0.05) higher compared with that in cells from large follicles. In a second study, granulosa cells from each genotype were pooled over all follicle sizes (greater than or equal to 1 mm diameter, one pool per genotype) and the rates of cAMP hydrolysis tested over a range of substrate concentrations (0-16 mumol/l) but no gene-specific differences with respect to the Michaelis constant and maximum velocity were noted.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    SciTech Connect

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

    1988-05-01

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

  12. Transcriptional regulation of inhibin beta B messenger ribonucleic acid levels in TM.4 or primary rat Sertoli cells by 8-bromo-cyclic adenosine monophosphate.

    PubMed

    Najmabadi, H; Rosenberg, L A; Yuan, Q X; Reyaz, G; Bhasin, S

    1993-04-01

    FSH, a major regulator of inhibin production in the testis, is believed to exert its effects via cAMP second messenger system. Inhibin alpha-subunit gene appears to be regulated by cAMP and has a palindromic cAMP response element sequence TGACGTCA. However, the regulation of the inhibin beta B-subunit gene by cAMP has been less clear. It has been assumed that beta B may not be regulated by cAMP, based mainly on observations that FSH stimulates only alpha, not beta B, mRNA levels, and that the 5'-up-stream regulatory region of the beta B gene does not contain the classical cAMP response element. However, we have observed that 8-bromo-cAMP stimulates beta B mRNA levels in both primary Sertoli (approximately 2-fold) and TM.4 cells (approximately 5-fold). We examined whether this cAMP-induced increase in beta B mRNA levels is the result of increased transcription or altered mRNA stability. Data from nuclear run-on assays demonstrate about a 2-fold increase in relative mRNA synthesis rates in primary Sertoli-cells and about a 4- to 5-fold increase in TM.4 cells. Transfection studies in TM.4 and JEG.3 cell lines with beta B:luciferase chimeric reporter gene constructs containing 1.5 kilobases of the beta B 5'-up-stream regulatory region revealed marked cAMP induction of reporter gene activity in both cell types.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Compartmentation of cAMP signalling in cardiomyocytes in health and disease.

    PubMed

    Perera, R K; Nikolaev, V O

    2013-04-01

    3',5'-cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger critically involved in the regulation of heart function. It has been shown to act in discrete subcellular signalling compartments formed by differentially localized receptors, phosphodiesterases and protein kinases. Cardiac diseases such as hypertrophy or heart failure are associated with structural and functional remodelling of these microdomains which leads to changes in cAMP compartmentation. In this review, we will discuss recent key findings which provided new insights into cAMP compartmentation in cardiomyocytes with a particular focus on its alterations in heart disease.

  14. 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

  15. Complex structure and biochemical characterization of the Staphylococcus aureus cyclic diadenylate monophosphate (c-di-AMP)-binding protein PstA, the founding member of a new signal transduction protein family.

    PubMed

    Campeotto, Ivan; Zhang, Yong; Mladenov, Miroslav G; Freemont, Paul S; Gründling, Angelika

    2015-01-30

    Signaling nucleotides are integral parts of signal transduction systems allowing bacteria to cope with and rapidly respond to changes in the environment. The Staphylococcus aureus PII-like signal transduction protein PstA was recently identified as a cyclic diadenylate monophosphate (c-di-AMP)-binding protein. Here, we present the crystal structures of the apo- and c-di-AMP-bound PstA protein, which is trimeric in solution as well as in the crystals. The structures combined with detailed bioinformatics analysis revealed that the protein belongs to a new family of proteins with a similar core fold but with distinct features to classical PII proteins, which usually function in nitrogen metabolism pathways in bacteria. The complex structure revealed three identical c-di-AMP-binding sites per trimer with each binding site at a monomer-monomer interface. Although distinctly different from other cyclic-di-nucleotide-binding sites, as the half-binding sites are not symmetrical, the complex structure also highlighted common features for c-di-AMP-binding sites. A comparison between the apo and complex structures revealed a series of conformational changes that result in the ordering of two anti-parallel β-strands that protrude from each monomer and allowed us to propose a mechanism on how the PstA protein functions as a signaling transduction protein.

  16. Daily rhythm in pineal phosphodiesterase (PDE) activity reflects adrenergic/3',5'-cyclic adenosine 5'-monophosphate induction of the PDE4B2 variant.

    PubMed

    Kim, Jong-So; Bailey, Michael J; Ho, Anthony K; Møller, Morten; Gaildrat, Pascaline; Klein, David C

    2007-04-01

    The pineal gland is a photoneuroendocrine transducer that influences circadian and circannual dynamics of many physiological functions via the daily rhythm in melatonin production and release. Melatonin synthesis is stimulated at night by a photoneural system through which pineal adenylate cyclase is adrenergically activated, resulting in an elevation of cAMP. cAMP enhances melatonin synthesis through actions on several elements of the biosynthetic pathway. cAMP degradation also appears to increase at night due to an increase in phosphodiesterase (PDE) activity, which peaks in the middle of the night. Here, it was found that this nocturnal increase in PDE activity results from an increase in the abundance of PDE4B2 mRNA (approximately 5-fold; doubling time, approximately 2 h). The resulting level is notably higher (>6-fold) than in all other tissues examined, none of which exhibit a robust daily rhythm. The increase in PDE4B2 mRNA is followed by increases in PDE4B2 protein and PDE4 enzyme activity. Results from in vivo and in vitro studies indicate that these changes are due to activation of adrenergic receptors and a cAMP-dependent protein kinase A mechanism. Inhibition of PDE4 activity during the late phase of adrenergic stimulation enhances cAMP and melatonin levels. The evidence that PDE4B2 plays a negative feedback role in adrenergic/cAMP signaling in the pineal gland provides the first proof that cAMP control of PDE4B2 is a physiologically relevant control mechanism in cAMP signaling.

  17. A reassessment of the modulatory role of cyclic AMP in catecholamine secretion by chromaffin cells.

    PubMed Central

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

    1995-01-01

    1. The role of adenosine 3':5'-cyclic monophosphate (cyclic AMP) in the regulation of catecholamine (CA) secretion in chromaffin cells remains equivocal from previous studies. 2. In the present study the effect of this cyclic nucleotide on basal CA secretion, as well as on intracellular calcium and membrane potential has been examined. 3. Forskolin and the permeable cyclic AMP analogue, 8-(4-chlorphenylthio)-adenosine-3'-5' monophosphate cyclic (pClpcAMP), increased basal CA secretion in a dose-dependent manner. The EC50s were 0.43 +/- 0.10 microM for forskolin and 39 +/- 9 microM for pClpcAMP. Other agonists with adenylate cyclase activity such as stimulants of adenosine receptors, beta-adrenoceptors, GABAB receptors and intestinal vasoactive peptide (VIP), also increased basal CA secretion in a highly significant manner. However, when they were added together with forskolin, CA secretion was not affected although an additive increase in cyclic AMP levels was produced. 4. Statistical analysis of the correlation between cyclic AMP levels and CA secretion evoked by these cyclic AMP increasing compounds showed that a significant direct correlation between both parameters existed only when low levels of cyclic AMP were produced by secretagogue stimulation. When the increase in intracellular cyclic AMP concentrations exceeded approximately 8 times the basal cyclic AMP levels the correlation was not significant. These results indicate a dual dose-dependent effect of cyclic AMP on basal CA secretion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7881750

  18. Cyclic AMP (cAMP) confers drug resistance against DNA damaging agents via PKAIA in CML cells.

    PubMed

    Xiao, Ling-Yi; Kan, Wai-Ming

    2017-01-05

    Cyclic adenosine monophosphate (cAMP) regulates many vital functions such as metabolism, proliferation, differentiation and death. Depending on cell types and stimulators, cAMP could either promote or attenuate cell death. cAMP signal can be transduced by protein kinase A (PKA) and/or exchange protein directly activated by cAMP (EPAC). In CML cells, cAMP may suppress their proliferation and enhance their differentiation. However, the role of cAMP on DNA damaging agent toxicity and the mechanism involved has not been studied. In this study, we studied the effect of cAMP on the sensitivity of CML cells to DNA damaging agents. We observed that forskolin (FSK) and dibutyryl-cAMP (DBcAMP) decreased cisplatin and etoposide-induced cell death in K562 cells. Moreover, PKA activator prevented K562 cells from DNA damaging agent-induced cell death while EPAC activator had no effect. Furthermore, we found that the PKA subtype, PKAIA, was involved in cAMP-attenuated resistance in K562 cells. Taken together, our results suggest that increased cAMP level confers CML cells to acquire a novel mechanism against DNA damaging agent toxicity via PKAIA. Thus, PKAIA inhibitor may be helpful in overcoming the resistance to DNA damaging agents in CML cells.

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

    DOE PAGES

    Tian, Yuanyuan; Cui, Wenjun; Huang, Manna; ...

    2014-08-05

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

  20. Dual Specificity and Novel Structural Folding of Yeast Phosphodiesterase-1 for Hydrolysis of Second Messengers Cyclic Adenosine and Guanosine 3′,5′-Monophosphate

    PubMed Central

    2015-01-01

    Cyclic nucleotide phosphodiesterases (PDEs) decompose second messengers cAMP and cGMP that play critical roles in many physiological processes. PDE1 of Saccharomyces cerevisiae has been subcloned and expressed in Escherichia coli. Recombinant yPDE1 has a KM of 110 μM and a kcat of 16.9 s–1 for cAMP and a KM of 105 μM and a kcat of 11.8 s–1 for cGMP. Thus, the specificity constant (kcat/KMcAMP)/(kcat/KMcGMP) of 1.4 indicates a dual specificity of yPDE1 for hydrolysis of both cAMP and cGMP. The crystal structures of unliganded yPDE1 and its complex with GMP at 1.31 Å resolution reveal a new structural folding that is different from those of human PDEs but is partially similar to that of some other metalloenzymes such as metallo-β-lactamase. In spite of their different structures and divalent metals, yPDE1 and human PDEs may share a common mechanism for hydrolysis of cAMP and cGMP. PMID:25050706

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  2. Specific inhibition by prostaglandins E2 and I2 of histamine-stimulated [14C]aminopyrine accumulation and cyclic adenosine monophosphate generation by isolated canine parietal cells.

    PubMed Central

    Soll, A H

    1980-01-01

    The effects of prostaglandins E2 and I2 on accumulation of [14C]aminopyrine and the generation of cyclic AMP by fractions of dispersed canine gastric mucosal cells, enriched in their content of parietal cells, have been studied. The parietal cell content of the fractions was enriched to between 43 and 70% using an elutriator rotor. The accumulation of [14C]aminopyrine was used as the index of parietal cell response to stimulation. Prostaglandin E2 (PGE2, 0.1 nM-0.1 mM) inhibited histamine stimulated aminopyrine uptake but did not block the response to carbachol, gastrin, or dibuturyl cyclic AMP. PGE2 did, however, inhibit aminopyrine uptake stimulated by carbachol and gastrin when the response to these agents was potentiated by histamine. PGE2 (0.1 NM-0.1 mM) inhibited histamine-stimulated cyclic AMP production in a dose-dependent fashion with maximal inhibition at 1 microM PGE2. Prostacyclin also inhibited both histamine-stimulated aminopyrine accumulation and histamine-stimulated cyclic AMP production. In the absence of added histamine, PGE2 in concentrations above 1 microM and prostacyclin in concentrations above 10 microM stimulated cyclic AMP production, probably by acting on the nonparietal cells as shown in previous studies. These present data are consistent with the hypothesis that prostaglandins E2 and I2 inhibit the response of isolated parietal cells to histamine by specifically blocking histamine-stimulated cyclic AMP production. PMID:6154063

  3. Measurement of cAMP in an undergraduate teaching laboratory, using ALPHAscreen technology.

    PubMed

    Bartho, Joseph D; Ly, Kien; Hay, Debbie L

    2012-02-14

    Adenosine 3',5'-monophosphate (cAMP) is a cellular second messenger with central relevance to pharmacology, cell biology, and biochemistry teaching programs. cAMP is produced from adenosine triphosphate by adenylate cyclase, and its production is reduced or enhanced upon activation of many G protein-coupled receptors. Therefore, the measurement of cAMP serves as an indicator of receptor activity. Although there are many assays available for measuring cAMP, few are suitable for large class teaching, and even fewer seem to have been adapted for this purpose. Here, we describe the use of bead-based ALPHAscreen (Amplified Luminescent Proximity Homogenous Assay) technology for teaching a class of more than 300 students the practical aspects of detecting signal transduction. This technology is applicable to the measurement of many different signaling pathways. This resource is designed to provide a practical guide for instructors and a useful model for developing other classes using similar technologies.

  4. A Biphasic and Brain-Region Selective Down-Regulation of Cyclic Adenosine Monophosphate Concentrations Supports Object Recognition in the Rat

    PubMed Central

    Hotte, Maïte; Dauphin, François; Freret, Thomas; Boulouard, Michel; Levallet, Guenaëlle

    2012-01-01

    Background We aimed to further understand the relationship between cAMP concentration and mnesic performance. Methods and Findings Rats were injected with milrinone (PDE3 inhibitor, 0.3 mg/kg, i.p.), rolipram (PDE4 inhibitor, 0.3 mg/kg, i.p.) and/or the selective 5-HT4R agonist RS 67333 (1 mg/kg, i.p.) before testing in the object recognition paradigm. Cyclic AMP concentrations were measured in brain structures linked to episodic-like memory (i.e. hippocampus, prefrontal and perirhinal cortices) before or after either the sample or the testing phase. Except in the hippocampus of rolipram treated-rats, all treatment increased cAMP levels in each brain sub-region studied before the sample phase. After the sample phase, cAMP levels were significantly increased in hippocampus (1.8 fold), prefrontal (1.3 fold) and perirhinal (1.3 fold) cortices from controls rat while decreased in prefrontal cortex (∼0.83 to 0.62 fold) from drug-treated rats (except for milrinone+RS 67333 treatment). After the testing phase, cAMP concentrations were still increased in both the hippocampus (2.76 fold) and the perirhinal cortex (2.1 fold) from controls animals. Minor increase were reported in hippocampus and perirhinal cortex from both rolipram (respectively, 1.44 fold and 1.70 fold) and milrinone (respectively 1.46 fold and 1.56 fold)-treated rat. Following the paradigm, cAMP levels were significantly lower in the hippocampus, prefrontal and perirhinal cortices from drug-treated rat when compared to controls animals, however, only drug-treated rats spent longer time exploring the novel object during the testing phase (inter-phase interval of 4 h). Conclusions Our results strongly suggest that a “pre-sample” early increase in cAMP levels followed by a specific lowering of cAMP concentrations in each brain sub-region linked to the object recognition paradigm support learning efficacy after a middle-term delay. PMID:22359674

  5. Convergence of 3',5'-cyclic adenosine 5'-monophosphate/protein kinase A and glycogen synthase kinase-3beta/beta-catenin signaling in corpus luteum progesterone synthesis.

    PubMed

    Roy, Lynn; McDonald, Claudia A; Jiang, Chao; Maroni, Dulce; Zeleznik, Anthony J; Wyatt, Todd A; Hou, Xiaoying; Davis, John S

    2009-11-01

    Progesterone secretion by the steroidogenic cells of the corpus luteum (CL) is essential for reproduction. Progesterone synthesis is under the control of LH, but the exact mechanism of this regulation is unknown. It is established that LH stimulates the LH receptor/choriogonadotropin receptor, a G-protein coupled receptor, to increase cAMP and activate cAMP-dependent protein kinase A (PKA). In the present study, we tested the hypothesis that cAMP/PKA-dependent regulation of the Wnt pathway components glycogen synthase kinase (GSK)-3beta and beta-catenin contributes to LH-dependent steroidogenesis in luteal cells. We observed that LH via a cAMP/PKA-dependent mechanism stimulated the phosphorylation of GSK3beta at N-terminal Ser9 causing its inactivation and resulted in the accumulation of beta-catenin. Overexpression of N-terminal truncated beta-catenin (Delta90 beta-catenin), which lacks the phosphorylation sites responsible for its destruction, significantly augmented LH-stimulated progesterone secretion. In contrast, overexpression of a constitutively active mutant of GSK3beta (GSK-S9A) reduced beta-catenin levels and inhibited LH-stimulated steroidogenesis. Chromatin immunoprecipitation assays demonstrated the association of beta-catenin with the proximal promoter of the StAR gene, a gene that expresses the steroidogenic acute regulatory protein, which is a cholesterol transport protein that controls a rate-limiting step in steroidogenesis. Collectively these data suggest that cAMP/PKA regulation of GSK3beta/beta-catenin signaling may contribute to the acute increase in progesterone production in response to LH.

  6. Regulatory T-Cell-Mediated Suppression of Conventional T-Cells and Dendritic Cells by Different cAMP Intracellular Pathways.

    PubMed

    Rueda, Cesar M; Jackson, Courtney M; Chougnet, Claire A

    2016-01-01

    Regulatory T-cells (Tregs) mediate their suppressive action by acting directly on conventional T-cells (Tcons) or dendritic cells (DCs). One mechanism of Treg suppression is the increase of cyclic adenosine 3',5'-monophosphate (cAMP) levels in target cells. Tregs utilize cAMP to control Tcon responses, such as proliferation and cytokine production. Tregs also exert their suppression on DCs, diminishing DC immunogenicity by downmodulating the expression of costimulatory molecules and actin polymerization at the immunological synapse. The Treg-mediated usage of cAMP occurs through two major mechanisms. The first involves the Treg-mediated influx of cAMP in target cells through gap junctions. The second is the conversion of adenosine triphosphate into adenosine by the ectonucleases CD39 and CD73 present on the surface of Tregs. Adenosine then binds to receptors on the surface of target cells, leading to increased intracellular cAMP levels in these targets. Downstream, cAMP can activate the canonical protein kinase A (PKA) pathway and the exchange protein activated by cyclic AMP (EPAC) non-canonical pathway. In this review, we discuss the most recent findings related to cAMP activation of PKA and EPAC, which are implicated in Treg homeostasis as well as the functional alterations induced by cAMP in cellular targets of Treg suppression.

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

    SciTech Connect

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

    1988-09-01

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

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

    SciTech Connect

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

    1985-06-01

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

  9. Adenosine Signaling Increases Proinflammatory and Profibrotic Mediators through Activation of a Functional Adenosine 2B Receptor in Renal Fibroblasts.

    PubMed

    Wilkinson, Patrick F; Farrell, Francis X; Morel, Diane; Law, William; Murphy, Suzanne

    2016-07-01

    Interstitial renal fibrosis is a major pathophysiological manifestation of patients diagnosed with Chronic Kidney Disease (CKD), Diabetic Nephropathy (DN) and other inflammatory diseases. Adenosine signaling is an innate autocrine and paracrine cellular signaling pathway involving several key mediators that are elevated in the blood and kidneys of patients with DN. In these studies, we hypothesized that extracellular adenosine signals through one or more functional adenosine GPCRs on renal fibroblasts which increases profibrotic and proinflammatory mediators by inducing an activated fibroblast phenotype. Utilizing the renal fibroblast cell line NRK-49F, the presence and relative abundance of adenosine receptors (AR) A1, A2A, A2B, and A3 were quantified by RT-PCR. Under normal homeostatic conditions, only AR1 and AR2B were detected. The functionality of each receptor was then assessed by receptor specific pharmacological agonism and antagonism and assessed for modulation of the GPCR associated secondary messenger molecule, cyclic adenosine monophosphate (cAMP). Agonism of the AR2B receptor resulted in increased intracellular cAMP while agonism of the AR1 receptor inhibited cAMP modulation. Upon direct agonism of the AR2B receptor, transcripts for profibrotic and inflammatory mediators including SMA-α, IL-6, TGF-β, CTGF, and fibronectin were elevated between 2-4 fold. These data indicate that renal fibroblasts express a functional AR1 receptor that inhibits cAMP upon stimulation, leading to a functional AR2B receptor that increases cAMP upon stimulation and also induces an activated fibroblast phenotype resulting in increased fibrotic and inflammatory mediators.

  10. Differential involvement of 3', 5'-cyclic adenosine monophosphate-dependent protein kinase in regulation of Fos and tyrosine hydroxylase expression in the heart after naloxone induced morphine withdrawal.

    PubMed

    Almela, Pilar; Cerezo, Manuela; González-Cuello, A; Milanés, M Victoria; Laorden, M Luisa

    2007-01-01

    We previously demonstrated that morphine withdrawal induced hyperactivity of the heart by the activation of noradrenergic pathways innervating the left and right ventricle, as evaluated by noradrenaline (NA) turnover and Fos expression. We investigated whether cAMP-dependent protein kinase (PKA) plays a role in this process by estimating changes in PKA immunoreactivity and the influence of inhibitor of PKA on Fos protein expression, tyrosine hydroxylase (TH) immunoreactivity levels and NA turnover in the left and right ventricle. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (5 mg/kg). When opioid withdrawal was precipitated, an increase in PKA immunoreactivity and phospho-CREB (cyclic AMP response element protein) levels were observed in the heart. Moreover, morphine withdrawal induces Fos expression, an enhancement of NA turnover and an increase in the total TH levels. When the selective PKA inhibitor HA-1004 was infused, concomitantly with morphine pellets, it diminished the increase in NA turnover and the total TH levels observed in morphine-withdrawn rats. However, this inhibitor neither modifies the morphine withdrawal induced Fos expression nor the increase of nonphosphorylated TH levels. The present findings indicate that an up-regulated PKA-dependent transduction pathway might contribute to the activation of the cardiac catecholaminergic neurons in response to morphine withdrawal and suggest that Fos is not a target of PKA at heart levels.

  11. Advanced glycation end-products impair Na⁺/K⁺-ATPase activity in diabetic cardiomyopathy: role of the adenosine monophosphate-activated protein kinase/sirtuin 1 pathway.

    PubMed

    Yuan, Qiong; Zhou, Qian-Yi; Liu, Du; Yu, Lun; Zhan, Lin; Li, Xiao-Jing; Peng, Hong-Yan; Zhang, Xiu-Ling; Yuan, Xin-Chu

    2014-02-01

    Decreased Na(+) /K(+) -ATPase activity, and both sirtuin 1 (SIRT1) and adenosine monophosphate-activated protein kinase (AMPK) have been reported to be involved in the development of diabetic cardiomyopathy (DCM). The present study aimed to investigate the advanced glycation end-products (AGE) that impair Na(+) /K(+) -ATPase stability by regulating the AMPK/SIRT1 pathway during progression of DCM. To study type 1 diabetic mellitus (T1DM), a disease model in rats was established by a single intraperitoneal injection of streptozotocin (STZ; 65 mg/kg), and neonatal rat cardiomyocytes were also cultured. Heart function was detected by Doppler, and SIRT1 and AMPK protein expression were detected by immunohistochemistry and western blotting. Na(+) /K(+) -ATPase activity was also monitored. Using in vivo rat models of DCM, we showed that Na(+) /K(+) -ATPase activity decreased when both AMPK and SIRT1 expression were downregulated. In vitro, AGE impaired Na(+) /K(+) -ATPase activity and decreased the AMPK and SIRT1 expression. Sirtuin 1 overexpression increased Na(+) /K(+) -ATPase activity. 5-aminoimidazole-4-carboxamide-3-ribonucleoside (AICAR) upregulated SIRT1 expression and increased Na(+) /K(+) -ATPase activity, which could be partially abolished by splitomicin. Our results suggest that the dysfunction of DCM is related to AGE-induced Na(+) /K(+) -ATPase activity impairment through a mechanism involving the AMPK/SIRT1 pathway.

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

    PubMed

    Ido, Yasuo

    2016-07-01

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

  13. Genistein promotes insulin action through adenosine monophosphate-activated protein kinase activation and p70 ribosomal protein S6 kinase 1 inhibition in the skeletal muscle of mice fed a high energy diet.

    PubMed

    Arunkumar, Elumalai; Anuradha, Carani Venkatraman

    2012-08-01

    Genistein (GEN), a soy isoflavone, exerts insulin-sensitizing actions in animals; however, the underlying mechanisms have not been determined. Because GEN is a known activator of adenosine monophosphate-activated protein kinase (AMPK), we hypothesize that GEN activates insulin signaling through AMPK activation. To test this hypothesis, a high fat-high fructose diet (HFFD)-fed mice model of insulin resistance was administered GEN, and the insulin signaling pathway proteins in the skeletal muscle were examined. Hyperglycemia and hyperinsulinemia observed in HFFD-fed mice were significantly lowered by GEN. GEN increased insulin-stimulated tyrosine phosphorylation of insulin receptor-β and insulin receptor substrate (IRS) 1 but down-regulated IRS-1 serine phosphorylation in the skeletal muscle of HFFD-fed mice. Furthermore, GEN treatment improved muscle IRS-1-associated phospatidylinositol-3 kinase expression, phosphorylation of Akt at Ser(473), and translocation of glucose transporter subtype 4. Phosphorylation of AMPK at Thr(172) and acetyl coenzyme A carboxylase (ACC) at Ser(79) was augmented, whereas phosphorylation of p70 ribosomal protein S6 kinase 1 at Thr(389) was significantly decreased after GEN treatment in the skeletal muscle of HFFD-fed mice. These results suggest that GEN might improve insulin action in the skeletal muscle by targeting AMPK.

  14. The fruit of Acanthopanax senticosus (Rupr. et Maxim.) Harms improves insulin resistance and hepatic lipid accumulation by modulation of liver adenosine monophosphate-activated protein kinase activity and lipogenic gene expression in high-fat diet-fed obese mice.

    PubMed

    Saito, Tetsuo; Nishida, Miyako; Saito, Masafumi; Tanabe, Akari; Eitsuka, Takahiro; Yuan, Shi-Hua; Ikekawa, Nobuo; Nishida, Hiroshi

    2016-10-01

    Obesity-associated insulin resistance is a major risk factor for most metabolic diseases, including dyslipidemia and type 2 diabetes. Acanthopanax senticosus (Rupr. et Maxim.) Harms (Goka) root has been used in traditional Chinese medicine for treatment of diabetes and other conditions; however, little is known about the effects of Goka fruit (GF). Goka fruit is rich in anthocyanin, which has beneficial effects on obesity and insulin resistance via activation of adenosine monophosphate-activated protein kinase (AMPK). We hypothesized that GF can improve obesity-associated insulin resistance. The aim of the present study was to investigate whether GF improves insulin resistance in high-fat diet (HFD)-induced obese mice. High-fat diet mice treated with GF (500 and 1000 mg/kg) for 12 weeks showed an improved glucose tolerance and insulin sensitivity, as well as reduced plasma insulin and liver lipid accumulation. Moreover, GF administration to HFD mice resulted in down-regulation of fatty acid synthase expression and up-regulation of cholesterol 7-alpha-hydroxylase expression in the liver. Notably, AMPK phosphorylation in the liver increased after GF administration. In summary, GF supplementation improved obesity-associated insulin resistance and hepatic lipid accumulation through modulation of AMPK activity and lipid metabolism-associated gene expression.

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

    PubMed

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

    2015-03-01

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

  16. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  18. Cyclic AMP Mimics the Anti-ageing Effects of Calorie Restriction by Up-Regulating Sirtuin.

    PubMed

    Wang, Zhuoran; Zhang, Lu; Liang, Yaru; Zhang, Chi; Xu, Zhiyu; Zhang, Lang; Fuji, Ryosuke; Mu, Wei; Li, Liyuan; Jiang, Junjun; Ju, Yong; Wang, Zhao

    2015-07-08

    Cyclic adenosine monophosphate (cAMP) plays an important role in many biological processes as a second messenger, and cAMP treatment has been reported to extend the lifespan of wild-type Drosophila melanogaster. Our study showed that exogenous cAMP improved ageing-related phenotypes by increasing the protein level of Sirtuins, which prevented metabolic disorders to mimic the effect of calorie restriction. Experiments in vitro showed that cAMP directly bound to SIRT1 and SIRT3 and consequently increased their activity. These findings suggest that cAMP slows the ageing process and is a good candidate to mimic calorie restriction. Our research provides a promising therapeutic strategy to target metabolic disorder-induced ageing-related diseases.

  19. The pleiotropic role of exchange protein directly activated by cAMP 1 (EPAC1) in cancer: implications for therapeutic intervention.

    PubMed

    Almahariq, Muayad; Mei, Fang C; Cheng, Xiaodong

    2016-01-01

    The pleiotropic second messenger adenosine 3',5'-cyclic monophosphate (cAMP) regulates a myriad of biological processes under both physiological and pathophysiological conditions. Exchange protein directly activated by cAMP 1 (EPAC1) mediates the intracellular functions of cAMP by acting as a guanine nucleotide exchange factor for the Ras-like Rap small GTPases. Recent studies suggest that EPAC1 plays important roles in immunomodulation, cancer cell migration/metastasis, and metabolism. These results, coupled with the successful development of EPAC-specific small molecule inhibitors, identify EPAC1 as a promising therapeutic target for cancer treatments.

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

    SciTech Connect

    Theibert, W.E.A.B.

    1985-01-01

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

  1. Expression of adenosine 5'-monophosphate-Activated protein kinase (AMPK) in ovine testis (Ovis aries): In vivo regulation by nutritional state.

    PubMed

    Taibi, N; Dupont, J; Bouguermouh, Z; Froment, P; Ramé, C; Anane, A; Amirat, Z; Khammar, F

    2017-03-01

    In the present study, we identified AMPK and investigated its potential role in steroidogenesis in vivo in the ovine testis in response to variation in nutritional status (fed control vs. restricted). We performed immunoblotting to show that both active and non-active forms of AMPK exist in ovine testis and liver. In testis, we confirmed these results by immunohistochemistry. We found a correlation between ATP (Adenosine-Triphosphate) levels and the expression of AMPK in liver. Also, low and high caloric diets induce isoform-dependent AMPK expression, with an increase in α2, ß1ß2 and γ1 activity levels. Although the restricted group exhibited an increase in lipid balance, only the triglyceride and HC-VLDL (Cholesterol-Very low density lipoprotein) fractions showed significant differences between groups, suggesting an adaptive mechanism. Moreover, the relatively low rate of non-esterified fatty acid released into the circulation implies re-esterification to compensate for the physiological need. In the fed control group, AMPK activates the production of testosterone in Leydig cells; this is, in turn, associated with an increase in the expression of 3ß-HSD (3 beta hydroxy steroid deshydrogenase), p450scc (Cholesterol side-chain cleavage enzyme) and StAR (Steroidogenic acute regulatory protein) proteins induced by decreased MAPK ERK½ (Extracellular signal-regulated kinase -Mitogen-activated protein kinase) phosphorylation. In contrast, in the restricted group, testosterone secretion was reduced but intracellular cholesterol concentration was not. Furthermore, the combination of high levels of lipoproteins and emergence of the p38 MAP kinase pathway suggest the involvement of pro-inflammatory cytokines, as confirmed by transcriptional repression of the StAR protein. Taken together, these results suggest that AMPK expression is tissue dependent.

  2. Adenosine monophosphate activated protein kinase (AMPK), a mediator of estradiol-induced apoptosis in long-term estrogen deprived breast cancer cells.

    PubMed

    Chen, Haiyan; Wang, Ji-Ping; Santen, Richard J; Yue, Wei

    2015-06-01

    Estrogens stimulate growth of hormone-dependent breast cancer but paradoxically induce tumor regress under certain circumstances. We have shown that long-term estrogen deprivation (LTED) enhances the sensitivity of hormone dependent breast cancer cells to estradiol (E2) so that physiological concentrations of estradiol induce apoptosis in these cells. E2-induced apoptosis involve both intrinsic and extrinsic pathways but precise mechanisms remain unclear. We found that exposure of LTED MCF-7 cells to E2 activated AMP activated protein kinase (AMPK). In contrast, E2 inhibited AMPK activation in wild type MCF-7 cells where E2 prevents apoptosis. As a result of AMPK activation, the transcriptional activity of FoxO3, a downstream factor of AMPK, was up-regulated in E2 treatment of LTED. Increased activity of FoxO3 was demonstrated by up-regulation of three FoxO3 target genes, Bim, Fas ligand (FasL), and Gadd45α. Among them, Bim and FasL mediate intrinsic and extrinsic apoptosis respectively and Gadd45α causes cell cycle arrest at the G2/M phase. To further confirm the role of AMPK in apoptosis, we used AMPK activator AICAR in wild type MCF-7 cells and examined apoptosis, proliferation and expression of Bim, FasL, and Gadd45α. The effects of AICAR on these parameters recapitulated those observed in E2-treated LTED cells. Activation of AMPK by AICAR also increased expression of Bax in MCF-7 cells and its localization to mitochondria, which is a required process for apoptosis. These results reveal that AMPK is an important factor mediating E2-induced apoptosis in LTED cells, which is implicative of therapeutic potential for relapsing breast cancer after hormone therapy.

  3. Prognostic value of coexistence of abnormal expression of micro-RNA-200b and cyclic adenosine monophosphate-responsive element-binding protein 1 in human astrocytoma.

    PubMed

    Zhang, Jun-qing; Yao, Qing-he; Kuang, Yong-qin; Ma, Yuan; Yang, Li-bin; Huang, Hai-dong; Cheng, Jing-ming; Yang, Tao; Liu, En-yu; Liang, Liang; Fan, Ke-xia; Zhao, Kai; Xia, Xun; Gu, Jian-wen

    2014-10-01

    Our aim was to investigate the expression of micro-RNA-200b (miR-200b) and cAMP-responsive element-binding protein 1 (CREB-1) in astrocytoma and its efficacy for predicting outcome. Both miR-200b and CREB-1 messenger RNA expression was measured in 122 astrocytomas and 30 nonneoplastic brain specimens by quantitative real-time polymerase chain reaction. Expression of miR-200b was significantly lower in astrocytoma than in nonneoplastic brain (P < .001), whereas CREB-1 messenger RNA expression was significantly elevated in the tumors (P < .001). Both miR-200b down-regulation and CREB-1 up-regulation were significantly associated with advanced pathologic grade (P = .002 and P = .006, respectively). Low miR-200b expression correlated negatively with Karnofsky performance score (P = .03), and high CREB-1 expression correlated positively with mean tumor diameter (P = .03). By Kaplan-Meier analysis, low miR-200b, high CREB-1, and coexistence of abnormal miR-200b and CREB-1 expression (low miR-200b/high CREB-1) were predictive of shorter progression-free survival and overall survival in both grade III and grade IV astrocytoma. By multivariate analysis, only low miR-200b/high CREB-1 expression was an independent prognostic factor for poor prognosis in astrocytoma of advanced grade. Both miR-200b and CREB-1 may play important cooperative roles in the progression of human astrocytoma. The efficacy of miR-200b and CREB-1 together as a predictor of prognosis in astrocytoma patients is shown for the first time.

  4. Inhibition of AMP deaminase as therapeutic target in cardiovascular pathology.

    PubMed

    Zabielska, Magdalena A; Borkowski, Tomasz; Slominska, Ewa M; Smolenski, Ryszard T

    2015-08-01

    AMP deaminase (AMPD; EC 3.5.4.6) catalyzes hydrolysis of the amino group from the adenine ring of AMP resulting in production of inosine 5'-monophosphate (IMP) and ammonia. This reaction helps to maintain healthy cellular energetics by removing excess AMP that accumulates in energy depleted cells. Furthermore, AMPD permits the synthesis of guanine nucleotides from the larger adenylate pool. This enzyme competes with cytosolic 5'-nucleotidases (c5NT) for AMP. Adenosine, a product of c5NT is a vasodilator, antagonizes inotropic effects of catecholamines and exerts anti-platelet, anti-inflammatory and immunosuppressive activities. The ratio of AMPD/c5NT defines the amount of adenosine produced in adenine nucleotide catabolic pathway. Inhibition of AMPD could alter this ratio resulting in increased adenosine production. Besides the potential effect on adenosine production, elevation of AMP due to inhibition of AMPD could also lead to activation of AMP regulated protein kinase (AMPK) with myriad of downstream events including enhanced energetic metabolism, mitochondrial biogenesis and cytoprotection. While the benefits of these processes are well appreciated in cells such as skeletal or cardiac myocytes its role in protection of endothelium could be even more important. Therapeutic use of AMPD inhibition has been limited due to difficulties with obtaining compounds with adequate characteristics. However, endothelium seems to be the easiest target as effective inhibition of AMPD could be achieved at much lower concentration than in the other types of cells. New generation of AMPD inhibitors has recently been established and its testing in context of endothelial and organ protection could provide important basic knowledge and potential therapeutic tools.

  5. Intracellular cAMP signaling by soluble adenylyl cyclase.

    PubMed

    Tresguerres, Martin; Levin, Lonny R; Buck, Jochen

    2011-06-01

    Soluble adenylyl cyclase (sAC) is a recently identified source of the ubiquitous second messenger cyclic adenosine 3',5' monophosphate (cAMP). sAC is distinct from the more widely studied source of cAMP, the transmembrane adenylyl cyclases (tmACs); its activity is uniquely regulated by bicarbonate anions, and it is distributed throughout the cytoplasm and in cellular organelles. Due to its unique localization and regulation, sAC has various functions in a variety of physiological systems that are distinct from tmACs. In this review, we detail the known functions of sAC, and we reassess commonly held views of cAMP signaling inside cells.

  6. Cyclic AMP Signaling: A Molecular Determinant of Peripheral Nerve Regeneration

    PubMed Central

    Knott, Eric P.; Assi, Mazen; Pearse, Damien D.

    2014-01-01

    Disruption of axonal integrity during injury to the peripheral nerve system (PNS) sets into motion a cascade of responses that includes inflammation, Schwann cell mobilization, and the degeneration of the nerve fibers distal to the injury site. Yet, the injured PNS differentiates itself from the injured central nervous system (CNS) in its remarkable capacity for self-recovery, which, depending upon the length and type of nerve injury, involves a series of molecular events in both the injured neuron and associated Schwann cells that leads to axon regeneration, remyelination repair, and functional restitution. Herein we discuss the essential function of the second messenger, cyclic adenosine monophosphate (cyclic AMP), in the PNS repair process, highlighting the important role the conditioning lesion paradigm has played in understanding the mechanism(s) by which cyclic AMP exerts its proregenerative action. Furthermore, we review the studies that have therapeutically targeted cyclic AMP to enhance endogenous nerve repair. PMID:25177696

  7. 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.

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2015-12-15

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

  10. Irisin ameliorates hepatic glucose/lipid metabolism and enhances cell survival in insulin-resistant human HepG2 cells through adenosine monophosphate-activated protein kinase signaling.

    PubMed

    So, Wing Yan; Leung, Po Sing

    2016-09-01

    Irisin is a newly identified myokine that promotes the browning of white adipose tissue, enhances glucose uptake in skeletal muscle and modulates hepatic metabolism. However, the signaling pathways involved in the effects on hepatic glucose and lipid metabolism have not been resolved. This study aimed to examine the role of irisin in the regulation of hepatic glucose/lipid metabolism and cell survival, and whether adenosine monophosphate-activated protein kinase (AMPK), a master metabolic regulator in the liver, is involved in irisin's actions. Human liver-derived HepG2 cells were cultured in normal glucose-normal insulin (NGNI) or high glucose-high insulin (HGHI/insulin-resistant) condition. Hepatic glucose and lipid metabolism was evaluated by glucose output and glycogen content or triglyceride accumulation assays, respectively. Our results showed that irisin stimulated phosphorylation of AMPK and acetyl-CoA-carboxylase (ACC) via liver kinase B1 (LKB1) rather than Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) in HepG2 cells. Irisin ameliorated hepatic insulin resistance induced by HGHI condition. Irisin reduced hepatic triglyceride content and glucose output, but increased glycogen content, with those effects reversed by dorsomorphin, an AMPK inhibitor. Furthermore, irisin also stimulated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and promoted cell survival in an AMPK-dependent manner. In conclusion, our data indicate that irisin ameliorates dysregulation of hepatic glucose/lipid metabolism and cell death in insulin-resistant states via AMPK activation. These findings reveal a novel irisin-mediated protective mechanism in hepatic metabolism which provides a scientific basis for irisin as a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes mellitus.

  11. Muscarinic receptor-independent activation of cyclic adenosine monophosphate-dependent protein kinase in rostral ventrolateral medulla underlies the sympathoexcitatory phase of cardiovascular responses during mevinphos intoxication in the rat.

    PubMed

    Tsai, Ching-Yi; Wu, Carol H Y; Chan, Samuel H H; Chang, Alice Y W

    2007-05-01

    As inhibitors of acetylcholinesterase, clinical presentations of poisoning from organophosphate compounds are generally believed to entail overstimulation by the accumulated acetylcholine on muscarinic receptors at peripheral and central synapses. That some patients still yielded to acute organophosphate poisoning despite repeated dosing of atropine suggests that cellular mechanisms that are independent of muscarinic receptor activation may also be engaged in organophosphate poisoning. The present study was undertaken to test the hypothesis that muscarinic receptor-independent activation of cyclic adenosine monophosphate-dependent protein kinase A (PKA) in rostral ventrolateral medulla (RVLM), a medullary site where sympathetic vasomotor tone originates and where the organophosphate poison mevinphos (Mev) acts, is involved in the cardiovascular responses exhibited during organophosphate intoxication. In Sprague-Dawley rats, microinjection bilaterally of Mev (10 nmol) into the RVLM significantly augmented PKA activity in ventrolateral medulla that was not antagonized by coadministration of an equimolar concentration (1 nmol) of atropine or selective muscarinic receptor type M1 (pirenzepine), M2 (methoctramine), M3 (4-diphenyl-acetoxy-N-dimethylpiperidinium), or M4 (tropicamide) inhibitor. Comicroinjection of two selective PKA antagonists (100 pmol), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide and (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolol[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-1][1,6]benzodiazocine-10-carboxylic acid, significantly blunted the initial sympathoexcitatory cardiovascular response and the accompanying augmentation of nitric oxide synthase (NOS I) expression in the ventrolateral medulla exhibited during Mev intoxication; the secondary sympathoinhibitory phase and associated elevation in NOS II expression were unaffected. We conclude that whereas a muscarinic receptor-independent augmentation of PKA

  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. Cyclic AMP and the regeneration of retinal ganglion cell axons.

    PubMed

    Hellström, Mats; Harvey, Alan R

    2014-11-01

    In this paper we present a brief review of studies that have reported therapeutic benefits of elevated cAMP on plasticity and regeneration after injury to the central nervous system (CNS). We also provide new data on the cellular mechanisms by which elevation of cyclic adenosine monophosphate (cAMP) promotes cytokine driven regeneration of adult CNS axons, using the visual system as the experimental model. cAMP is a second messenger for many intracellular signalling pathways. Elevation of cAMP in the eye by intravitreal injection of the cell permeant analogue (8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphate; CPT-cAMP), when added to recombinant ciliary neurotrophic factor (rCNTF), significantly enhances rCNTF-induced regeneration of adult rat retinal ganglion cell (RGC) axons into peripheral nerve (PN) grafted onto transected optic nerve. This effect is mediated to some extent by protein kinase A (PKA) signalling, but CPT-cAMP also acts via PI3K/Akt signalling to reduce suppressor of cytokine signalling protein 3 (SOCS3) activity in RGCs. Another target for cAMP is the exchange protein activated by cAMP (Epac), which can also mediate cAMP-induced axonal growth. Here we describe some novel results and discuss to what extent the pro-regenerative effects of CPT-cAMP on adult RGCs are mediated via Epac as well as via PKA-dependent pathways. We used the established PN-optic nerve graft model and quantified the survival and regenerative growth of adult rat RGCs after intravitreal injection of rCNTF in combination with a selective activator of PKA and/or a specific activator of Epac. Viable RGCs were identified by βIII-tubulin immunohistochemistry and regenerating RGCs retrogradely labelled and quantified after an injection of fluorogold into the distal end of the PN grafts, 4 weeks post-transplantation. The specific agonists of either PKA or Epac were both effective in enhancing the effects of rCNTF on RGC axonal regeneration, but interestingly, injections

  14. In vivo model with targeted cAMP biosensor reveals changes in receptor-microdomain communication in cardiac disease.

    PubMed

    Sprenger, Julia U; Perera, Ruwan K; Steinbrecher, Julia H; Lehnart, Stephan E; Maier, Lars S; Hasenfuss, Gerd; Nikolaev, Viacheslav O

    2015-04-28

    3',5'-cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger that regulates physiological functions by acting in distinct subcellular microdomains. Although several targeted cAMP biosensors are developed and used in single cells, it is unclear whether such biosensors can be successfully applied in vivo, especially in the context of disease. Here, we describe a transgenic mouse model expressing a targeted cAMP sensor and analyse microdomain-specific second messenger dynamics in the vicinity of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). We demonstrate the biocompatibility of this targeted sensor and its potential for real-time monitoring of compartmentalized cAMP signalling in adult cardiomyocytes isolated from a healthy mouse heart and from an in vivo cardiac disease model. In particular, we uncover the existence of a phosphodiesterase-dependent receptor-microdomain communication, which is affected in hypertrophy, resulting in reduced β-adrenergic receptor-cAMP signalling to SERCA.

  15. Involvement of cAMP-PKA pathway in adenosine A1 and A2A receptor-mediated regulation of acetaldehyde-induced activation of HSCs.

    PubMed

    Yang, Yaru; Wang, He; Lv, Xiongwen; Wang, Qi; Zhao, Han; Yang, Feng; Yang, Yan; Li, Jun

    2015-08-01

    The present study was undertaken to investigate the mechanism by which adenosine receptors (ARs)-mediated the cAMP/PKA/CREB signal pathway regulates the activation of acetaldehyde-induced hepatic stellate cells (HSCs). Primary HSCs were isolated from SD rats, cultured in vitro, and activated with different concentrations of acetaldehyde at different time points. Quantitative real-time PCR and Western blotting were used to quantify both protein and mRNA levels of the four AR (A1R, A2AR, A2BR, and A3R) in rat HSCs. Selective inhibitors of PDEs and the Gi/o protein pathway, general AR agonists, and AR subtype specific agents were used to study the AR signaling. The level of cAMP was measured by radio-immunoassay, and the expression of α-SMA, collagen type I and III, PKA and p-CREB were also detected by Western blotting. Acetaldehyde could significantly promote HSC proliferation, with a maximum stimulatory effect observed at 48 h after exposure to 200 μM acetaldehyde. All four AR subtypes could be present in rat HSCs, and the mRNA and protein expression levels for A2AR and A1R in much greater abundance than those for A2BR and A3R. The expression of A2AR and A1R was significantly increased in acetaldehyde-induced HSCs as compared with that of control group, whereas the expression of A2BR and A3R remained unaffected by the addition of acetaldehyde. Curiously, there is coupling of A2AR to the Gs-AC signaling, as well as coupling of A1R to the Gi/o-AC signaling pathway in acetaldehyde-induced HSCs. Both the A2AR and A1R antagonists could suppress the activation of HSC, although they have opposing effects on cAMP signal transduction. These results suggested that a combination of cAMP/PKA/CREB signals via A2AR and A1R likely mediate the activation of acetaldehyde-induced HSCs, and A1R coupled to the Gi/o-AC signaling pathway may be masked by the more predominant A2AR that coupled to the Gs-AC signaling pathway.

  16. Cell-cell contact mediates cAMP secretion in Dictyostelium discoideum.

    PubMed

    Fontana, D R; Price, P L; Phillips, J C

    1991-01-01

    Cyclic adenosine 3':5' monophosphate (cAMP) and cell-cell contact regulate developmental gene expression in Dictyostelium discoideum. Developing D. discoideum amoebae synthesize and secrete cAMP following the binding of cAMP to their surface cAMP receptor, a response called cAMP signaling. We have demonstrated two responses of developing D. discoideum amoebae to cell-cell contact. Cell-cell contact elicits cAMP secretion and alters the amount of cAMP secreted in a subsequent cAMP signaling response. Depending upon experimental conditions, bacterial-amoebal contact and amoebal-amoebal contact can enhance or diminish the amount of cAMP secreted during a subsequent cAMP signaling response. We have hypothesized that cell-cell contact regulates D. discoideum development by altering cellular and extracellular levels of cAMP. To begin testing this hypothesis, these responses were further characterized. The two responses to cell-cell contact are independent, i.e., they can each occur in the absence of the other. The responses to cell-cell contact also have unique temperature dependences when compared to each other, cAMP signaling, and phagocytosis. This suggests that these four responses have unique steps in their transduction mechanisms. The secretion of cAMP in response to cell-cell contact appears to be a non-specific response; contact between D. discoideum amoebae and Enterobacter aerogenes, latex beads, or other amoebae elicits cAMP secretion. Despite the apparent similarities of the effects of bacterial-amoebal and amoebal-amoebal contact on the cAMP signaling response, this contact-induced response appears to be specific. Latex beads addition does not alter the magnitude of a subsequent cAMP signaling response.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2016-09-08

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

  19. The breakdown of adenosine triphosphate in the contraction cycle of the frog sartorius muscle

    PubMed Central

    Mommaerts, W. F. H. M.; Wallner, A.

    1967-01-01

    1. It is confirmed that a fluorodinitrobenzene (FDNB)-treated frog sartorius muscle does not split phosphorylcreatine in the course of its contraction cycle, but does use adenosine triphosphate (ATP). 2. Good stoicheiometric relations between the diminution of ATP and the formation of adenosine diphosphate (ADP), adenosine monophosphate (AMP) and phosphate are obtained, and in a 0·2 sec tetanus at 0° C the net break-down of ATP amounts to 0·27, the total equivalent break-down to 0·34 μmoles/g. 3. There is no difference in this quantity between muscles interrupted at the height of contraction and those that have also relaxed, and, in experiments specifically designed to determine relaxation metabolism separately, no such metabolism is found. Thus, all the ATP-break-down occurs in the contraction phase. PMID:6065882

  20. Revisiting cAMP signaling in the carotid body

    PubMed Central

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

    2014-01-01

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

  1. cAMP and forskolin decrease. gamma. -aminobutyric acid-gated chloride flux in rat brain synaptoneurosomes

    SciTech Connect

    Heuschneider, G.; Schwartz, R.D. )

    1989-04-01

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

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

    SciTech Connect

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

    2009-04-03

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2016-11-15

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

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

    PubMed

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

    2002-01-01

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

  6. Purine metabolism in adenosine deaminase deficiency.

    PubMed Central

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

    1976-01-01

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

  7. 8-OH-DPAT facilitated memory consolidation and increased hippocampal and cortical cAMP production.

    PubMed

    Manuel-Apolinar, L; Meneses, A

    2004-01-05

    Animals were submitted to an associative learning task named Pavlovian/instrumental autoshaping (P/I-A) and treated with selective 5-HT1A and 5-HT7 receptor agonists and antagonists. Next, they were sacrificed, their brains removed, dissected and changes on cortical and hippocampal cyclic adenosine monophosphate (cAMP) production were determined. Results revealed that, the 8-OH-DPAT treatment facilitated memory consolidation of autoshaping and that effect was blocked completely by WAY100635 and partially by DR4004. WAY100635 or DR4004 alone had no effect on autoshaping. The cAMP results were complex and yielded no clear relationship to the memory results. Thus, cortical and hippocampal increased on cAMP production was observed following administration of the 5-HT(1A/7) agonist 8-OH-DPAT. The memory effect was, completely or partially, reversed by the selective antagonists WAY100635 (5-HT1A) or DR4004 (5-HT7), respectively.

  8. Pharmacological targeting of AKAP-directed compartmentalized cAMP signalling.

    PubMed

    Dema, Alessandro; Perets, Ekaterina; Schulz, Maike Svenja; Deák, Veronika Anita; Klussmann, Enno

    2015-12-01

    The second messenger cyclic adenosine monophosphate (cAMP) can bind and activate protein kinase A (PKA). The cAMP/PKA system is ubiquitous and involved in a wide array of biological processes and therefore requires tight spatial and temporal regulation. Important components of the safeguard system are the A-kinase anchoring proteins (AKAPs), a heterogeneous family of scaffolding proteins defined by its ability to directly bind PKA. AKAPs tether PKA to specific subcellular compartments, and they bind further interaction partners to create local signalling hubs. The recent discovery of new AKAPs and advances in the field that shed light on the relevance of these hubs for human disease highlight unique opportunities for pharmacological modulation. This review exemplifies how interference with signalling, particularly cAMP signalling, at such hubs can reshape signalling responses and discusses how this could lead to novel pharmacological concepts for the treatment of disease with an unmet medical need such as cardiovascular disease and cancer.

  9. 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

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

    PubMed

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

    2015-01-01

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

  11. cAMP analogs and their metabolites enhance TREK-1 mRNA and K+ current expression in adrenocortical cells.

    PubMed

    Enyeart, Judith A; Liu, Haiyan; Enyeart, John J

    2010-03-01

    bTREK-1 K(+) channels set the resting membrane potential of bovine adrenal zona fasciculata (AZF) cells and function pivotally in the physiology of cortisol secretion. Adrenocorticotropic hormone controls the function and expression of bTREK-1 channels through signaling mechanisms that may involve cAMP and downstream effectors including protein kinase A (PKA) and exchange protein 2 directly activated by cAMP (Epac2). Using patch-clamp and Northern blot analysis, we explored the regulation of bTREK-1 mRNA and K(+) current expression by cAMP analogs and several of their putative metabolites in bovine AZF cells. At concentrations sufficient to activate both PKA and Epac2, 8-bromoadenosine-cAMP enhanced the expression of both bTREK-1 mRNA and K(+) current. N(6)-Benzoyladenosine-cAMP, which activates PKA but not Epac, also enhanced the expression of bTREK-1 mRNA and K(+) current measured at times from 24 to 96 h. An Epac-selective cAMP analog, 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (8CPT-2'-OMe-cAMP), potently stimulated bTREK-1 mRNA and K(+) current expression, whereas the nonhydrolyzable Epac activator 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, Sp-isomer was ineffective. Metabolites of 8CPT-2'-OMe-cAMP, including 8-(4-chlorophenylthio)-2'-O-methyladenosine-5'-O-monophosphate and 8CPT-2'-OMe-adenosine, promoted the expression of bTREK-1 transcripts and ion current with a temporal pattern, potency, and effectiveness resembling that of the parent compound. Likewise, at low concentrations, 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP; 30 microM) but not its nonhydrolyzable analog 8-(4-chlorophenylthio)-cAMP, Sp-isomer, enhanced the expression of bTREK-1 mRNA and current. 8CPT-cAMP metabolites, including 8CPT-adenosine and 8CPT-adenine, also increased bTREK-1 expression. These results indicate that cAMP increases the expression of bTREK-1 mRNA and K(+) current through a cAMP-dependent but Epac2-independent mechanism. They further demonstrate that one or more metabolites of 8

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

    PubMed Central

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

    2015-01-01

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

  13. Activation of cyclic amp/protein kinase: a signaling pathway enhances osteoblast cell adhesion on biomaterials for regenerative engineering.

    PubMed

    Lo, Kevin W-H; Ashe, Keshia M; Kan, Ho Man; Lee, Duron A; Laurencin, Cato T

    2011-04-01

    Osteoblast cell adhesion on biomaterials is an important goal for implants to be useful in bone regeneration technologies. The adhesion of osteoblastic cells to biomaterials has been investigated in the field of bone regenerative engineering. Previous work from our group demonstrated that osteoblastic cells adhering to biodegradable biomaterials require the expression of integrins on the cell surface. However, the underlying molecular signaling mechanism is still not fully clear. We report here that cyclic adenosine monophosphate (cAMP), a small signaling molecule, regulates osteoblast cell adhesion to biomaterial surfaces. We used an in vitro cell adhesion assay to demonstrate that at 0.1 mM, 8-Br-cAMP, a cell-permeable cAMP analog, significantly enhances osteoblast-like cells' (MC3T3-E1) adherence to biomaterials. Moreover, we demonstrate that a commonly used cAMP-elevating agent, forskolin, promotes cell adhesion similar to that of the cell permeable cAMP analog. By using different target-specific cAMP analogs: 8-CPT-2Me-cAMP which specifically activates exchange protein activated by cAMP (Epac), and 6-Bnz-cAMP which specifically activates protein kinase A (PKA), we observed that the PKA signaling pathway plays a dominant role in this process. Thus, this report suggests a new method to enhance osteoblast cell adhesion on biodegradable biomaterials for bone regenerative engineering applications.

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

    SciTech Connect

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

    1982-09-01

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

  15. Neuronal activity promotes myelination via a cAMP pathway.

    PubMed

    Malone, Misti; Gary, Devin; Yang, In Hong; Miglioretti, Anna; Houdayer, Thierry; Thakor, Nitish; McDonald, John

    2013-06-01

    Neuronal activity promotes myelination in vivo and in vitro. However, the molecular events that mediate activity-dependent myelination are not completely understood. Seven, daily 1 h sessions of patterned electrical stimulation (ESTIM) promoted myelin segment formation in mixed cultures of dorsal root ganglion (DRG) neurons and oligodendrocytes (OLs); the increase in myelination was frequency-dependent. Myelin segment formation was also enhanced following exposure of DRGs to ESTIM prior to OL addition, suggesting that ESTIM promotes myelination in a manner involving neuron-specific signaling. Cyclic adenosine monophosphate (cAMP) levels in DRGs were increased three-fold following ESTIM, and artificially increasing cAMP mimicked the ability of ESTIM to promote myelination. Alternatively, inhibiting the cAMP pathway suppressed ESTIM-induced myelination. We used compartmentalized, microfluidic platforms to isolate DRG soma from OLs and assessed cell-type specific effects of ESTIM on myelination. A selective increase or decrease in DRG cAMP levels resulted in enhanced or suppressed myelination, respectively. This work describes a novel role for the cAMP pathway in neurons that results in enhanced myelination.

  16. Fast determination of adenosine 5'-triphosphate (ATP) and its catabolites in royal jelly using ultraperformance liquid chromatography.

    PubMed

    Zhou, Ling; Xue, XiaoFeng; Zhou, JinHui; Li, Yi; Zhao, Jing; Wu, LiMing

    2012-09-12

    To obtain insight into the metabolic regulation of adenosine 5'-triphosphate (ATP) in royal jelly and to determine whether ATP and its catabolites can be used as objective parameters to evaluate the freshness and quality of royal jelly (RJ), a rapid ultraperformance liquid chromatography (UPLC) method has been developed for feasible separation and quantitation of ATP and its catabolites in RJ, namely, adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), inosine monophosphate (IMP), inosine (HxR), and hypoxanthine (Hx). The analytes in the sample were extracted using 5% precooled perchloric acid. Chromatographic separation was performed on a Waters Acquity UPLC system with a Waters BEH Shield RP18 column and gradient elution based on a mixture of two solvents: solvent A, 50 mM phosphate buffer (pH 6.5); and solvent B, acetonitrile. The recoveries were in the range of 86.0-102.3% with RSD of no more than 3.6%. The correlation coefficients of six analytes were high (r(2) ≥ 0.9988) and within the test ranges. The limits of detection and quantification for the investigated compounds were lower, at 0.36-0.68 and 1.22-2.30 mg/kg, respectively. The overall intra- and interday RSDs were no more than 1.8%. The developed method was successfully applied to the analysis of the analytes in samples. The results showed that ATP in RJ sequentially degrades to ADP, AMP, IMP, HxR, and Hx during storage.

  17. Alpha-Lipoic acid increases energy expenditure by enhancing adenosine monophosphate-activated protein kinase-peroxisome proliferator-activated receptor-gamma coactivator-1alpha signaling in the skeletal muscle of aged mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Skeletal muscle mitochondrial dysfunction is associated with aging and diabetes, which decreases respiratory capacity and increases reactive oxygen species. Lipoic acid (LA) possesses antioxidative and antidiabetic properties. Metabolic action of LA is mediated by activation of adenosine monophospha...

  18. [Interaction of 8-substituted derivatives and adenosine-3',5'-cyclophosphate esters with protein kinase from pig brain].

    PubMed

    Guliaev, N N; Tunitskaia, V L; Nesterova, M V; Mazurova, L A; Murtuzaev, I M

    1977-11-01

    A synthesis of previously unknown 8-substituted derivatives and alkyl esters of cyclic adenosine-3',5'-monophosphate, containing reactive groups, was carried out. The interaction of the compounds obtained with a homogeneous preparation of protein kinase from pig brain was studied. It was found that all compounds, with the exception of neutral esters of 3',5'-AMP, activate the enzyme and competitively inhibit 3H-labelled 3',5'-cAMP binding by the regulatory subunit of protein kinase. The activating effect and affinity of 8-(beta-aminoethylamino)-3',5'-cAMP for protein kinase was 10 times lower than that for 3',5'-cAMP and other 8-substituted derivatives of the cyclic nucleotide. It was found that 8-(N-chloroacetylaminoethylamino)-3',5'-cAMP interaction with the enzyme is of irreversible type, which suggest covalent blocking of the nucleophilic group of the 3',5'-cAMP binding site of protein kinase. The data obtained indicate that the 3',5'-cAMP molecule is bound to the regulatory site of protein kinase in the syn-conformation. The previously made assumption on the crucial importance of the negative charge in the 3',5'-cyclophosphate system for the interaction of cyclic AMP with the regulatory subunit of protein kinase has been thus confirmed.

  19. (S)-α-Chlorohydrin Inhibits Protein Tyrosine Phosphorylation through Blocking Cyclic AMP - Protein Kinase A Pathway in Spermatozoa

    PubMed Central

    Zheng, Weiwei; Yang, Bei; Pi, Jingbo; He, Gengsheng; Qu, Weidong

    2012-01-01

    α-Chlorohydrin is a common contaminant in food. Its (S)-isomer, (S)-α-chlorohydrin (SACH), is known for causing infertility in animals by inhibiting glycolysis of spermatozoa. The aim of present work was to examine the relationship between SACH and protein tyrosine phosphorylation (PTP), which plays a critical role in regulating mammalian sperm capacitation. In vitro exposure of SACH 50 µM to isolated rat epididymal sperm inhibited PTP. Sperm-specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) activities, the intracellular adenosine 5′-triphosphate (ATP) levels, 3′-5′-cyclic adenosine monophosphate (cAMP) levels and phosphorylation of protein kinase A (PKA) substrates in rat sperm were diminished dramatically, indicating that both glycolysis and the cAMP/PKA signaling pathway were impaired by SACH. The inhibition of both PTP and phosphorylation of PKA substrates by SACH could be restored by addition of cAMP analog dibutyryl-cAMP (dbcAMP) and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Moreover, addition of glycerol protected glycolysis, ATP levels, phosphorylation of PKA substrates and PTP against the influence of SACH. These results suggested SACH inhibited PTP through blocking cAMP/PKA pathway in sperm, and PTP inhibition may play a role in infertility associated with SACH. PMID:22916194

  20. Abnormal Mitochondrial cAMP/PKA Signaling Is Involved in Sepsis-Induced Mitochondrial and Myocardial Dysfunction

    PubMed Central

    Neviere, Remi; Delguste, Florian; Durand, Arthur; Inamo, Jocelyn; Boulanger, Eric; Preau, Sebastien

    2016-01-01

    Adrenergic receptors couple to Gs-proteins leading to transmembrane adenylyl cyclase activation and cytosolic cyclic adenosine monophosphate (cAMP) production. Cyclic AMP is also produced in the mitochondrial matrix, where it regulates respiration through protein kinase A (PKA)-dependent phosphorylation of respiratory chain complexes. We hypothesized that a blunted mitochondrial cAMP-PKA pathway would participate in sepsis-induced heart dysfunction. Adult male mice were subjected to intra-abdominal sepsis. Mitochondrial respiration of cardiac fibers and myocardial contractile performance were evaluated in response to 8Br-cAMP, PKA inhibition (H89), soluble adenylyl cyclase inhibition (KH7), and phosphodiesterase inhibition (IBMX; BAY60-7550). Adenosine diphosphate (ADP)-stimulated respiratory rates of cardiac fibers were reduced in septic mice. Compared with controls, stimulatory effects of 8Br-cAMP on respiration rates were enhanced in septic fibers, whereas inhibitory effects of H89 were reduced. Ser-58 phosphorylation of cytochrome c oxidase subunit IV-1 was reduced in septic hearts. In vitro, incubation of septic cardiac fibers with BAY60-7550 increased respiratory control ratio and improved cardiac MVO2 efficiency in isolated septic heart. In vivo, BAY60-7550 pre-treatment of septic mice have limited impact on myocardial function. Mitochondrial cAMP-PKA signaling is impaired in the septic myocardium. PDE2 phosphodiesterase inhibition by BAY60-7550 improves mitochondrial respiration and cardiac MVO2 efficiency in septic mice. PMID:27973394

  1. Identification of a Specific Assembly of the G Protein Golf as a Critical and Regulated Module of Dopamine and Adenosine-Activated cAMP Pathways in the Striatum

    PubMed Central

    Hervé, Denis

    2011-01-01

    In the principal neurons of striatum (medium spiny neurons, MSNs), cAMP pathway is primarily activated through the stimulation of dopamine D1 and adenosine A2A receptors, these receptors being mainly expressed in striatonigral and striatopallidal MSNs, respectively. Since cAMP signaling pathway could be altered in various physiological and pathological circumstances, including drug addiction and Parkinson’s disease, it is of crucial importance to identify the molecular components involved in the activation of this pathway. In MSNs, cAMP pathway activation is not dependent on the classical Gs GTP-binding protein but requires a specific G protein subunit heterotrimer containing Gαolf/β2/γ7 in particular association with adenylyl cyclase type 5. This assembly forms an authentic functional signaling unit since loss of one of its members leads to defects of cAMP pathway activation in response to D1 or A2A receptor stimulation, inducing dramatic impairments of behavioral responses dependent on these receptors. Interestingly, D1 receptor (D1R)-dependent cAMP signaling is modulated by the neuronal levels of Gαolf, indicating that Gαolf represents the rate-limiting step in this signaling cascade and could constitute a critical element for regulation of D1R responses. In both Parkinsonian patients and several animal models of Parkinson’s disease, the lesion of dopamine neurons produces a prolonged elevation of Gαolf levels. This observation gives an explanation for the cAMP pathway hypersensitivity to D1R stimulation, occurring despite an unaltered D1R density. In conclusion, alterations in the highly specialized assembly of Gαolf/β2/γ7 subunits can happen in pathological conditions, such as Parkinson’s disease, and it could have important functional consequences in relation to changes in D1R signaling in the striatum. PMID:21886607

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

    PubMed Central

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

    2014-01-01

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

  3. Evidence for cAMP as a mediator of gonadotropin secretion from male pituitaries

    SciTech Connect

    Bourne, G.A.; Baldwin, D.M.

    1987-09-01

    The purpose of this study was to use sodium flufenamate, a compound that inhibits gonadotropin-releasing hormone (GnRH)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production in the pituitary, to evaluate the potential role of cAMP as a mediator of GnRH-stimulated gonadotropin secretion from male pituitaries. Quartered male pituitaries were perifused at 37/sup 0/C and sequential effluent fractions collected every 10 min. Infusions of GnRH resulted in a twofold increase in luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. Cycloheximide, 5 ..mu..M, completely inhibited the GnRH-stimulated LH and FSH secretion. Infusions of 0.1 mM flufenamate had similar effects on gonadotropin secretion as cycloheximide, whereas the administration of 5 mM dibutyryl cAMP in combination with GnRH and flufenamate restored the secretory responses of both hormones. The flufenamate-inhibited GnRH stimulated LH and FSH release, which was restored by DBcAMP and appeared to be protein synthesis dependent and specific for cAMP.These results suggest an indirect role for cAMP as a mediator of gonadotropin secretion from male pituitaries. However, in contrast to female pituitaries, the secretion of these hormones form male pituitaries is completely dependent on cAMP and de novo protein synthesis.

  4. Effects of selective phosphodiesterase inhibition on cyclic AMP hydrolysis in rat cerebral cortical slices.

    PubMed Central

    Challiss, R. A.; Nicholson, C. D.

    1990-01-01

    1. The effects of selective inhibition of phosphodiesterase activities on the concentration and rate of hydrolysis of adenosine 3':5' cyclic-monophosphate (cyclic AMP) in rat cerebral cortical slices has been studied. 2. Isoprenaline caused a rapid, concentration-dependent increase in cyclic AMP concentration to new steady-state levels (basal: 7.1 +/- 0.7; 10 microM isoprenaline: 14.3 +/- 1.4 pmol mg-1 protein). Addition of a beta-adrenoceptor antagonist to isoprenaline-stimulated cerebral cortical slices caused a rapid decrease in cyclic AMP concentration to basal levels (t1/2: 58 +/- 18 s). 3. Preincubation of slices for 30 min with the phosphodiesterase inhibitors 1-methyl-3-isobutylxanthine, denbufylline, rolipram or Ro20,1724 caused concentration-dependent increases in basal and isoprenaline-stimulated cyclic AMP concentrations and decreased the rate of cyclic AMP hydrolysis measured after addition of a beta-adrenoceptor antagonist. However, SKF 94120 and zaprinast had none of these effects. 4. The results are discussed with respect to previous studies of phosphodiesterase isozymic activities isolated from cerebrum and it is suggested that the Ca2+/calmodulin-independent, low Km cyclic AMP phosphodiesterase isozyme, which is selectively inhibited by denbufylline, rolipram and Ro20,1724, and is present in cerebrum is of critical importance to the regulation of cyclic AMP concentration in this tissue. PMID:2158837

  5. [The inhibiting effect of 8-Cl-adenosine-3',5'-cyclophosphate on the growth of melanoma B-16 in mice].

    PubMed

    Nesterov, M V; Baranova, L A; Sologub, V K; Khropov, Iu V; Severin, E S

    1992-01-01

    A site-selective analogue of the cyclic adenosine monophosphate 8-chloro-adenosine-3',5'-cyclophosphate was studied for its effects on the growth of transplanted murine melanoma B-16. When the agent was given to the mice, a substantial effect on the growth of the tumor was produced by a number of factors, which included the route of administration, concentration of the agent, the time and duration of therapy. Intraperitoneal injections of the agent in a dose of 20 mg/kg/day which were made during three consecutive days, beginning from day 5 after tumor transplantation caused a 58% decrease in tumor growth as compared to the controls. An examination of tumour biopsy specimen revealed that after a course of the injections there was a significant suppression of the activity of cAMP-dependent protein kinase, type I, and a drastic increase in that of cAMP-dependent protein kinase, type II.

  6. Termination of cAMP signals by Ca2+ and Gαi via extracellular Ca2+ sensors

    PubMed Central

    Gerbino, Andrea; Ruder, Warren C.; Curci, Silvana; Pozzan, Tullio; Zaccolo, Manuela; Hofer, Aldebaran M.

    2005-01-01

    Termination of cyclic adenosine monophosphate (cAMP) signaling via the extracellular Ca2+-sensing receptor (CaR) was visualized in single CaR-expressing human embryonic kidney (HEK) 293 cells using ratiometric fluorescence resonance energy transfer–dependent cAMP sensors based on protein kinase A and Epac. Stimulation of CaR rapidly reversed or prevented agonist-stimulated elevation of cAMP through a dual mechanism involving pertussis toxin–sensitive Gαi and the CaR-stimulated increase in intracellular [Ca2+]. In parallel measurements with fura-2, CaR activation elicited robust Ca2+ oscillations that increased in frequency in the presence of cAMP, eventually fusing into a sustained plateau. Considering the Ca2+ sensitivity of cAMP accumulation in these cells, lack of oscillations in [cAMP] during the initial phases of CaR stimulation was puzzling. Additional experiments showed that low-frequency, long-duration Ca2+ oscillations generated a dynamic staircase pattern in [cAMP], whereas higher frequency spiking had no effect. Our data suggest that the cAMP machinery in HEK cells acts as a low-pass filter disregarding the relatively rapid Ca2+ spiking stimulated by Ca2+-mobilizing agonists under physiological conditions. PMID:16247029

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  9. Double electron–electron resonance reveals cAMP-induced conformational change in HCN channels

    PubMed Central

    Zagotta, William N.; Stoll, Stefan

    2014-01-01

    Binding of 3′,5′-cyclic adenosine monophosphate (cAMP) to hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels regulates their gating. cAMP binds to a conserved intracellular cyclic nucleotide-binding domain (CNBD) in the channel, increasing the rate and extent of activation of the channel and shifting activation to less hyperpolarized voltages. The structural mechanism underlying this regulation, however, is unknown. We used double electron–electron resonance (DEER) spectroscopy to directly map the conformational ensembles of the CNBD in the absence and presence of cAMP. Site-directed, double-cysteine mutants in a soluble CNBD fragment were spin-labeled, and interspin label distance distributions were determined using DEER. We found motions of up to 10 Å induced by the binding of cAMP. In addition, the distributions were narrower in the presence of cAMP. Continuous-wave electron paramagnetic resonance studies revealed changes in mobility associated with cAMP binding, indicating less conformational heterogeneity in the cAMP-bound state. From the measured DEER distributions, we constructed a coarse-grained elastic-network structural model of the cAMP-induced conformational transition. We find that binding of cAMP triggers a reorientation of several helices within the CNBD, including the C-helix closest to the cAMP-binding site. These results provide a basis for understanding how the binding of cAMP is coupled to channel opening in HCN and related channels. PMID:24958877

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

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

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

  11. Structural requirements for parathyroid hormone action in mature bone. Effects on release of cyclic adenosine monophosphate and bone gamma-carboxyglutamic acid-containing protein from perfused rat hindquarters.

    PubMed Central

    Calvo, M S; Fryer, M J; Laakso, K J; Nissenson, R A; Price, P A; Murray, T M; Heath, H

    1985-01-01

    To determine the structural requirements for parathyroid hormone (PTH) activity in mature bone, we perfused the surgically isolated hindquarters of adult male rats with either native bovine PTH-(1-84) [bPTH-(1-84)] or the synthetic amino-terminal fragment, bovine PTH-(1-34) [bPTH-(1-34)]. Changes in the release of cyclic AMP (cAMP) and bone Gla protein (BGP) were monitored as evidence of bone-specific response to PTH; tissue specificity of the cAMP response was confirmed through in vitro examination on nonskeletal tissue response to PTH. Biologically active, monoiodinated 125I-bPTH-(1-84) was administered to determine if mature murine bone cleaves native hormone. We found that perfused rat bone continuously releases BGP, and that both bPTH-(1-84) and bPTH-(1-34) acutely suppress this release. In addition, both hormones stimulate cAMP release from perfused rat hindquarters. When examined on a molar basis, the magnitude of the cAMP response was dose-dependent and similar for both hormones, with doses yielding half-maximal cAMP responses. The response for bPTH-(1-34) was 0.5 nmol and for bPTH-(1-84) was 0.7 nmol. Moreover, biologically active 125I-bPTH-(1-84) was not metabolized in our hindquarter perfusion system. These findings indicate that PTH-(1-84) does not require extraskeletal or skeletal cleavage to an amino-terminal fragment in order to stimulate cAMP generation in, or suppress BGP release from, mature rat bone. PMID:3001148

  12. AMP metabolism in the marine bacterium Beneckea natriegens.

    PubMed

    Pickard, M A; Whelihan, J A; Knowles, C J

    1980-05-01

    The catabolism of AMP by preparations from Beneckea natriegens has been reexamined. In the absence of ATP, cell-free extracts catabolized AMP via adenosine to inosine. When ATP was present, adenylate kinase converted AMP to ADP, lowering the rate of AMP catabolism. Particle-free supernatants (225,000 x g) metabolized AMP alone slowly, but adenylate kinase was active when ATP was added. Washed particulate fractions contained AMP nucleotidase activity which converted AMP to adenosine; in the presence of ATP, adenosine formation was reduced by residual adenylate kinase associated with the particulate fraction. IMP was not detected as a metabolite in these experiments.

  13. Cardiovascular selectivity of adenosine receptor agonists in anaesthetized dogs.

    PubMed Central

    Gerencer, R. Z.; Finegan, B. A.; Clanachan, A. S.

    1992-01-01

    1. In order to determine the relevance of adenosine (Ado) receptor classification obtained from in vitro methods to the cardiovascular actions of Ado agonists in vivo, the cardiovascular effects of adenosine 5'-monophosphate (AMP), N6-cyclohexyladenosine (CHA, 400 fold A1-selective), 5'-N-ethyl-carboxamidoadenosine (NECA, A1 approximately A2) and 2-phenylaminoadenosine (PAA, 5 fold A2-selective) were compared in open-chest, fentanyl-pentobarbitone anaesthetized dogs. 2. Graded doses of CHA (10 to 1000 micrograms kg-1), NECA (0.5 to 100 micrograms kg-1) or PAA (0.1 to 20 micrograms kg-1) were administered intravenously and changes in haemodynamics and myocardial contractility were assessed 10 min following each dose. The effects of graded infusions of AMP (200 to 1000 micrograms kg-1 min-1) were also evaluated. 3. AMP and each of the Ado analogues (NECA > PAA > CHA) increased the systemic vascular conductance index (SVCI) in a dose-dependent manner and reduced mean arterial pressure (MAP). At doses causing similar increases in SVCI, these agonists caused (i) similar reflex increases in heart rate (HR) and cardiac index (CI) and decreases in AV conduction interval (AVi) and (ii) similar increases in coronary vascular conductance (CVC). 4. After cardiac autonomic blockade with atropine (0.2 mg kg-1) and propranolol (1 mg kg-1), AMP, CHA and PAA still increased SVCI and CVC and decreased MAP. CHA and PAA had no marked effects on HR, CI or AVi. As in the absence of cardiac autonomic blockade, equieffective vasodilator doses of CHA and PAA had identical effects on CVC, CI and AVi.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1467827

  14. Triple negative breast cancer development can be selectively suppressed by sustaining an elevated level of cellular cyclic AMP through simultaneously blocking its efflux and decomposition

    PubMed Central

    Wang, Wei; Li, Yue; Zhu, Jessica Y.; Fang, Dongdong; Ding, Han-Fei; Dong, Zheng; Jing, Qing; Su, Shi-Bing; Huang, Shuang

    2016-01-01

    Triple negative breast cancer (TNBC) has the highest mortality among all breast cancer types and lack of targeted therapy is a key factor contributing to its high mortality rate. In this study, we show that 8-bromo-cAMP, a cyclic adenosine monophosphate (cAMP) analog at high concentration (> 1 mM) selectively suppresses TNBC cell growth. However, commonly-used cAMP-elevating agents such as adenylyl cyclase activator forskolin and pan phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) are ineffective. Inability of cAMP elevating agents to inhibit TNBC cell growth is due to rapid diminution of cellular cAMP through efflux and decomposition. By performing bioinformatics analyses with publically available gene expression datasets from breast cancer patients/established breast cancer cell lines and further validating using specific inhibitors/siRNAs, we reveal that multidrug resistance-associated protein 1/4 (MRP1/4) mediate rapid cAMP efflux while members PDE4 subfamily facilitate cAMP decomposition. When cAMP clearance is prevented by specific inhibitors, forskolin blocks TNBC's in vitro cell growth by arresting cell cycle at G1/S phase. Importantly, cocktail of forskolin, MRP inhibitor probenecid and PDE4 inhibitor rolipram suppresses TNBC in vivo tumor development. This study suggests that a TNBC-targeted therapeutic strategy can be developed by sustaining an elevated level of cAMP through simultaneously blocking its efflux and decomposition. PMID:27901486

  15. Sustained delivery of dbcAMP by poly(propylene carbonate) micron fibers promotes axonal regenerative sprouting and functional recovery after spinal cord hemisection.

    PubMed

    Xia, Tongliang; Ni, Shilei; Li, Xingang; Yao, Jun; Qi, Hongxu; Fan, Xiaoyong; Wang, Jiangang

    2013-11-13

    This study describes the use of poly(propylene carbonate) (PPC) electrospun fibers as vehicle for the sustained delivery of dibutyryl cyclic adenosine monophosphate (dbcAMP) to the hemisected spinal cord. The dbcAMP and PPC were uniformly mixed with acetonitrile; then, electrospinning was used to generate micron fibers. The release of dbcAMP was assessed by ELISA in vitro. Our results showed that the encapsulation of dbcAMP in the fibers led to stable and prolonged release in vitro. The PPC micron fibers containing dbcAMP and the PPC micron fibers without dbcAMP were then implanted into the hemisected thoracic spinal cord, followed by testing of the functional recovery and immunohistochemistry. Compared with the control group, sustained delivery of dbcAMP promoted axonal regenerative sprouting and functional recovery and reduced glial scar formation, and the PPC micron fibers without dbcAMP did not have these effects. Our findings demonstrated the feasibility of using PPC electrospun fibers containing dbcAMP for spinal cord injury. The approach described here also will provide a platform for the potential delivery of other axon-growth-promoting or scar-inhibiting agents.

  16. cAMP Promotes Cell Migration Through Cell Junctional Complex Dynamics and Actin Cytoskeleton Remodeling: Implications in Skin Wound Healing.

    PubMed

    Kim, Mi Ok; Ryu, Jung Min; Suh, Han Na; Park, Soo Hyun; Oh, Yeon-Mok; Lee, Sang Hun; Han, Ho Jae

    2015-11-01

    Stem cells have attracted great interest for their therapeutic capacity in tissue regeneration. Cyclic adenosine 3',5'-monophosphate (cAMP), existing in high concentration at wound sites, mediated various signaling pathways such as cytoskeleton dynamics, cell adhesion, and cell migration in stem cells, which suggest the critical roles of cAMP in the wound healing process through functional regulation of stem cells. However, the mechanisms behind the effect of cAMP on mouse embryonic stem cell (mESC) motility and its roles on skin wound healing remain to be fully elucidated. In the present study, 8-Bromo cAMP-treated mESCs showed significant wound closure and improved neovascularization. Moreover, 8-Bromo cAMP stimulated mESC migration into the wound bed. 8-Bromo cAMP also increased ESC motility in in vitro migration assay. 8-Bromo cAMP induced myosin light chain phosphorylation through Rac1 and Cdc42 signaling, which were involved in 8-Bromo cAMP-induced decrease in expression of junction proteins (connexin 43, E-cadherin, and occludin) at the plasma membrane. Subsequently, 8-Bromo cAMP induced the disruption of cell junctions (including gap junctions, adherens junctions, and tight junctions), which reduced the function of the gap junctions and cell adhesion. In addition, 8-Bromo cAMP-induced Rac1 and Cdc42 activation increased Arp3, TOCA, PAK, and N-WASP expression, but decreased cofilin phosphorylation level, which elicited actin cytoskeleton remodeling. In contrast to the control, 8-Bromo cAMP evoked a substantial migration of cells into the denuded area, which was blocked by the small interfering RNAs of the signaling pathway-related molecules or by inhibitors. In conclusion, cAMP enhanced the migration of mESCs through effective coordination of junctional disruption and actin cytoskeleton remodeling, which increased the wound healing capacity of ESCs.

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

    PubMed

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

    2014-10-01

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

  18. The Mucosal Adjuvant Cyclic di-AMP Exerts Immune Stimulatory Effects on Dendritic Cells and Macrophages

    PubMed Central

    Libanova, Rimma; Lienenklaus, Stefan; Weiss, Siegfried; Guzmán, Carlos A.

    2014-01-01

    The cyclic di-nucleotide bis-(3′,5′)-cyclic dimeric adenosine monophosphate (c-di-AMP) is a candidate mucosal adjuvant with proven efficacy in preclinical models. It was shown to promote specific humoral and cellular immune responses following mucosal administration. To date, there is only fragmentary knowledge on the cellular and molecular mode of action of c-di-AMP. Here, we report on the identification of dendritic cells and macrophages as target cells of c-di-AMP. We show that c-di-AMP induces the cell surface up-regulation of T cell co-stimulatory molecules as well as the production of interferon-β. Those responses were characterized by in vitro experiments with murine and human immune cells and in vivo studies in mice. Analyses of dendritic cell subsets revealed conventional dendritic cells as principal responders to stimulation by c-di-AMP. We discuss the impact of the reported antigen presenting cell activation on the previously observed adjuvant effects of c-di-AMP in mouse immunization studies. PMID:24755640

  19. PKA and Epac activation mediates cAMP-induced vasorelaxation by increasing endothelial NO production.

    PubMed

    García-Morales, Verónica; Cuíñas, Andrea; Elíes, Jacobo; Campos-Toimil, Manuel

    2014-03-01

    Vascular relaxation induced by 3',5'-cyclic adenosine monophosphate (cAMP) is both endothelium-dependent and endothelium-independent, although the underlying signaling pathways are not fully understood. Aiming to uncover potential mechanisms, we performed contraction-relaxation experiments on endothelium-denuded and intact rat aorta rings and measured NO levels in isolated human endothelial cells using single cell fluorescence imaging. The vasorelaxant effect of forskolin, an adenylyl cyclase activator, was decreased after selective inhibitor of protein kinase A (PKA), a cAMP-activated kinase, or L-NAME, an endothelial nitric oxide synthase (eNOS) inhibitor, only in intact aortic rings. Both selective activation of PKA with 6-Bnz-cAMP and exchange protein directly activated by cAMP (Epac) with 8-pCPT-2'-O-Me-cAMP significantly relaxed phenylephrine-induced contractions. The vasorelaxant effect of the Epac activator, but not that of the PKA activator, was reduced by endothelium removal. Forskolin, dibutyryl cAMP (a cAMP analogue), 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP increased NO levels in endothelial cells and the forskolin effect was significantly inhibited by inactivation of both Epac and PKA, and eNOS inhibition. Our results indicate that the endothelium-dependent component of forskolin/cAMP-induced vasorelaxation is partially mediated by an increase in endothelial NO release due to an enhanced eNOS activity through PKA and Epac activation in endothelial cells.

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

    PubMed Central

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

    2011-01-01

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

  1. 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.

  2. Growth rate regulation of lac operon expression in Escherichia coli is cyclic AMP dependent.

    PubMed

    Kuo, Jong-Tar; Chang, Yu-Jen; Tseng, Ching-Ping

    2003-10-23

    In contrast to the ribosomal RNA gene expression increasing with growth rate, transcription of the lac operon is downregulated by cell growth rate. In continuous culture, growth rate regulation of lac promoter was independent of carbon substrate used and its location on the chromosome. Since the lac operon is activated by cyclic adenosine monophosphate (cAMP), which decreases with increasing cell growth rate, expression of plac-lacZ reporter fusion was analyzed in cya mutant under various growth conditions. The results demonstrated that expression of plac-lacZ in cya mutant was both lower and growth rate independent. In addition, ppGpp (guanosine tetraphosphate) was not involved in the mechanism of growth rate regulation of the lac promoter. Thus, the results of this study indicate that cAMP mediates the growth rate-dependent regulation of lac operon expression in Escherichia coli.

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

    PubMed

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

    2013-02-15

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

  4. Development and application of an LC-MS/MS method for measuring the effect of (partial) agonists on cAMP accumulation in vitro.

    PubMed

    Goutier, W; Spaans, P A; van der Neut, M A W; McCreary, A C; Reinders, J H

    2010-04-30

    Cyclic-adenosine monophosphate (cAMP) plays an important role in cell signalling and is widely used as a marker for receptor activation and as a target for treating various diseases. In this paper we present the development and validation of a new method for the determination of cAMP and ATP (adenosine triphosphate) and other nucleotides in a biological system by combining zwitterionic hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectrometry (MS/MS). The HILIC-MS/MS method was developed for the simultaneous quantitative analysis of cAMP and ATP, and was validated by assessment of linearity (over a range from 0.5 to 100nM for cAMP and 50 nM to 50 microM for ATP (r(2)>0.999)), resolution, limit of detection (0.5 and 50 nM for cAMP and ATP, respectively) and reproducibility. Furthermore, the method was validated and applied in vitro to determine cAMP accumulation in biological samples. The effect of several dopamine D(2) (partial) agonists and antagonists on cAMP accumulation was assessed by determination of the cAMP/ATP ratio in cells transfected with the human dopamine D(2L) receptor. Quinpirole, dopamine and ropinirole produced agonist effects on cAMP accumulation, with a potency of quinpirole>ropinirole>dopamine. Lisuride, terguride and bifeprunox were found to be partial agonists with efficacies of lisuride>terguride>bifeprunox. As expected, haloperidol, (-)-sulpiride and LY-741626 were antagonists. These results demonstrate that the present analytical method was robust, fast, sensitive, and selective. Moreover, it showed utility in determining cAMP/ATP in biological systems and the ability to study the effect of (partial) agonists and antagonists which makes it a useful tool for drug discovery.

  5. Transcriptional activation of the SH2D2A gene is dependent on a cyclic adenosine 5'-monophosphate-responsive element in the proximal SH2D2A promoter.

    PubMed

    Dai, Ke-Zheng; Johansen, Finn-Eirik; Kolltveit, Kristin Melkevik; Aasheim, Hans-Christian; Dembic, Zlatko; Vartdal, Frode; Spurkland, Anne

    2004-05-15

    The SH2D2A gene, encoding the T cell-specific adapter protein (TSAd), is rapidly induced in activated T cells. In this study we investigate the regulation of the SH2D2A gene in Jurkat T cells and in primary T cells. Reporter gene assays demonstrated that the proximal 1-kb SH2D2A promoter was constitutively active in Jurkat TAg T cells and, to a lesser extent, in K562 myeloid cells, Reh B cells, and 293T fibroblast cells. The minimal SH2D2A promoter was located between position -236 and -93 bp from the first coding ATG, and transcriptional activity in primary T cells depended on a cAMP response element (CRE) centered around position -117. Nuclear extracts from Jurkat TAg cells and activated primary T cells contained binding activity to this CRE, as observed in an EMSA. Consistent with this observation, we found that a cAMP analog was a very potent inducer of SH2D2A mRNA expression in primary T cells as measured by real-time RT-PCR. Furthermore, activation of SH2D2A expression by CD3 stimulation required cAMP-dependent protein kinase activity. Thus, transcriptional regulation of the SH2D2A gene in activated T cells is critically dependent on a CRE in the proximal promoter region.

  6. 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.

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

    PubMed

    Alqurashi, May; Gehring, Chris; Marondedze, Claudius

    2016-06-01

    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.

  8. 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

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

    PubMed Central

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

    2016-01-01

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

  10. Quantitative Measurement of cAMP Concentration Using an Exchange Protein Directly Activated by a cAMP-Based FRET-Sensor

    PubMed Central

    Salonikidis, Petrus S.; Zeug, André; Kobe, Fritz; Ponimaskin, Evgeni; Richter, Diethelm W.

    2008-01-01

    Förster resonance energy transfer (FRET)-based biosensors for the quantitative analysis of intracellular signaling, including sensors for monitoring cyclic adenosine monophosphate (cAMP), are of increasing interest. The measurement of the donor/acceptor emission ratio in tandem biosensors excited at the donor excitation wavelength is a commonly used technique. A general problem, however, is that this ratio varies not only with the changes in cAMP concentration but also with the changes of the ionic environment or other factors affecting the folding probability of the fluorophores. Here, we use a spectral FRET analysis on the basis of two excitation wavelengths to obtain a reliable measure of the absolute cAMP concentrations with high temporal and spatial resolution by using an “exchange protein directly activated by cAMP”. In this approach, FRET analysis is simplified and does not require additional calibration routines. The change in FRET efficiency (E) of the biosensor caused by [cAMP] changes was determined as ΔE = 15%, whereas E varies between 35% at low and 20% at high [cAMP], allowing quantitative measurement of cAMP concentration in the range from 150 nM to 15 μM. The method described is also suitable for other FRET-based biosensors with a 1:1 donor/acceptor stoichiometry. As a proof of principle, we measured the specially resolved cAMP concentration within living cells and determined the dynamic changes of cAMP levels after stimulation of the Gs-coupled serotonin receptor subtype 7 (5-HT7). PMID:18708470

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

    PubMed

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

    2009-02-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  13. Probing the cyclic nucleotide binding sites of cAMP-dependent protein kinases I and II with analogs of adenosine 3',5'-cyclic phosphorothioates.

    PubMed

    Dostmann, W R; Taylor, S S; Genieser, H G; Jastorff, B; Døskeland, S O; Ogreid, D

    1990-06-25

    A set of cAMP analogs were synthesized that combined exocyclic sulfur substitutions in the equatorial (Rp) or the axial (Sp) position of the cyclophosphate ring with modifications in the adenine base of cAMP. The potency of these compounds to inhibit the binding of [3H]cAMP to sites A and B from type I (rabbit skeletal muscle) and type II (bovine myocardium) cAMP-dependent protein kinase was determined quantitatively. On the average, the Sp isomers had a 5-fold lower affinity for site A and a 30-fold lower affinity for site B of isozyme I than their cyclophosphate homolog. The mean reduction in affinities for the equivalent sites of isozyme II were 20- and 4-fold, respectively. The Rp isomers showed a decrease in affinity of approximately 400-fold and 200-fold for site A and B, respectively, of isozyme I, against 200-fold and 45-fold for site A and B of isozyme II. The Sp substitutions therefore increased the relative preference for site A of isozyme I and site B of isozyme II. The Rp substitution, on the other hand, increased the relative preference for site B of both isozymes. These data show that the Rp and Sp substitutions are tolerated differently by the two intrachain sites of isozymes I and II. They also support the hypothesis that it is the axial, and not the previously proposed equatorial oxygen that contributes the negative charge for the ionic interaction with an invariant arginine in all four binding sites. In addition, they demonstrate that combined modifications in the adenine ring and the cyclic phosphate ring of cAMP can enhance the ability to discriminate between site A and B of one isozyme as well as to discriminate between isozyme I and II. Since Rp analogs of cAMP are known to inhibit activation of cAMP-dependent protein kinases, the findings of the present study have implications for the synthesis of analogs having a very high selectivity for isozyme I or II.

  14. Regulation of rhythmic melatonin production in pineal cells of chick embryo by cyclic AMP.

    PubMed

    Macková, M; Lamosová, D; Zeman, M

    1998-05-01

    The pineal cells of chick embryos incubated in vitro exhibited a daily rhythm of melatonin synthesis under a 12:12 light:dark (LD) cycle at the embryonic days 16 and 19. In order to elucidate whether cyclic adenosine monophosphate (cAMP)--a component of the melatonin generating system--is already at work in the embryonic period, we measured the effects of forskolin and isobuthylmethylxantine (IBMX) on melatonin production, cAMP efflux and accumulation. Forskolin (after 10, 20, 30, 45, 60 and 90 min of administration) and IBMX (6 h), when applied during the light phase of LD cycle, stimulated melatonin production and cAMP efflux and accumulation during the embryonic period (at days 16 and 19 fo development). Our results suggest that the biochemical pathway involving cAMP, which controls melatonin production in the postnatal period, is developed before hatching and already on embryonic day 19 works in a way similar to that in post-hatched chicks. Differences in response to cAMP stimulation between 16- and 19-day-old pinealocytes seem to be mostly quantitative.

  15. Modulation of cAMP metabolism in Mycobacterium tuberculosis and its effect on host infection.

    PubMed

    Barba, Jeannette; Alvarez, Angel H; Flores-Valdez, Mario Alberto

    2010-05-01

    Mycobacterium tuberculosis remains the single most relevant bacterial infectious agent as Tuberculosis is estimated to affect one-third of the world population. Like other microorganisms, M. tuberculosis needs to sense and adapt to changes in the several niches where it is found, ranging from the environment to a number of host-adapted programs, including infection of cell types such as macrophages, dendritic cells, epithelial cells and adipocytes. A strategy commonly used by cells to respond to such changes consists of producing small molecules known as second messengers. 3',5'-cyclic adenosine monophosphate (cAMP) is one of the best-studied second messengers in many organisms, and in recent years its participation during the M. tuberculosis infection cycle has just begun to be thoroughly considered. In this work, we aimed to provide a perspective of how cAMP metabolism proceeds in M. tuberculosis, which genes are activated in response to cAMP signaling in this organism, and discuss the evidence for bacterially produced cAMP use during infection. Furthermore, key issues needing to be addressed for better understanding cAMP physiology in slow-growing pathogenic mycobacteria are presented.

  16. Evidence for cAMP as a mediator of gonadotropin secretion from female pituitaries

    SciTech Connect

    Bourne, G.A.; Baldwin, D.M.

    1987-09-01

    Sodium flufenamate, which inhibited gonadotropin-releasing hormone (GnRH)-stimulated increases in adenosine 3',5'-cyclic monophosphate (cAMP), was used to evaluate the potential role of cAMP as a mediator of GnRH-stimulated gonadotropin secretion. Quartered pituitaries from diestrous II female rats were perifused at 37/sup 0/C, and sequential effluent fractions were collected every 10 min. Administration of GnRH resulted in a characteristic biphasic response for both luteinizing hormone (LH) and follicle-stimulating hormone (FSH), whereas 5 ..mu..M cycloheximide inhibited the secondary augmented responses (phase II) of both hormones. Infusions of 0.1 mM flufenamate inhibited GnRH-stimulated gonadotropin secretion in a manner similar to that of cycloheximide, whereas the administration of 5 mM dibutyryl cAMP in combination with GnRH and flufenamate resulted in the restoration of LH and FSH secretion. The dibutyryl cAMP-restored response appeared to be protein synthesis dependent and specific for cAMP. These results suggest that although the cyclic nucleotide is not involved in the acute release of LH and FSH, it does appear to play a pivotal but indirect role in phase II release of the hormones, by effects involving the stimulation of de novo protein synthesis.

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

    PubMed

    Raslan, Zaher; Naseem, Khalid M

    2015-01-01

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

  18. Ultrastructural localization of 5'AMP odorant receptor sites on the dendrites of olfactory receptor neurons of the spiny lobster.

    PubMed

    Blaustein, D N; Simmons, R B; Burgess, M F; Derby, C D; Nishikawa, M; Olson, K S

    1993-07-01

    A unique probe--biotinylated adenosine-5'-monophosphate (5'AMP-biotin)--was used in transmission electron microscopic (TEM) studies to localize 5'AMP odorant binding sites on the dendrites of olfactory receptor neurons in the aesthetasc sensilla of the spiny lobster, Panulirus argus. This probe is capable of both binding to and exciting 5'AMP-sensitive olfactory receptor neurons, as revealed through biochemical and electrophysiological assays. TEM studies showed that 5'AMP-biotin binding sites are distributed along the entire dendritic region that is exposed to odorants, including the transitional zone (between the inner and outer dendritic segments, including the ciliary segment) and all of the outer dendritic segment. The density of 5'AMP binding sites per micron2 of membrane is similar along the length of the olfactory dendrite. However, the relative number of 5'AMP-biotin binding sites per micron2 of sensillar area diminishes in the distal 30% of the aesthetasc due to a decrease in the amount of dendritic membrane in that region. The distribution of these 5'AMP binding sites is therefore much more extensive than that of enzymes that inactivate 5'AMP--5'ectonucleotidase/phosphatase--which are restricted to the transitional zone (Gleeson et al., 1991). Taken together, these results suggest that 5'AMP-biotin is labeling 5'AMP-specific olfactory receptor sites that are located along the entire outer dendritic segment and that can be coupled to olfactory transduction. This study represents the first in situ localization of specific olfactory receptor sites using a specific, functionally defined ligand.

  19. CSF Concentrations of cAMP and cGMP Are Lower in Patients with Creutzfeldt-Jakob Disease but Not Parkinson's Disease and Amyotrophic Lateral Sclerosis

    PubMed Central

    Oeckl, Patrick; Steinacker, Petra; Lehnert, Stefan; Jesse, Sarah; Kretzschmar, Hans A.; Ludolph, Albert C.; Otto, Markus; Ferger, Boris

    2012-01-01

    Background The cyclic nucleotides cyclic adenosine-3′,5′-monophosphate (cAMP) and cyclic guanosine-3′,5′-monophosphate (cGMP) are important second messengers and are potential biomarkers for Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and Creutzfeldt-Jakob disease (CJD). Methodology/Principal Findings Here, we investigated by liquid chromatography/tandem mass spectrometry (LC-MS/MS) the cerebrospinal fluid (CSF) concentrations of cAMP and cGMP of 82 patients and evaluated their diagnostic potency as biomarkers. For comparison with a well-accepted biomarker, we measured tau concentrations in CSF of CJD and control patients. CJD patients (n = 15) had lower cAMP (−70%) and cGMP (−55%) concentrations in CSF compared with controls (n = 11). There was no difference in PD, PD dementia (PDD) and ALS cases. Receiver operating characteristic (ROC) curve analyses confirmed cAMP and cGMP as valuable diagnostic markers for CJD indicated by the area under the curve (AUC) of 0.86 (cAMP) and 0.85 (cGMP). We calculated a sensitivity of 100% and specificity of 64% for cAMP and a sensitivity of 67% and specificity of 100% for cGMP. The combination of both nucleotides increased the sensitivity to 80% and specificity to 91% for the term cAMPxcGMP (AUC 0.92) and to 93% and 100% for the ratio tau/cAMP (AUC 0.99). Conclusions/Significance We conclude that the CSF determination of cAMP and cGMP may easily be included in the diagnosis of CJD and could be helpful in monitoring disease progression as well as in therapy control. PMID:22396786

  20. Hindbrain A2 noradrenergic neuron adenosine 5'-monophosphate-activated protein kinase activation, upstream kinase/phosphorylase protein expression, and receptivity to hormone and fuel reporters of short-term food deprivation are regulated by estradiol.

    PubMed

    Briski, Karen P; Alenazi, Fahaad S H; Shakya, Manita; Sylvester, Paul W

    2016-09-12

    Estradiol (E) mitigates acute and postacute adverse effects of 12 hr-food deprivation (FD) on energy balance. Hindbrain 5'-monophosphate-activated protein kinase (AMPK) regulates hyperphagic and hypothalamic metabolic neuropeptide and norepinephrine responses to FD in an E-dependent manner. Energy-state information from AMPK-expressing hindbrain A2 noradrenergic neurons shapes neural responses to metabolic imbalance. Here we investigate the hypothesis that FD causes divergent changes in A2 AMPK activity in E- vs. oil (O)-implanted ovariectomized female rats, alongside dissimilar adjustments in circulating metabolic fuel (glucose, free fatty acids [FFA]) and energy deficit-sensitive hormone (corticosterone, glucagon, leptin) levels. FD decreased blood glucose in oil (O)- but not E-implanted ovariectomized female rats and elevated and reduced glucagon levels in O and E, respectively. FD decreased circulating leptin in O and E, but increased corticosterone and FFA concentrations in E only. Western blot analysis of laser-microdissected A2 neurons showed that glucocorticoid receptor type II and very-long-chain acyl-CoA synthetase 3 protein profiles were amplified in FD/E vs. FD/O. A2 total AMPK protein was elevated without change in activity in FD/O, whereas FD/E exhibited increased AMPK activation along with decreased upstream phosphatase expression. The catecholamine biosynthetic enzyme dopamine-β-hydroxylase (DβH) was increased in FD/O but not FD/E A2 cells. The data show discordance between A2 AMPK activation and glycemic responses to FD; sensor activity was refractory to glucose decrements in FD/O but augmented in FD/E despite stabilized glucose and elevated FFA levels. E-dependent amplification of AMPK activity may reflect adaptive conversion to fatty acid oxidation and/or glucocorticoid stimulation. FD augmentation of A2 DβH protein profiles in FD/O but not FD/E animals suggests that FD may correspondingly regulate NE synthesis vs. metabolism/release in the

  1. Dual recognition unit strategy improves the specificity of the adenosine triphosphate (ATP) aptamer biosensor for cerebral ATP assay.

    PubMed

    Yu, Ping; He, Xiulan; Zhang, Li; Mao, Lanqun

    2015-01-20

    Adenosine triphosphate (ATP) aptamer has been widely used as a recognition unit for biosensor development; however, its relatively poor specificity toward ATP against adenosine-5'-diphosphate (ADP) and adenosine-5'-monophosphate (AMP) essentially limits the application of the biosensors in real systems, especially in the complex cerebral system. In this study, for the first time, we demonstrate a dual recognition unit strategy (DRUS) to construct a highly selective and sensitive ATP biosensor by combining the recognition ability of aptamer toward A nucleobase and of polyimidazolium toward phosphate. The biosensors are constructed by first confining the polyimidazolium onto a gold surface by surface-initiated atom transfer radical polymerization (SI-ATRP), and then the aptamer onto electrode surface by electrostatic self-assembly to form dual-recognition-unit-functionalized electrodes. The constructed biosensor based on DRUS not only shows an ultrahigh sensitivity toward ATP with a detection limit down to the subattomole level but also an ultrahigh selectivity toward ATP without interference from ADP and AMP. The constructed biosensor is used for selective and sensitive sensing of the extracellular ATP in the cerebral system by combining in vivo microdialysis and can be used as a promising neurotechnology to probing cerebral ATP concentration.

  2. Phosphorylation of Rap1 by cAMP-dependent Protein Kinase (PKA) Creates a Binding Site for KSR to Sustain ERK Activation by cAMP.

    PubMed

    Takahashi, Maho; Li, Yanping; Dillon, Tara J; Stork, Philip J S

    2017-01-27

    Cyclic adenosine monophosphate (cAMP) is an important mediator of hormonal stimulation of cell growth and differentiation through its activation of the extracellular signal-regulated kinase (ERK) cascade. Two small G proteins, Ras and Rap1 have been proposed to mediate this activation. Using HEK293 cells as a model system, we have recently shown that both Ras and Rap1 are required for cAMP signaling to ERKs. However, cAMP-dependent Ras signaling to ERKs is transient and rapidly terminated by PKA phosphorylation of the Raf isoforms C-Raf and B-Raf. In contrast, cAMP-dependent Rap1 signaling to ERKs and Rap1 is potentiated by PKA. We show that this is due to sustained binding of B-Raf to Rap1. One of the targets of PKA is Rap1 itself, directly phosphorylating Rap1a on serine 180 and Rap1b on serine 179. We show that these phosphorylations create potential binding sites for the adaptor protein 14-3-3 that links Rap1 to the scaffold protein KSR. These results suggest that Rap1 activation of ERKs requires PKA phosphorylation and KSR binding. Because KSR and B-Raf exist as heterodimers within the cell, this binding also brings B-Raf to Rap1, allowing Rap1 to couple to ERKs through B-Raf binding to Rap1 independently of its Ras-binding domain.

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  4. Activation of extracellular signal-regulated kinases, NF-kappa B, and cyclic adenosine 5'-monophosphate response element-binding protein in lung neutrophils occurs by differing mechanisms after hemorrhage or endotoxemia.

    PubMed

    Abraham, E; Arcaroli, J; Shenkar, R

    2001-01-01

    Acute lung injury is frequently associated with sepsis or blood loss and is characterized by a proinflammatory response and infiltration of activated neutrophils into the lungs. Hemorrhage or endotoxemia result in activation of cAMP response element-binding protein (CREB) and NF-kappa B in lung neutrophils as well as increased expression of proinflammatory cytokines, such as TNF-alpha and macrophage-inflammatory peptide-2, by these cells. Activation of the extracellular regulated kinase (ERK) pathway occurs in stress responses and is involved in CREB activation. In the present experiments, hemorrhage or endotoxemia produced increased activation of mitogen-activated protein kinase kinase (MEK)1/2 and ERK2 (p42), but not of ERK1 (p44), in lung neutrophils. ERK1, ERK2, and MEK1/2 were not activated in peripheral blood neutrophils after hemorrhage or endotoxemia. Inhibition of xanthine oxidase led to further increase in the activation of MEK1/2 and ERK2 in lung neutrophils after hemorrhage, but not after endotoxemia. Alpha-adrenergic blockade before hemorrhage resulted in increased activation in lung neutrophils of MEK1/2, ERK1, ERK2, and CREB, but decreased activation of NF-kappa B. In contrast, alpha-adrenergic blockade before endotoxemia was associated with decreased activation of MEK1/2, ERK2, and CREB, but increased activation of NF-kappa B. Beta-adrenergic blockade before hemorrhage did not alter MEK1/2 or ERK1 activation in lung neutrophils, but decreased activation of ERK2 and CREB, while increasing activation of NF-kappa B. Beta-adrenergic inhibition before endotoxemia did not affect activation of MEK1/2, ERK1, ERK2, CREB, or NF-kappa B. These data indicate that the pathways leading to lung neutrophil activation after hemorrhage are different from those induced by endotoxemia.

  5. The small molecule PKA-specific cyclic AMP analogue as an inducer of osteoblast-like cells differentiation and mineralization.

    PubMed

    Lo, Kevin W-H; Kan, Ho Man; Ashe, Keshia M; Laurencin, Cato T

    2012-01-01

    Osteoblastic differentiation is an important landmark for bone formation, bone repair and regeneration; however, it is a very complex process controlled by different signalling mechanisms. Several groups have reported that the cyclic adenosine monophosphate (cAMP) signalling system is responsible for regulating osteoblast cell differentiation. Nonetheless, to date, the principle role of the cAMP molecules related to this process remains controversial. Moreover, the underlying cAMP-dependent signalling cascade governing the osteoblastic differentiation has not been clarified. In this study we investigated the roles of the cAMP-dependent protein kinase A (PKA) signalling in proliferation, differentiation and mineralization of osteoblast-like MC3T3-E1 cells, using the PKA-specific small molecule cAMP analogue, 6-Bnz-cAMP, at 100 µM. Alkaline phosphatase (ALP) activity, runt transcription factor 2 (Runx2), osteopontin (OPN) and osteocalcin (OCN) protein expressions were used as osteoblast-specific markers to demonstrate osteoblastic differentiation. Further, calcium measurement of the extracellular matrix was employed as the hallmark of matrix mineralization or calcification. We report here that activation of PKA by the small molecule 6-Bnz-cAMP induces osteoblastic differentiation and matrix mineralization of osteoblast-like MC3T3-E1 cells. Moreover, 6-Bnz-cAMP does not induce cytotoxicity to the cells, as revealed by our cell proliferation studies. Therefore, based on these findings, we propose that the PKA-specific small molecule 6-Bnz-cAMP may serve as a novel bone-inducing growth factor for repairing and regenerating bone tissues during bone-regenerative engineering.

  6. 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.

  7. Nitric oxide-dependent vasodilatation of rabbit femoral artery by beta(2)-adrenergic stimulation or cyclic AMP elevation in vivo.

    PubMed

    Xu, B; Li, J; Gao, L; Ferro, A

    2000-03-01

    Some studies suggest that beta-adrenoceptor-mediated vasorelaxation is in part mediated through nitric oxide (NO) release. We wished to determine the contribution of the L-arginine / NO system to vasodilatation in response to beta-adrenoceptor stimulation with isoprenaline or cyclic adenosine-3',5'-monophosphate (cyclic AMP) elevation with forskolin and dibutyryl cyclic AMP in vivo, using a rabbit femoral artery constant perfusion model. Baseline femoral artery pressure was similar in rabbits receiving isoprenaline, forskolin or dibutyryl cyclic AMP. Isoprenaline, forskolin and dibutyryl cyclic AMP each decreased femoral artery pressure in a dose-dependent manner. The doses (mol kg(-1)) of isoprenaline, forskolin and dibutyryl cyclic AMP which decreased pressure by 10% from baseline, expressed as a negative logarithm (-log ED(10)) were: 10.0+/-0.2, 9.5+/-0.1 and 4.9+/-0.1 respectively (P<0.0001 for each). Use of beta-adrenoceptor subtype-selective antagonists showed that the vascular response to isoprenaline was purely due to stimulation of the beta(2)-adrenoceptor subtype. Injection of 1 micromol kg(-1) N(G)-nitro-L-arginine methyl ester (L-NAME) did not alter baseline pressure. However, it abolished the pressure response to isoprenaline (P<0.0001), and significantly attenuated the pressure responses to forskolin and dibutyryl cyclic AMP: -log ED(10) values for forskolin and dibutyryl cyclic AMP, in the presence of L-NAME, were 7.9+/-0.1 and 3.5+/-0.3 respectively (P<0.0001 for each, as compared with values in the absence of L-NAME). These results indicate that beta(2)-adrenergic stimulation and cylic AMP elevation activate the L-arginine/NO system in rabbit femoral artery in vivo, and that NO generation contributes importantly to the changes in vascular tone induced by agents which modulate beta-adrenoceptors or cyclic AMP.

  8. GABAB and adenosine receptors mediate enhancement of the K+ current, IAHP, by reducing adenylyl cyclase activity in rat CA3 hippocampal neurons.

    PubMed

    Gerber, U; Gähwiler, B H

    1994-11-01

    1. Gamma-aminobuturic acid-B (GABAB) and adenosine A1 receptors, which are expressed in hippocampal pyramidal cells, are linked to pertussis toxin-sensitive G-proteins known to be coupled negatively to the enzyme adenylyl cyclase. This study investigates the electrophysiological consequences of adenylyl cyclase inhibition in response to stimulation of these receptors. 2. Single-electrode voltage-clamp recordings were obtained from CA3 pyramidal cells in rat hippocampal slice cultures in presence of tetrodotoxin. The calcium-dependent potassium current (IAHP), which is very sensitive to intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP), was used as an electrophysiological indicator of adenylyl cyclase activity. 3. Application of baclofen (10 microM), a selective agonist at GABAB receptors, or adenosine (50 microM) each resulted in a transient decrease followed by a significant enhancement in the amplitude of evoked IAHP. The initial reduction in amplitude of IAHP probably reflects inadequacies in voltage clamp of electronically distant dendritic sites, due to the shunting caused by concomitant activation of potassium conductance by baclofen/adenosine. Comparable increases in membrane conductance in response to the GABAA agonist, muscimol, caused a similar reduction in IAHP. The enhancement of IAHP is consistent with an inhibition of constitutively active adenylyl cyclase. 4. The receptor mediating the responses to adenosine was identified as belonging to the A1 subtype on the basis of its sensitivity to the selective antagonist 8-cyclopentyl-1,3-dipropylxanthine.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    PubMed

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

    2009-09-29

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

  11. Mutations affecting the cAMP transduction pathway modify olfaction in Drosophila.

    PubMed

    Martín, F; Charro, M J; Alcorta, E

    2001-06-01

    The rutabaga and dunce genes, encode two enzymes of the cyclic adenosine monophosphate transduction pathway in Drosophila, adenylyl cyclase and cyclic adenosine monophosphate phosphodiesterase, respectively. Two main second messenger systems, depending on inositol 1,4,5-triphosphate and cyclic adenosine monophosphate, have been associated with olfaction in vertebrates as well as invertebrates. A relationship between the cyclic adenosine monophosphate signaling pathway and olfactory reception in Drosophila is suggested by the presence of cyclic nucleotide gated channels and cyclic-nucleotide modulated K+ channels in the antennae, the main olfactory organs. In this report, molecular, electrophysiological and behavioral data support the role of cyclic adenosine monophosphate in olfactory function for this species. Expression of both genes in the antennae has been shown by messenger ribonucleic acid analysis. Changes in the electroantennogram kinetics have been observed specifically on the slope of the initial rising phase, as predicted for processes that affect cyclic adenosine monophosphate concentration. Olfactory behavior changes due to both mutations were coherent with a functional meaning of the reported electrophysiological phenotype in olfactory perception. Sensitivity level increases or decreases for the mutants compared to the control line depending on the odorant. These results are compatible with some olfactory coding at the reception level by differential activation of a dual transduction system involving the inositol 1,4,5-triphosphate and cyclic adenosine monophosphate cascades.

  12. Adenyl cyclases and cAMP in plant signaling - past and present.

    PubMed

    Gehring, Chris

    2010-06-25

    In lower eukaryotes and animals 3'-5'-cyclic adenosine monophosphate (cAMP) and adenyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, have long been established as key components and second messengers in many signaling pathways. In contrast, in plants, both the presence and biological role of cAMP have been a matter of ongoing debate and some controversy. Here we shall focus firstly on the discovery of cellular cAMP in plants and evidence for a role of this second messenger in plant signal transduction. Secondly, we shall review current evidence of plant ACs, analyse aspects of their domain organisations and the biological roles of candidate molecules. In addition, we shall assess different approaches based on search motifs consisting of functionally assigned amino acids in the catalytic centre of annotated and/or experimentally tested nucleotide cyclases that can contribute to the identification of novel candidate molecules with AC activity such as F-box and TIR proteins.

  13. Phosphodiesterase DosP increases persistence by reducing cAMP which reduces the signal indole.

    PubMed

    Kwan, Brian W; Osbourne, Devon O; Hu, Ying; Benedik, Michael J; Wood, Thomas K

    2015-03-01

    Persisters are bacteria that are highly tolerant to antibiotics due to their dormant state and are of clinical significance owing to their role in infections. Given that the population of persisters increases in biofilms and that cyclic diguanylate (c-di-GMP) is an intracellular signal that increases biofilm formation, we sought to determine whether c-di-GMP has a role in bacterial persistence. By examining the effect of 30 genes from Escherichia coli, including diguanylate cyclases that synthesize c-di-GMP and phosphodiesterases that breakdown c-di-GMP, we determined that DosP (direct oxygen sensing phosphodiesterase) increases persistence by over a thousand fold. Using both transcriptomic and proteomic approaches, we determined that DosP increases persistence by decreasing tryptophanase activity and thus indole. Corroborating this effect, addition of indole reduced persistence. Despite the role of DosP as a c-di-GMP phosphodiesterase, the decrease in tryptophanase activity was found to be a result of cyclic adenosine monophosphate (cAMP) phosphodiesterase activity. Corroborating this result, the reduction of cAMP via CpdA, a cAMP-specific phosphodiesterase, increased persistence and reduced indole levels similarly to DosP. Therefore, phosphodiesterase DosP increases persistence by reducing the interkingdom signal indole via reduction of the global regulator cAMP.

  14. New perspectives in cyclic AMP-mediated axon growth and guidance: The emerging epoch of Epac.

    PubMed

    Peace, Andrew G; Shewan, Derryck A

    2011-03-10

    In the search for a cure to brain and spinal cord injury much has been learned about the inhibitory environment of the central nervous system (CNS), and yet a clinical therapy remains elusive. In recent years great advances have been made in understanding intracellular molecular mechanisms that transduce cell surface receptor-mediated signals that neurons receive from their environment. Many of these signalling pathways share common mechanisms, which presents the possibility that manipulating activities of key cell signalling molecules such as those regulated by 3'-5'-cyclic adenosine monophosphate (cAMP) might allow axons to simultaneously overcome the inhibitory effects of a number of extracellular ligands. The identification of Epac, a novel direct intracellular target for cAMP, has opened up a new avenue of research that is beginning to explain how cAMP can mediate a range of neuronal functions including distinct axon growth and guidance decisions. With current research tools that allow more specific activation of proteins or knock-down of their expression, as well as quantitation of protein activities in live cells, it is already becoming clear that Epac plays highly important roles in the development and function of the nervous system. Here, we focus on emerging evidence that Epac mediates cAMP-regulated axon growth and chemoattraction, and thus represents a novel target for overcoming axon growth inhibition and promoting CNS regeneration.

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

    PubMed

    Hsu, Chia George; Burkholder, Thomas J

    2016-12-01

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

  16. Selective β2 adrenergic agonist increases Cx43 and miR-451 expression via cAMP-Epac.

    PubMed

    Mostafavi, Hossein; Khaksarian, Mojtaba; Joghataei, Mohammad Taghi; Soleimani, Masoud; Hassanzadeh, Gholamreza; Eftekhari, Sanaz; Soleimani, Mansooreh; Mousavizadeh, Kazem; Estiri, Hajar; Ahmadi, Sedighesadat; Hadjighassem, Mahmoud Reza

    2014-06-01

    It has been demonstrated that connexin 43 (Cx43) and microRNAs have significant roles in glioma. Cyclic adenosine monophosphate (cAMP) is suggested to be a regulator of connexins and microRNAs. However, it remains elusive whether cAMP and exchange protein directly activated by cAMP (Epac2), have a regulatory effect on Cx43 and microRNA-451 (miR-451) in astrocytoma cells. We treated 1321N1 astrocytoma cells with a selective β2 adrenergic agonist and a selective Epac activator with and without adenyl cyclase and protein kinase A inhibition. Cx43 and miR-451 expression were measured. Next, we evaluated the effect of miR-451 overexpression on Cx43 expression. Cell proliferation was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results demonstrated that cAMP-Epac2 increased Cx43 and miR-451 expression. However, the alteration of miR-451 expression required a higher dose of drugs. Overexpression of miR-451 had no significant effect on Cx43 expression. The MTT assay showed that cAMP-Epac stimulation and miR-451 overexpression had a synergic inhibitory effect on cell proliferation. These findings may expand our understanding of the molecular biology of glioma and provide new potential therapeutic targets.

  17. cAMP Signaling Regulates Synchronised Growth of Symbiotic Epichloë Fungi with the Host Grass Lolium perenne

    PubMed Central

    Voisey, Christine R.; Christensen, Michael T.; Johnson, Linda J.; Forester, Natasha T.; Gagic, Milan; Bryan, Gregory T.; Simpson, Wayne R.; Fleetwood, Damien J.; Card, Stuart D.; Koolaard, John P.; Maclean, Paul H.; Johnson, Richard D.

    2016-01-01

    The seed-transmitted fungal symbiont, Epichloë festucae, colonizes grasses by infecting host tissues as they form on the shoot apical meristem (SAM) of the seedling. How this fungus accommodates the complexities of plant development to successfully colonize the leaves and inflorescences is unclear. Since adenosine 3′, 5′-cyclic monophosphate (cAMP)-dependent signaling is often essential for host colonization by fungal pathogens, we disrupted the cAMP cascade by insertional mutagenesis of the E. festucae adenylate cyclase gene (acyA). Consistent with deletions of this gene in other fungi, acyA mutants had a slow radial growth rate in culture, and hyphae were convoluted and hyper-branched suggesting that fungal apical dominance had been disrupted. Nitro blue tetrazolium (NBT) staining of hyphae showed that cAMP disruption mutants were impaired in their ability to synthesize superoxide, indicating that cAMP signaling regulates accumulation of reactive oxygen species (ROS). Despite significant defects in hyphal growth and ROS production, E. festucae ΔacyA mutants were infectious and capable of forming symbiotic associations with grasses. Plants infected with E. festucae ΔacyA were marginally less robust than the wild-type (WT), however hyphae were hyper-branched, and leaf tissues heavily colonized, indicating that the tight regulation of hyphal growth normally observed in maturing leaves requires functional cAMP signaling. PMID:27833620

  18. cAMP Signaling Regulates Synchronised Growth of Symbiotic Epichloë Fungi with the Host Grass Lolium perenne.

    PubMed

    Voisey, Christine R; Christensen, Michael T; Johnson, Linda J; Forester, Natasha T; Gagic, Milan; Bryan, Gregory T; Simpson, Wayne R; Fleetwood, Damien J; Card, Stuart D; Koolaard, John P; Maclean, Paul H; Johnson, Richard D

    2016-01-01

    The seed-transmitted fungal symbiont, Epichloë festucae, colonizes grasses by infecting host tissues as they form on the shoot apical meristem (SAM) of the seedling. How this fungus accommodates the complexities of plant development to successfully colonize the leaves and inflorescences is unclear. Since adenosine 3', 5'-cyclic monophosphate (cAMP)-dependent signaling is often essential for host colonization by fungal pathogens, we disrupted the cAMP cascade by insertional mutagenesis of the E. festucae adenylate cyclase gene (acyA). Consistent with deletions of this gene in other fungi, acyA mutants had a slow radial growth rate in culture, and hyphae were convoluted and hyper-branched suggesting that fungal apical dominance had been disrupted. Nitro blue tetrazolium (NBT) staining of hyphae showed that cAMP disruption mutants were impaired in their ability to synthesize superoxide, indicating that cAMP signaling regulates accumulation of reactive oxygen species (ROS). Despite significant defects in hyphal growth and ROS production, E. festucae ΔacyA mutants were infectious and capable of forming symbiotic associations with grasses. Plants infected with E. festucae ΔacyA were marginally less robust than the wild-type (WT), however hyphae were hyper-branched, and leaf tissues heavily colonized, indicating that the tight regulation of hyphal growth normally observed in maturing leaves requires functional cAMP signaling.

  19. Isolation and properties of AMP deaminase from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California, Mexico.

    PubMed

    Marquez-Rios, E; Pacheco-Aguilar, R; Castillo-Yañez, F J; Figueroa-Soto, C G; Ezquerra-Brauer, J M; Gollas-Galvan, T

    2008-09-01

    Adenosine monophosphate (AMP) deaminase was purified from jumbo squid mantle muscle by chromatography in cellulose phosphate, Q-Fast and 5'-AMP sepharose. Specific activity of 2.5U/mg protein, 4.5% recovery and 133.68 purification fold were obtained at the end of the experiment. SDS-PAGE showed a single band with 87kDa molecular mass, native PAGE proved a band of 178kDa, whereas gel filtration detected a 180kDa protein, suggesting the homodimeric nature of this enzyme, in which subunits are not linked by covalent forces. Isoelectric focusing of this enzyme showed a pI of 5.76, which agrees with pI values of AMP deaminase from other invertebrate organisms. AMP deaminase presented a kinetic sigmoidal plot with Vmax of 1.16μM/min/mg, Km of 13mM, Kcat of 3.48μM.s(-1) and a Kcat/Km of 267 (mol/L)(-1).s(-1). The apparent relative low catalytic activity of jumbo squid muscle AMP deaminase in the absence of positive effectors is similar to that reported for homologous enzymes in other invertebrate organisms.

  20. Inhibin alpha gene expression in human trophoblasts is regulated by interactions between TFAP2 and cAMP signaling pathways.

    PubMed

    Depoix, Christophe L; Debiève, Frédéric; Hubinont, Corinne

    2014-11-01

    Inhibin α (Inha) gene expression is regulated, in rat granulosa cells, via a cyclic 3',5'-adenosine monophosphate (AMP)-response element (CRE) found in a region of the promoter that is homologous to the human INHA promoter. We previously found that during in vitro cytotrophoblast differentiation, human INHA gene expression was regulated by TFAP2A via association with an AP-2 site located upstream of this CRE. The aim of this study was to evaluate if the human INHA gene was also regulated by cAMP in trophoblasts, and to investigate the possible crosstalk between TFAP2 and cAMP signaling pathways in the regulation of INHA gene expression. Treatment with cAMP or forskolin increased INHA mRNA expression by 7- and 2-fold in primary cytotrophoblasts and choriocarcinoma-derived BeWo cells, respectively. Treatment with the protein kinase A inhibitor H-89 reduced forskolin-induced luciferase activity by ∼40% in BeWo cells transfected with an INHA promoter-driven luciferase reporter vector. TFAP2 overexpression increased basal luciferase activity, whereas the dominant repressor KCREB abolished it. Surprisingly, mutation of the CRE also eliminated the TFAP2-induced transcription, although TFAP2 overexpression was still able to increase forskolin-induced luciferase activity when the AP-2 binding site, but not the CRE site, was mutated. Thus, INHA gene expression is upregulated by cAMP via CRE in human trophoblasts, and TFAP2 regulates this expression by interacting with CRE.

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

    NASA Technical Reports Server (NTRS)

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

    1993-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  3. Inhibition of the cAMP/PKA/CREB Pathway Contributes to the Analgesic Effects of Electroacupuncture in the Anterior Cingulate Cortex in a Rat Pain Memory Model

    PubMed Central

    Sun, Jing; Liu, Bo-Yi; Shen, Zui; Fang, Fang; Wang, Jia-Ling

    2016-01-01

    Pain memory is considered as endopathic factor underlying stubborn chronic pain. Our previous study demonstrated that electroacupuncture (EA) can alleviate retrieval of pain memory. This study was designed to observe the different effects between EA and indomethacin (a kind of nonsteroid anti-inflammatory drugs, NSAIDs) in a rat pain memory model. To explore the critical role of protein kinase A (PKA) in pain memory, a PKA inhibitor was microinjected into anterior cingulate cortex (ACC) in model rats. We further investigated the roles of the cyclic adenosine monophosphate (cAMP), PKA, cAMP response element-binding protein (CREB), and cAMP/PKA/CREB pathway in pain memory to explore the potential molecular mechanism. The results showed that EA alleviates the retrieval of pain memory while indomethacin failed. Intra-ACC microinjection of a PKA inhibitor blocked the occurrence of pain memory. EA reduced the activation of cAMP, PKA, and CREB and the coexpression levels of cAMP/PKA and PKA/CREB in the ACC of pain memory model rats, but indomethacin failed. The present findings identified a critical role of PKA in ACC in retrieval of pain memory. We propose that the proper mechanism of EA on pain memory is possibly due to the partial inhibition of cAMP/PKA/CREB signaling pathway by EA. PMID:28090359

  4. Inhibitory effect of luteolin on the odorant-induced cAMP level in HEK293 cells expressing the olfactory receptor.

    PubMed

    Yoon, Yeo Cho; Hwang, Jin-Teak; Sung, Mi-Jeong; Wang, Shuaiyu; Munkhtugs, Davaatseren; Rhyu, Mee-Ra; Park, Jae-Ho

    2012-01-01

    Luteolin is a flavonoid in many fruits and vegetables. Although luteolin has important biological functions, including antioxidant, anti-inflammatory, antimicrobial, and neuroprotective activities, little is known about the functions of luteolin in the olfactory system. Various odorants can be detected and distinguished by using several molecular processes, including the binding of odorants to odorant receptors, activation of adenylyl cyclase (AC), changes of cyclic adenosine monophosphate (cAMP) and Ca(2+) levels in olfactory sensory neurons, as well as changes in membrane potentials and the transmission of electric signals to the brain. Because AC-cAMP signal transduction plays a pivotal role in the olfactory system, we evaluated the effects of luteolin on the AC-cAMP pathway that had been stimulated by the odorant eugenol. We demonstrated that eugenol caused an upregulation of the cAMP level and the phosphorylation of phosphokinase A (PKA, a downstream target of cAMP) in human embryonic kidney 293 (HEK293) cells expressing the murine eugenol receptor. This upregulation significantly decreased in the presence of luteolin, suggesting that luteolin inhibited the odorant-induced production of cAMP and affected the downstream phosphorylation of PKA.

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

    SciTech Connect

    Not Available

    1986-03-01

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

  6. Regulation of mitochondrial poly(ADP-Ribose) polymerase activation by the β-adrenoceptor/cAMP/protein kinase A axis during oxidative stress.

    PubMed

    Brunyanszki, Attila; Olah, Gabor; Coletta, Ciro; Szczesny, Bartosz; Szabo, Csaba

    2014-10-01

    We investigated the regulation of mitochondrial poly(ADP-ribose) polymerase 1 (PARP1) by the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) system during oxidative stress in U937 monocytes. Oxidative stress induced an early (10 minutes) mitochondrial DNA damage, and concomitant activation of PARP1 in the mitochondria. These early events were followed by a progressive mitochondrial oxidant production and nuclear PARP1 activation (by 6 hours). These processes led to a functional impairment of mitochondria, culminating in cell death of mixed (necrotic/apoptotic) type. β-Adrenoceptor blockade with propranolol or inhibition of its downstream cAMP/PKA signaling attenuated, while β-adrenoceptor agonists and cAMP/PKA activators enhanced, the oxidant-mediated PARP1 activation. In the presence of cAMP, recombinant PKA directly phosphorylated recombinant PARP1 on serines 465 (in the automodification domain) and 782 and 785 (both in the catalytic domain). Inhibition of the β-adrenergic receptor/cAMP/PKA axis protected against the oxidant-mediated cell injury. Propranolol also suppressed PARP1 activation in peripheral blood leukocytes during bacterial lipopolysaccharide (LPS)-induced systemic inflammation in mice. We conclude that the activation of mitochondrial PARP1 is an early, active participant in oxidant-induced cell death, which is under the control of β-adrenoceptor/cAMP/PKA axis through the regulation of PARP1 activity by PARP1 phosphorylation.

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

    PubMed Central

    Stratakis, Constantine A.

    2013-01-01

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

  8. Mucosal adenosine stimulates chloride secretion in canine tracheal epithelium

    SciTech Connect

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

    1986-08-01

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

  9. cAMP induction by ouabain promotes endothelin-1 secretion via MAPK/ERK signaling in beating rabbit atria.

    PubMed

    Peng, Li-Qun; Li, Ping; Zhang, Qiu-Li; Hong, Lan; Liu, Li-Ping; Cui, Xun; Cui, Bai-Ri

    2016-01-01

    Adenosine 3',5'-cyclic monophosphate (cAMP) participates in the regulation of numerous cellular functions, including the Na(+)-K(+)-ATPase (sodium pump). Ouabain, used in the treatment of several heart diseases, is known to increase cAMP levels but its effects on the atrium are not understood. The aim of the present study was to examine the effect of ouabain on the regulation of atrial cAMP production and its roles in atrial endothelin-1 (ET-1) secretion in isolated perfused beating rabbit atria. Our results showed that ouabain (3.0 µmol/L) significantly increased atrial dynamics and cAMP levels during recovery period. The ouabain-increased atrial dynamics was blocked by KB-R7943 (3.0 µmol/L), an inhibitor for reverse mode of Na(+)-Ca(2+) exchangers (NCX), but did not by L-type Ca(2+) channel blocker nifedipine (1.0 µmol/L) or protein kinase A (PKA) selective inhibitor H-89 (3.0 µmol/L). Ouabain also enhanced atrial intracellular cAMP production in response to forskolin and theophyline (100.0 µmol/L), an inhibitor of phosphodiesterase, potentiated the ouabain-induced increase in cAMP. Ouabain and 8-Bromo-cAMP (0.5 µmol/L) markedly increased atrial ET-1 secretion, which was blocked by H-89 and by PD98059 (30 µmol/L), an inhibitor of extracellular-signal-regulated kinase (ERK) without changing ouabain-induced atrial dynamics. Our results demonstrated that ouabain increases atrial cAMP levels and promotes atrial ET-1 secretion via the mitogen-activated protein kinase (MAPK)/ERK signaling pathway. These findings may explain the development of cardiac hypertrophy in response to digitalis-like compounds.

  10. Inhibition by simulated ischemia or hypoxia of delayed afterdepolarizations provoked by cyclic AMP: significance for ischemic and reperfusion arrhythmias.

    PubMed

    Saman, S; Coetzee, W A; Opie, L H

    1988-02-01

    Controversy exists about the role of an increased level of tissue cyclic adenosine 3'-5' monophosphate (cAMP) in the genesis of early ischemic ventricular arrhythmias. Evidence for an arrhythmogenic role for cAMP was proposed by Podzuweit et al. (1978) and Opie et al. (1979) who argued that ischemic ventricular fibrillation was associated with increased levels of tissue cAMP in the ischemic zone. Lubbe et al. (1978) found that infusion of dibutyryl (dBcAMP), or the beta-adrenergic stimulant epinephrine, or the phosphodiesterase inhibitor theophylline, all produced a marked fall in the ventricular fibrillation threshold and an increase in the duration of the vulnerable period of the isolated perfused rat heart. In contrast, Muller et al. (1986) recently showed that prevention of ventricular fibrillation by beta-adrenergic blockade is not directly associated with decreased levels of cAMP, while Manning et al. (1985) used forskolin to stimulate adenylate cyclase and found that the markedly elevated tissue cAMP levels in the rat heart did not promote ischemic or reperfusion arrhythmias. Some of these contradictions could be resolved if the electrophysiological mechanisms by which increased levels of cAMP might predispose to arrhythmias were better understood. It is known that intracellular injection of cAMP into cardiac myocytes can enhance delayed afterdepolarizations (DADs; Matsuda et al. 1982) and that DADs may explain certain arrhythmias such as those evoked by digitalis toxicity (Ferrier, 1977) or reperfusion (Ferrier et al. 1985).(ABSTRACT TRUNCATED AT 250 WORDS)

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

    PubMed

    Hu, Shouye; Cao, Qingwen; Xu, Peng; Ji, Wenchen; Wang, Gang; Zhang, Yuelin

    2016-03-01

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

  12. Long-term cilostazol administration ameliorates memory decline in senescence-accelerated mouse prone 8 (SAMP8) through a dual effect on cAMP and blood-brain barrier.

    PubMed

    Yanai, Shuichi; Toyohara, Jun; Ishiwata, Kiichi; Ito, Hideki; Endo, Shogo

    2017-04-01

    Phosphodiesterases (PDEs), which hydrolyze and inactivate 3', 5'-cyclic adenosine monophosphate (cAMP) and 3', 5'-cyclic guanosine monophosphate (cGMP), play an important role in synaptic plasticity that underlies memory. Recently, several PDE inhibitors were assessed for their possible therapeutic efficacy in treating cognitive disorders. Here, we examined how cilostazol, a selective PDE3 inhibitor, affects brain functions in senescence-accelerated mouse prone 8 (SAMP8), an animal model of age-related cognitive impairment. Long-term administration of cilostazol restored the impaired context-dependent conditioned fear memory of SAMP8 to match that in normal aging control substrain SAMR1. Cilostazol also increased the number of cells containing phosphorylated cAMP-responsive element binding protein (CREB), a downstream component of the cAMP pathway. Finally, cilostazol improves blood-brain barrier (BBB) integrity, demonstrated by reduced extravasation of 2-deoxy-2-(18)F-fluoro-d-glucose and Evans Blue dye in the brains of SAMP8. This improvement in BBB integrity was associated with an increased amount of zona occludens protein 1 (ZO-1) and occludin proteins, components of tight junctions integral to the BBB. The results suggest that long-term administration of cilostazol exerts its beneficial effects on age-related cognitive impairment through a dual mechanism: by enhancing the cAMP system in the brain and by maintaining or improving BBB integrity.

  13. PET measurements of cAMP-mediated phosphodiesterase-4 with (R)-[11C]rolipram.

    PubMed

    Kenk, Miran; Thomas, Adam; Lortie, Mireille; Dekemp, Rob; Beanlands, Rob S; Dasilva, Jean N

    2011-01-01

    Cyclic adenosine monophosphate (cAMP) is the common second messenger in signal-transduction cascades originating at a number of monoamine receptors involved in neurotransmission, cardiac function and smooth muscle contraction. Altered regulation of cAMP synthesis (at receptors, G-protein subunits or adenylyl cyclase) and breakdown by phosphodiesterase (PDE) enzymes have been implicated in a number of pathologies. The PDE4 inhibitor (R)-rolipram, and the less active (S)- enantiomer, have been labeled with carbon-11 and characterized by in vivo and in vitro experiments for use in the evaluation of altered PDE4 levels in the brain and cardiac tissues. (R)-[11C]Rolipram has been shown to bind selectively to PDE4 over other PDE isozymes, with specific binding reflecting approximately 80 and 40% of the total detected radioactivity in the rat brain and the heart, respectively. Tracer retention in PDE4-rich tissues is increased by cAMP-elevating treatments, as detected by in vivo PET studies and ex vivo biodistribution experiments. In vivo PET imaging studies display strong region-specific signal in the brain and heart, as evaluated in rats, pigs, monkeys and humans. Impaired cAMP-mediated signaling was observed in animal models of aging, obesity, anthracycline-induced cardiotoxicity and myocardial infarction using (R)-[11C]rolipram. Given the critical role of cAMP in multiple hormonal pathways, the good safety profile and well-characterized pharmacokinetics, (R)-[11C]rolipram PET imaging provides a novel tool for serial monitoring of cAMP-mediated signaling at the PDE4 level, yielding insight into pathological progression with potential for directing therapy.

  14. AIP inactivation leads to pituitary tumorigenesis through defective Gαi-cAMP signaling.

    PubMed

    Tuominen, I; Heliövaara, E; Raitila, A; Rautiainen, M-R; Mehine, M; Katainen, R; Donner, I; Aittomäki, V; Lehtonen, H J; Ahlsten, M; Kivipelto, L; Schalin-Jäntti, C; Arola, J; Hautaniemi, S; Karhu, A

    2015-02-26

    The aryl hydrocarbon receptor interacting protein (AIP) is a tumor-suppressor gene underlying the pituitary adenoma predisposition. Thus far, the exact molecular mechanisms by which inactivated AIP exerts its tumor-promoting action have been unclear. To better understand the role of AIP in pituitary tumorigenesis, we performed gene expression microarray analysis to examine changes between Aip wild-type and knockout mouse embryonic fibroblast (MEF) cell lines. Transcriptional analyses implied that Aip deficiency causes a dysfunction in cyclic adenosine monophosphate (cAMP) signaling, as well as impairments in signaling cascades associated with developmental and immune-inflammatory responses. In vitro experiments showed that AIP deficiency increases intracellular cAMP concentrations in both MEF and murine pituitary adenoma cell lines. Based on knockdown of various G protein α subunits, we concluded that AIP deficiency leads to elevated cAMP concentrations through defective Gαi-2 and Gαi-3 proteins that normally inhibit cAMP synthesis. Furthermore, immunostaining of Gαi-2 revealed that AIP deficiency is associated with a clear reduction in Gαi-2 protein expression levels in human and mouse growth hormone (GH)-secreting pituitary adenomas, thus indicating defective Gαi signaling in these tumors. By contrast, all prolactin-secreting tumors showed prominent Gαi-2 protein levels, irrespective of Aip mutation status. We additionally observed reduced expression of phosphorylated extracellular signal-regulated kinases 1/2 and cAMP response element-binding protein levels in mouse and human AIP-deficient somatotropinomas. This study implies for the first time that a failure to inhibit cAMP synthesis through dysfunctional Gαi signaling underlies the development of GH-secreting pituitary adenomas in AIP mutation carriers.

  15. Calcium regulates motility and protein phosphorylation by changing cAMP and ATP concentrations in boar sperm in vitro.

    PubMed

    Li, Xinhong; Wang, Lirui; Li, Yuhua; Zhao, Na; Zhen, Linqing; Fu, Jieli; Yang, Qiangzhen

    2016-09-01

    Considering the importance of calcium (Ca(2+)) in regulating sperm capacitation, hyperactivation and acrosome reaction, little is known about the molecular mechanism of action of this ion in this process. In the present study, assessment of the molecular mechanism from the perspective of energy metabolism occurred. Sperm motility variables were determined using computer-assisted sperm analysis (CASA) and the phosphorylation of PKA substrates, tyrosine residues and AMP-activated protein kinase (AMPK) were analyzed by Western blot. Moreover, intracellular sperm-specific glyceraldehyde 3-phosphatedehydrogenase (GAPDH) activity, 3'-5'-cyclic adenosine monophosphate (cAMP) and adenosine 5'-triphosphate (ATP) concentrations were assessed in boar sperm treated with Ca(2+). Results of the present study indicated that, under greater extracellular Ca(2+)concentrations (≥3.0mM), sperm motility and protein phosphorylation were inhibited. Interestingly, these changes were correlated with that of GAPDH activity, AMPK phosphorylation, cAMP and ATP concentrations. The negative effects of Ca(2+) on these intracellular processes were attenuated by addition of the calmodulin (CaM) inhibitor W7 and the inhibitor of calmodulin-dependent protein kinase (CaMK), KN-93. In the presence of greater extracellular Ca(2+), however, the phosphorylation pathway was suppressed by H-89. Taken together, these results suggested that Ca(2+) had a dual role in regulating boar sperm motility and protein phosphorylation due to the changes of cAMP and ATP concentrations, in response to cAMP-mediated signal transduction and the Ca(2+) signaling cascade. The present study provided some novel insights into the molecular mechanism underlying the effects of Ca(2+) on boar sperm as well as the involvement of energy metabolism in this mechanism.

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

    PubMed

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

    2015-01-01

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

  17. The characteristics of hepatic Gsα-cAMP axis in HSHF diet-fed obese insulin resistance rats and genetic diabetic mice.

    PubMed

    Xue, Nina; Wei, Chen; Zhang, Lihong; Liu, Hongying; Wang, Xiaojuan; Wang, Lili

    2017-03-04

    Stimulatory G protein α-subunit (Gsα) mediated cyclic adenosine monophosphate (cAMP) signal is required for elevated hepatic glucose production (HGP) in diabetic patients. However, it remains obscure of the exact characteristics of hepatic Gsα-cAMP signal axis (including Gsα, glucagon receptor, β2-adrenergic receptor, cAMP, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) in insulin resistance (IR) and type 2 diabetes mellitus (T2DM). In current study, we investigated the changing characteristics of hepatic Gsα-cAMP signal axis and blood glucose in high-sugar-high-fat (HSHF)-diet-induced IR wistar rats and db/db diabetic mice. As expected, the HSHF-diet rats were characterized by hyperinsulinemia, hyperglycemia and impaired glucose tolerance. According to a threshold (1.7) of HOMA-R, the process of IR in HSHF-diet rats could be divided into slight and high IR stages, with the week-23 as the cut-off point. In early slight IR stage, key molecules expressions of hepatic Gsα-cAMP signal axis in HSHF-diet rats were up-regulated with significantly elevated fasting blood glucose (FBG) from 18 to 23 weeks. Unexpectedly, in high IR stage, hepatic Gsα-cAMP signal axis was recovered comparatively to that of normal chow-diet rats, and no significant differences in FBG levels were found. However, in diabetic db/db mice, up-regulation of hepatic Gsα-cAMP signal axis was responsible for its severely increased fasting hyperglycaemia. Our data revealed a positive correlation between hepatic Gsα-cAMP signal axis and FBG in slight IR stage of HSHF-diet rats and diabetic db/db mice. The current finding thus suggested hepatic Gsα-cAMP signal axis plays a central role in regulating of FBG during the occurrence and development of T2DM.

  18. Ethologically based resolution of D2-like dopamine receptor agonist-versus antagonist-induced behavioral topography in dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein of 32 kDa "knockout" mutants congenic on the C57BL/6 genetic background.

    PubMed

    Nally, Rachel E; Kinsella, Anthony; Tighe, Orna; Croke, David T; Fienberg, Allen A; Greengard, Paul; Waddington, John L

    2004-09-01

    Given the critical role of dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein of 32 kDa (DARPP-32) in the regulation of dopaminergic function, DARPP-32-null mutant mice congenic on the inbred C57BL/6 strain for 10 generations were examined phenotypically for their ethogram of responsivity to the selective D2-like receptor agonist RU 24213 (N-n-propyl-N-phenylethyl-p-3-hydroxyphenylethylamine) and the selective D2-like receptor antagonist YM 09151-2 (cis-N-[1-benzyl-2-methyl-pyrrolidin-3-yl]-5-chloro-2-methoxy-4-methylaminobenzamide), using procedures that resolve all topographies of behavior in the natural repertoire. After vehicle challenge, levels of sniffing and rearing seated were reduced in DARPP-32 mutants; the injection procedure seems to constitute a "stressor" that reveals phenotypic effects of DARPP-32 deletion not apparent under natural conditions. Topographical effects of 0.3 to 10.0 mg/kg RU 24213, primarily induction of sniffing and ponderous locomotion with accompanying reductions in rearing, grooming, sifting and chewing, were not altered to any material extent in DARPP-32-null mice. However, topographical effects of 0.005 to 0.625 mg/kg YM 09151-2, namely, reduction in sniffing, locomotion, rearing, grooming, and chewing but not sifting, were essentially absent in DARPP-32 mutants. Thus, the D2-like receptor agonist-mediated ethogram was essentially conserved, whereas major elements of the corresponding D2-like receptor antagonist-mediated ethogram were essentially absent in DARPP-32-null mice. This suggests some relationship between 1) extent of tonic dopaminergic activation of DARPP-32 mechanisms and 2) compensatory mechanisms consequent to the developmental absence of DARPP-32, which may emerge to act differentially on individual elements of the DARPP-32 system. Critically, the present data indicate that phenotypic effects of a given gene deletion using an agonist acting on the system disrupted cannot be generalized to a

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

    PubMed

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

    2011-01-21

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

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

    PubMed

    Rose, Nicholas D; Regan, John M

    2015-12-01

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

  1. The cAMP-producing agonist beraprost inhibits human vascular smooth muscle cell migration via exchange protein directly activated by cAMP

    PubMed Central

    McKean, Jenny S.; Murray, Fiona; Gibson, George; Shewan, Derryck A.; Tucker, Steven J.; Nixon, Graeme F.

    2015-01-01

    Aims During restenosis, vascular smooth muscle cells (VSMCs) migrate from the vascular media to the developing neointima. Preventing VSMC migration is therefore a therapeutic target for restenosis. Drugs, such as prostacyclin analogues, that increase the intracellular concentration of cyclic adenosine monophosphate (cAMP) can inhibit VSMC migration, but the mechanisms via which this occurs are unknown. Two main downstream mediators of cAMP are protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). This study has examined the effects of the prostacyclin analogue beraprost on VSMC migration and investigated the intracellular pathways involved. Methods and results In a chemotaxis chamber, human saphenous vein VSMC migrated towards a platelet-derived growth-factor-BB (PDGF) chemogradient. Incubation with therapeutically relevant concentrations of cAMP-producing agonist beraprost significantly decreased PDGF-induced migration. Direct activation of either PKA or Epac inhibited migration whereas inhibition of PKA did not prevent the anti-migratory effect of beraprost. Direct activation of Epac also prevented hyperplasia in ex vivo serum-treated human veins. Using fluorescence resonance energy transfer, we demonstrated that beraprost activated Epac but not PKA. The mechanisms of this Epac-mediated effect involved activation of Rap1 with subsequent inhibition of RhoA. Cytoskeletal rearrangement at the leading edge of the cell was consequently inhibited. Interestingly, Epac1 was localized to the leading edge of migrating VSMC. Conclusions These results indicate that therapeutically relevant concentrations of beraprost can inhibit VSMC migration via a previously unknown mechanism involving the cAMP mediator Epac. This may provide a novel target that could blunt neointimal formation. PMID:26092100

  2. Protective Effect of a cAMP Analogue on Behavioral Deficits and Neuropathological Changes in Cuprizone Model of Demyelination.

    PubMed

    Vakilzadeh, Gelareh; Khodagholi, Fariba; Ghadiri, Tahereh; Darvishi, Marzieh; Ghaemi, Amir; Noorbakhsh, Farshid; Gorji, Ali; Sharifzadeh, Mohammad

    2015-08-01

    Multiple sclerosis (MS) is an inflammatory demyelinating disease that leads to neuronal cell loss. Cyclic AMP and its analogs are well known to decrease inflammation and apoptosis. In the present study, we examined the effects of bucladesine, a cell-permeable analogue of cyclic adenosine monophosphate (cAMP), on myelin proteins (PLP, PMP-22), inflammation, and apoptotic, as well as anti-apoptotic factors in cuprizone model of demyelination. C57BL/6J mice were fed with chow containing 0.2% copper chelator cuprizone or vehicle by daily oral gavage for 5 weeks to induce reversible demyelination predominantly of the corpus callosum. Bucladesine was administered intraperitoneally at different doses (0.24, 0.48, or 0.7 μg/kg body weight) during the last 7 days of 5-week cuprizone treatment. Bucladesine exhibited a protective effect on myelination. Furthermore, bucladesine significantly decreased the production of interleukin-6 pro-inflammatory mediator as well as nuclear factor-κB activation and reduced the mean number of apoptotic cells compared to cuprizone-treated mice. Bucladesine also decreased production of caspase-3 as well as Bax and increased Bcl-2 levels. Our data revealed that enhancement of intracellular cAMP prevents demyelination and plays anti-inflammatory and anti-apoptotic properties in mice cuprizone model of demyelination. This suggests the modulation of intracellular cAMP as a potential target for treatment of MS.

  3. Berberine attenuates cAMP-induced lipolysis via reducing the inhibition of phosphodiesterase in 3T3-L1 adipocytes.

    PubMed

    Zhou, Libin; Wang, Xiao; Yang, Ying; Wu, Ling; Li, Fengying; Zhang, Rong; Yuan, Guoyue; Wang, Ning; Chen, Mingdao; Ning, Guang

    2011-04-01

    Berberine, a hypoglycemic agent, has been shown to decrease plasma free fatty acids (FFAs) level in insulin-resistant rats. In the present study, we explored the mechanism responsible for the antilipolytic effect of berberine in 3T3-L1 adipocytes. It was shown that berberine attenuated lipolysis induced by catecholamines, cAMP-raising agents, and a hydrolyzable cAMP analog, but not by tumor necrosis factor α and a nonhydrolyzable cAMP analog. Unlike insulin, the inhibitory effect of berberine on lipolysis in response to isoproterenol was not abrogated by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, but additive to that of PD98059, an extracellular signal-regulated kinase kinase inhibitor. Prior exposure of adipocytes to berberine decreased the intracellular cAMP production induced by isoproterenol, forskolin, and 3-isobutyl-1-methylxanthine (IBMX), along with hormone-sensitive lipase (HSL) Ser-563 and Ser-660 dephosphorylation, but had no effect on perilipin phosphorylation. Berberine stimulated HSL Ser-565 as well as adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. However, compound C, an AMPK inhibitor, did not reverse the regulatory effect of berberine on HSL Ser-563, Ser-660, and Ser-565 phosphorylation, nor the antilipolytic effect of berberine. Knockdown of AMPK using RNA interference also failed to restore berberine-suppressed lipolysis. cAMP-raising agents increased AMPK activity, which was not additive to that of berberine. Stimulation of adipocytes with berberine increased phosphodiesterase (PDE) 3B and PDE4 activity measured by hydrolysis of (3)[H]cAMP. These results suggest that berberine exerts an antilipolytic effect mainly by reducing the inhibition of PDE, leading to a decrease in cAMP and HSL phosphorylation independent of AMPK pathway.

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

    PubMed

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

    2015-04-15

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

  5. Antidiabetic sulfonylureas and cAMP cooperatively activate Epac2A.

    PubMed

    Takahashi, Toshimasa; Shibasaki, Tadao; Takahashi, Harumi; Sugawara, Kenji; Ono, Aika; Inoue, Naoko; Furuya, Toshio; Seino, Susumu

    2013-10-22

    Sulfonylureas are widely used drugs for treating insulin deficiency in patients with type 2 diabetes. Sulfonylureas bind to the regulatory subunit of the pancreatic β cell potassium channel that controls insulin secretion. Sulfonylureas also bind to and activate Epac2A, a member of the Epac family of cyclic adenosine monophosphate (cAMP)-binding proteins that promote insulin secretion through activation of the Ras-like guanosine triphosphatase Rap1. Using molecular docking simulation, we identified amino acid residues in one of two cyclic nucleotide-binding domains, cNBD-A, in Epac2A predicted to mediate the interaction with sulfonylureas. We confirmed the importance of the identified residues by site-directed mutagenesis and analysis of the response of the mutants to sulfonylureas using two assays: changes in fluorescence resonance energy transfer (FRET) of an Epac2A-FRET biosensor and direct sulfonylurea-binding experiments. These residues were also required for the sulfonylurea-dependent Rap1 activation by Epac2A. Binding of sulfonylureas to Epac2A depended on the concentration of cAMP and the structures of the drugs. Sulfonylureas and cAMP cooperatively activated Epac2A through binding to cNBD-A and cNBD-B, respectively. Our data suggest that sulfonylureas stabilize Epac2A in its open, active state and provide insight for the development of drugs that target Epac2A.

  6. Oncogenic K-ras expression is associated with derangement of the cAMP/PKA pathway and forskolin-reversible alterations of mitochondrial dynamics and respiration.

    PubMed

    Palorini, R; De Rasmo, D; Gaviraghi, M; Sala Danna, L; Signorile, A; Cirulli, C; Chiaradonna, F; Alberghina, L; Papa, S

    2013-01-17

    The Warburg effect in cancer cells has been proposed to involve several mechanisms, including adaptation to hypoxia, oncogenes activation or loss of oncosuppressors and impaired mitochondrial function. In previous papers, it has been shown that K-ras transformed mouse cells are much more sensitive as compared with normal cells to glucose withdrawal (undergoing apoptosis) and present a high glycolytic rate and a strong reduction of mitochondrial complex I. Recent observations suggest that transformed cells have a derangement in the cyclic adenosine monophosphate/cAMP-dependent protein kinase (cAMP/PKA) pathway, which is known to regulate several mitochondrial functions. Herein, the derangement of the cAMP/PKA pathway and its impact on transformation-linked changes of mitochondrial functions is investigated. Exogenous stimulation of PKA activity, achieved by forskolin treatment, protected K-ras-transformed cells from apoptosis induced by glucose deprivation, enhanced complex I activity, intracellular adenosine triphosphate (ATP) levels, mitochondrial fusion and decreased intracellular reactive oxygen species (ROS) levels. Several of these effects were almost completely prevented by inhibiting the PKA activity. Short-time treatment with compounds favoring mitochondrial fusion strongly decreased the cellular ROS levels especially in transformed cells. These findings support the notion that glucose shortage-induced apoptosis, specific of K-ras-transformed cells, is associated to a derangement of PKA signaling that leads to mitochondrial complex I decrease, reduction of ATP formation, prevalence of mitochondrial fission over fusion, and thereby opening new approaches for development of anticancer drugs.

  7. Kindlin-2 regulates hemostasis by controlling endothelial cell–surface expression of ADP/AMP catabolic enzymes via a clathrin-dependent mechanism

    PubMed Central

    Pluskota, Elzbieta; Ma, Yi; Bledzka, Kamila M.; Bialkowska, Katarzyna; Soloviev, Dmitry A.; Szpak, Dorota; Podrez, Eugene A.; Fox, Paul L.; Hazen, Stanley L.; Dowling, James J.; Ma, Yan-Qing

    2013-01-01

    Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice. In the present study, we tested whether hemostasis might be perturbed in kindlin-2+/− mice. Bleeding time and carotid artery occlusion time were significantly prolonged in kindlin-2+/− mice. Whereas plasma concentrations/activities of key coagulation/fibrinolytic proteins and platelet counts and aggregation were similar in wild-type and kindlin-2+/− mice, kindlin-2+/− endothelial cells (ECs) showed enhanced inhibition of platelet aggregation induced by adenosine 5′-diphosphate (ADP) or low concentrations of other agonists. Cell-surface expression of 2 enzymes involved in ADP/adenosine 5′-monophosphate (AMP) degradation, adenosine triphosphate (ATP) diphosphohydrolase (CD39) and ecto-5′-nucleotidase (CD73) were increased twofold to threefold on kindlin-2+/− ECs, leading to enhanced ATP/ADP catabolism and production of adenosine, an inhibitor of platelet aggregation. Trafficking of CD39 and CD73 at the EC surface was altered in kindlin-2+/− mice. Mechanistically, this was attributed to direct interaction of kindlin-2 with clathrin heavy chain, thereby controlling endocytosis and recycling of CD39 and CD73. The interaction of kindlin-2 with clathrin was independent of its integrin binding site but still dependent on a site within its F3 subdomain. Thus, kindlin-2 regulates trafficking of EC surface enzymes that control platelet responses and hemostasis. PMID:23896409

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

    PubMed

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

    2007-03-29

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

  9. Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes

    PubMed Central

    Tamás, Peter; Hawley, Simon A.; Clarke, Rosemary G.; Mustard, Kirsty J.; Green, Kevin; Hardie, D. Grahame; Cantrell, Doreen A.

    2006-01-01

    The adenosine monophosphate (AMP)–activated protein kinase (AMPK) has a crucial role in maintaining cellular energy homeostasis. This study shows that human and mouse T lymphocytes express AMPKα1 and that this is rapidly activated in response to triggering of the T cell antigen receptor (TCR). TCR stimulation of AMPK was dependent on the adaptors LAT and SLP76 and could be mimicked by the elevation of intracellular Ca2+ with Ca2+ ionophores or thapsigargin. AMPK activation was also induced by energy stress and depletion of cellular adenosine triphosphate (ATP). However, TCR and Ca2+ stimulation of AMPK required the activity of Ca2+–calmodulin-dependent protein kinase kinases (CaMKKs), whereas AMPK activation induced by increased AMP/ATP ratios did not. These experiments reveal two distinct pathways for the regulation of AMPK in T lymphocytes. The role of AMPK is to promote ATP conservation and production. The rapid activation of AMPK in response to Ca2+ signaling in T lymphocytes thus reveals that TCR triggering is linked to an evolutionally conserved serine kinase that regulates energy metabolism. Moreover, AMPK does not just react to cellular energy depletion but also anticipates it. PMID:16818670

  10. Adenosine modulates cell growth in the human breast cancer cells via adenosine receptors.

    PubMed

    Panjehpour, Mojtaba; Karami-Tehrani, Fatemeh

    2007-01-01

    Adenosine modulates the proliferation, survival, and apoptosis of many different cell types. The present study was performed to investigate the role of adenosine receptors in the human breast cancer cell lines MCF-7 and MDA-MB468. The biological effects of adenosine on the cells were analyzed by adenylyl cyclase and cell viability assay as well as RT-PCR of adenosine receptors. RT-PCR results show the expression of the transcript of all adenosine receptors in both cell lines. By using adenosine and selective adenosine receptor agonists or antagonists, we found that A3 stimulation reduced cell viability, which was abolished by pretreatment with A3 receptor antagonist. Moreover, we demonstrated that adenosine (natural agonist) triggers a cytotoxic signal via A3 receptor activation that was not seen for other subclasses of adenosine receptors. Intracellular cAMP concentration was changed significantly only for A3 and A2B receptor-selective agonists, which indicates the functional form of these receptors on the cell surface. In conclusion, our findings revealed the role of adenosine receptors in breast cancer cell lines on growth modulation role of A3 and functional form of A2B, although its involvement in cell growth modulation was not seen. Theses findings as well as data by others may provide a possible application of adenosine receptor agonists/antagonists in breast malignancies.

  11. Cyclen-based bismacrocycles for biological anion recognition. A potentiometric and NMR study of AMP, ADP and ATP nucleotide complexation.

    PubMed

    Delépine, Anne-Sophie; Tripier, Raphaël; Handel, Henri

    2008-05-21

    The behaviour of two cyclen-based bismacrocycles linked by aromatic spacers as receptors of adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) anions is explored. The two bismacrocycles differ from one another by the nature of their spacers, which are respectively 1,3-dimethylbenzene (BMC), or 2,6-dimethylpyridine (BPyC). Potentiometric investigations supported by (1)H and (31)P NMR measurements were performed over a wide pH range to characterize and understand the driving forces implicated in the supramolecular assemblies. A comparison is also carried out with the results presented in this work and those obtained previously with these two ligands and inorganic phosphates. The comparison exhibits the importance of pi-stacking capability of the organic anions in the binding and hydrogen-bonding network. For BPyC, NMR studies highlight two coordination schemes depending on the protonation of the nitrogen atom of the pyridinyl spacer, which acts in acidic media as a supplementary anchoring point.

  12. Modulation by stimulation rate of basal and cAMP-elevated Ca2+ channel current in guinea pig ventricular cardiomyocytes

    PubMed Central

    1995-01-01

    The modulation of L-type Ca2+ current (ICa) by changes in stimulation frequency was investigated in single ventricular cardiomyocytes isolated from guinea pig hearts. Electrical recordings were carried out at 21-25 degrees C and at 33-37 degrees C with the whole-cell patch clamp method, under K(+)-free conditions. A comparison is made between the response to frequency changes for ICa in the basal state and after the application of drugs which elevate the level of adenosine-3',5'- cyclic monophosphate (cAMP) within the cells. Peak basal ICa was reduced with an increase in stimulation rate from 0.5 Hz to 1, 2, 3, 4, or 5 Hz. This frequency-induced reduction of ICa was enhanced by reduced temperature, was unchanged when Na+ or Ba2+ carried the basal Ca2+ channel current, and was greatly enhanced after elevating cAMP levels with forskolin, isoprenaline, or 8-(4-chlorophenylthio)-cyclic AMP. We examined the mechanism of the enhancement of the frequency- induced reduction of ICa by cAMP, and found two conditions which abolished it: (a) application of isoprenaline when Na+ carried the Ca2+ channel current in Ca(2+)-free solution, or (b) application of 3- isobutyl-1-methylxanthine, a broad-spectrum phosphodiesterase inhibitor. It was further shown that an elevation of both ICa and cAMP (induced by isoprenaline), and not an increase of ICa alone (induced by Bay K 8644), is required to produce the extra component of reduction by frequency. It is concluded that Ca2+ entry results in feedback regulation of ICa, through the activation of Ca(2+)-dependent phosphodiesterase(s). This is important in the context of sympathetic stimulation, which produces the companion conditions of an elevated heart rate and increases in cAMP levels and Ca2+ entry. PMID:8537815

  13. Anticancer effect of adenosine on gastric cancer via diverse signaling pathways.

    PubMed

    Tsuchiya, Ayako; Nishizaki, Tomoyuki

    2015-10-21

    Extracellular adenosine induces apoptosis in a variety of cancer cells via intrinsic and extrinsic pathways. In the former pathway, adenosine uptake into cells triggers apoptosis, and in the latter pathway, adenosine receptors mediate apoptosis. Extracellular adenosine also induces apoptosis of gastric cancer cells. Extracellular adenosine is transported into cells through an adenosine transporter and converted to AMP by adenosine kinase. In turn, AMP activates AMP-activated protein kinase (AMPK). AMPK is the factor responsible for caspase-independent apoptosis of GT3-TKB gastric cancer cells. Extracellular adenosine, on the other hand, induces caspase-dependent apoptosis of MKN28 and MKN45 gastric cancer cells by two mechanisms. Firstly, AMP, converted from intracellularly transported adenosine, initiates apoptosis, regardless of AMPK. Secondly, the A3 adenosine receptor, linked to Gi/Gq proteins, mediates apoptosis by activating the Gq protein effector, phospholipase Cγ, to produce inositol 1,4,5-trisphosphate and diacylglycerol, which activate protein kinase C. Consequently, the mechanisms underlying adenosine-induced apoptosis vary, depending upon gastric cancer cell types. Understand the contribution of each downstream target molecule of adenosine to apoptosis induction may aid the establishment of tailor-made chemotherapy for gastric cancer.

  14. 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.

  15. 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.

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

    PubMed Central

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

    2015-01-01

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

  17. [Effect of red light on activity of cAMP phosphodiesterases in photoperiodically different cereals and vernalized winter wheat].

    PubMed

    Fedenko, E P; Koksharova, T A

    2007-01-01

    Red light illumination of seedlings of photoperiodically different cereals had a different effect on the activity of multiple cyclic adenosine monophosphate phosphodiesterases. The response of all phosphodiesterase forms was reversed in fully vernalized winter wheat Triticum aestivum L.

  18. Ex vivo study of 5-HT(1A) and 5-HT(7) receptor agonists and antagonists on cAMP accumulation during memory formation and amnesia.

    PubMed

    Perez-García, G; Meneses, A

    2008-12-16

    The cyclic adenosine monophosphate (cAMP) is a second messenger and a central component of intracellular signaling pathways that regulate a wide range of biological functions, including memory. Hence, in this work, firstly the time-course of memory formation was determined in an autoshaping learning task, which had allowed the identification of testing times for increases or decreases in performance. Next, untrained, trained and overtrained groups were compared in cAMP production. Moreover, selective stimulation and antagonism of 5-HT(1A) and 5-HT(7) receptors during memory formation and cAMP production were determined. Finally, since there is scarce information about how pharmacological models of amnesia affect cAMP production, the cholinergic or glutamatergic antagonists, scopolamine and dizocilpine, were tested. The major findings of this work showed that when the time-course was determined inasmuch as training and testing sessions occurred, memory performance was graduate and progressive. Notably, for the fourth to seventh (i.e., 48-120 h following autoshaping training session) testing session performance was significantly higher from the previous ones. When animals received 5-HT(1A) and 5-HT(7) receptor agonists and antagonists or amnesic drugs significant increases or decrements in memory performance were observed at 24 and 48 h. Moreover, when ex vivo cAMP production from trained and overtrained groups were compared to untrained ones, significant differences were observed among groups and brain areas. Trained animals treated with 8-OHDPAT, AS19, 8-OHDPAT plus AS19, WAY100635, SB-269970, scopolamine or dizocilpine were compared to similar untrained groups, and eightfold-reduced cAMP production was evident, showing the importance of cAMP production in the signaling case in mammalian memory formation.

  19. The combination of db-cAMP and ChABC with poly(propylene carbonate) microfibers promote axonal regenerative sprouting and functional recovery after spinal cord hemisection injury.

    PubMed

    Xia, Tongliang; Huang, Bin; Ni, Shilei; Gao, Lei; Wang, Jiangang; Wang, Jian; Chen, Anjing; Zhu, Shaowei; Wang, Benlin; Li, Gang; Zhu, Shugan; Li, Xingang

    2017-02-01

    This study describes the use of poly(propylene carbonate) (PPC) electrospun microfibres impregnated with a combination of dibutyryl cyclic adenosine monophosphate (db-cAMP) and chondroitinase ABC (ChABC) in the treatment of right-side hemisected spinal cord injury (SCI). Release of db-cAMP and/or ChABC from the microfibres was assessed in vitro using high-performance liquid chromatography (HPLC). Drug-impregnated microfibres were implanted into the hemisected thoracic spinal cord of rats, and treatment was evaluated using functional recovery examinations and immunohistochemistry. Our results demonstrated that the microfibres containing db-cAMP and/or ChABC displayed a stable and prolonged release of each agent. Sustained delivery of db-cAMP and/or ChABC was found to promote axonal regenerative sprouting, functional recovery, and reduced glial scar formation when compared to untreated control animals. The combination of both db-cAMP and ChABC was determined to be more effective than using either drug alone in the treatment of SCI. These findings demonstrate the feasibility of using PPC electrospun microfibres for multi-drug combination therapy in SCI.

  20. Biomechanical forces promote blood development through prostaglandin E2 and the cAMP-PKA signaling axis.

    PubMed

    Diaz, Miguel F; Li, Nan; Lee, Hyun Jung; Adamo, Luigi; Evans, Siobahn M; Willey, Hannah E; Arora, Natasha; Torisawa, Yu-Suke; Vickers, Dwayne A; Morris, Samantha A; Naveiras, Olaia; Murthy, Shashi K; Ingber, Donald E; Daley, George Q; García-Cardeña, Guillermo; Wenzel, Pamela L

    2015-05-04

    Blood flow promotes emergence of definitive hematopoietic stem cells (HSCs) in the developing embryo, yet the signals generated by hemodynamic forces that influence hematopoietic potential remain poorly defined. Here we show that fluid shear stress endows long-term multilineage engraftment potential upon early hematopoietic tissues at embryonic day 9.5, an embryonic stage not previously described to harbor HSCs. Effects on hematopoiesis are mediated in part by a cascade downstream of wall shear stress that involves calcium efflux and stimulation of the prostaglandin E2 (PGE2)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling axis. Blockade of the PGE2-cAMP-PKA pathway in the aorta-gonad-mesonephros (AGM) abolished enhancement in hematopoietic activity. Furthermore, Ncx1 heartbeat mutants, as well as static cultures of AGM, exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element-binding protein (CREB). Similar to flow-exposed cultures, transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution. These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential.

  1. Resveratrol provides neuroprotection by inhibiting phosphodiesterases and regulating the cAMP/AMPK/SIRT1 pathway after stroke in rats.

    PubMed

    Wan, Dan; Zhou, Yehan; Wang, Ke; Hou, Yongying; Hou, Ruihang; Ye, Xiufeng

    2016-03-01

    Dysfunction of energy metabolism can be a significant and fundamental pathophysiological basis for strokes. In studies of both humans and rodents, resveratrol, a natural polyphenol, has been reported to provide protection from cerebral ischemic injury by regulating expression of silent mating type information regulation 2 homolog 1 (SIRT1). However, direct evidence demonstrating that resveratrol exerts neuroprotection from cerebral ischemia injury by decreasing energy consumption is still lacking. Therefore, the aim of this study was to elucidate the mechanisms and signaling pathways through which resveratrol regulates energy metabolism in the ischemic brain, and to identify potential targets of resveratrol. ATP levels in brain tissues were detected by high performance liquid chromatography. SIRT1 and the phosphorylation of adenosine-monophosphate-activated protein kinase (P-AMPK) expressiones were evaluated by western blot. Levels of phosphodiesterase (PDEs) and cAMP were quantitated by real-time PCR and ELISA, respectively. Results showed that resveratrol significantly reduced the harmful effects of cerebral ischemic injury in vivo. Moreover, levels of ATP, p-AMPK, SIRT1, and cAMP were increased by resveratrol and PDE inhibitors. In conclusion, our findings indicate that resveratrol provides neuroprotection by inhibiting PDEs and regulating the cAMP/AMPK/SIRT1 pathway, which reduces ATP energy consumption during ischemia.

  2. Adenosine Kinase: Exploitation for Therapeutic Gain

    PubMed Central

    2013-01-01

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

  3. Resveratrol attenuates vascular endothelial inflammation by inducing autophagy through the cAMP signaling pathway.

    PubMed

    Chen, Ming-Liang; Yi, Long; Jin, Xin; Liang, Xin-Yu; Zhou, Yong; Zhang, Ting; Xie, Qi; Zhou, Xi; Chang, Hui; Fu, Yu-Jie; Zhu, Jun-Dong; Zhang, Qian-Yong; Mi, Man-Tian

    2013-12-01

    Inflammation participates centrally in all stages of atherosclerosis (AS), which begins with inflammatory changes in the endothelium, characterized by expression of the adhesion molecules. Resveratrol (RSV) is a naturally occurring phytoalexin that can attenuate endothelial inflammation; however, the exact mechanisms have not been thoroughly elucidated. Autophagy refers to the normal process of cell degradation of proteins and organelles, and is protective against certain inflammatory injuries. Thus, we intended to determine the role of autophagy in the antiinflammatory effects of RSV in human umbilical vein endothelial cells (HUVECs). We found that RSV pretreatment reduced tumor necrosis factor ? (TNF/TNF?)-induced inflammation and increased MAP1LC3B2 (microtubule-associated protein 1 light chain 3 ? 2) expression and SQSTM1/p62 (sequestosome 1) degradation in a concentration-dependent manner. A bafilomycin A 1 (BafA1) challenge resulted in further accumulation of MAP1LC3B2 in HUVECs. Furthermore, autophagy inhibitors 3-methyladenine (3-MA), chloroquine as well as ATG5 and BECN1 siRNA significantly attenuated RSV-induced autophagy, which, subsequently, suppressed the downregulation of RSV-induced inflammatory factors expression. RSV also increased cAMP (cyclic adenosine monophosphate) content, the expression of PRKA (protein kinase A) and SIRT1 (sirtuin 1), as well as the activity of AMPK (AMP-activated protein kinase). RSV-induced autophagy in HUVECs was abolished in the presence of inhibitors of ADCY (adenylyl cyclase, KH7), PRKA (H-89), AMPK (compound C), or SIRT1 (nicotinamide and EX-527), as well as ADCY, PRKA, AMPK, and SIRT1 siRNA transfection, indicating that the effects of RSV on autophagy induction were dependent on cAMP, PRKA, AMPK and SIRT1. In conclusion, RSV attenuates endothelial inflammation by inducing autophagy, and the autophagy in part was mediated through the activation of the cAMP-PRKA-AMPK-SIRT1 signaling pathway.

  4. Cyclic AMP affects the haemocyte responses of larval Galleria mellonella to selected antigens.

    PubMed

    Marin, David; Dunphy, Gary B; Mandato, Craig A

    2005-05-01

    Signal transduction of the innate immediate responses of insect haemocytes to foreign matter is rarely considered. Herein using a combination of adenylate cyclase inhibitors and activators and phosphodiesterase inhibitors we determined that cyclic adenosine monophosphate (cAMP) at high levels normally impairs non-self response. Haemocyte contact with glass and bacteria lowered cAMP in vitro. Inactive phosphodiesterases, including type 4, impaired haemocyte reactions in vitro. Using the drugs in vivo to modulate adenylate cyclase and phosphodiesterases altered the total and types of haemocytes. Adenylate cyclase inhibitors and etazolate (a type 4 phosphodiesterase inhibitor) alone produced changes in the haemograms similar to those caused by Bacillus subtilis. Sequential injections of an enzyme modulator followed by B. subtilis impaired bacterial removal due (1) in the case of enzyme inhibitors, to the removal of haemocytes prior to bacterial challenge and (2) in the case of forskolin and IBMX to the shut-down of the haemocytes. Activating adenylate cyclase or inhibiting phosphodiesterase impaired bacterial removal when co-injecting the compounds and bacteria.

  5. Enhanced cAMP accumulation by a phorbol ester in cerebral cortical cells

    SciTech Connect

    Beeler, J.F.; Davis, C.W.

    1987-05-01

    Phorbol 12-myristate-13-acetate (PMA) was found to be selective in its ability to alter cAMP accumulations in cultured rat cerebral cortical cells. Basal levels of cAMP in cultured neuronal and nonneuronal cells preincubated in the absence or presence of PMA were 14 pmol/mg protein and 16 pmol/mg protein, respectively. Adenosine increased cAMP levels in a dose-dependent manner. cAMP accumulation in response to low concentrations of adenosine was not significantly altered by pretreatment with PMA but marked potentiation of adenosine elicited accumulations was observed at 10 and 100 ..mu..M adenosine. Longer preincubation with PMA resulted in a decreased ability of PMA to enhance adenosine elicited accumulations of cAMP. PMA did not significantly alter cAMP accumulation by forskolin (FOR) and enhanced norepinephrine stimulated cAMP by only 2-fold. For similarly potentiated adenosine/sub 2/ (A/sub 2/)- receptor elicited accumulation of cAMP which could be further enhanced by PMA. These results suggest that the effects of the phorbol ester are more specific for potentiating adenosine stimulated cAMP accumulation and may occur as a result of a more efficient coupling between the A/sub 2/-receptor, N-protein and adenylate cyclase.

  6. Intermittent parathyroid hormone (1-34) application regulates cAMP-response element binding protein activity to promote the proliferation and osteogenic differentiation of bone mesenchymal stromal cells, via the cAMP/PKA signaling pathway.

    PubMed

    Chen, Bailing; Lin, Tao; Yang, Xiaoxi; Li, Yiqiang; Xie, Denghui; Cui, Haowen

    2016-06-01

    The potential effects of intermittent parathyroid hormone (1-34) [PTH (1-34)] administration on bone formation have previously been investigated. A number of studies have suggested that the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway is associated with PTH-induced osteogenic differentiation. However, the precise signaling pathways and molecular mechanism by which PTH (1-34) induces the osteogenic differentiation of bone mesenchymal stromal cells (BMSCs) remain elusive. The purpose of the present study was to investigate the mechanism underlying the effect of intermittent PTH (1-34) application on the proliferation and osteogenic differentiation of BMSCs. BMSCs were randomly divided into four groups, as follows: Osteogenic medium (control group); osteogenic medium and intermittent PTH (1-34); osteogenic medium and intermittent PTH (1-34) plus the adenylyl cyclase activator forskolin; and osteogenic medium and intermittent PTH (1-34) plus the PKA inhibitor H-89. A cell proliferation assay revealed that PTH (1-34) stimulates BMSC proliferation via the cAMP/PKA pathway. Furthermore, reverse transcription-quantitative polymerase chain reaction, alkaline phosphatase activity testing and cell examination using Alizarin Red S staining demonstrated that PTH (1-34) administration promotes osteogenic differentiation and mineralization, mediated by the cAMP/PKA pathway. Crucially, the results of western blot analyses suggested that PTH (1-34) treatment and, to a greater degree, PTH (1-34) plus forskolin treatment caused an increase in phosphorylated cAMP response element binding protein (p-CREB) expression, but the effect of PTH on p-CREB expression was blocked by H-89. In conclusion, the current study demonstrated that intermittent PTH (1-34) administration regulates downstream proteins, particularly p-CREB, in the cAMP/PKA signaling pathway, to enhance the proliferation, osteogenic differentiation and mineralization of BMSCs.

  7. Regulation of adenosine transport by acute and chronic ethanol exposure

    SciTech Connect

    Nagy, L.E.; Casso, D.; Diamond, I.; Gordon, A.S. )

    1989-02-09

    Chronic exposure to ethanol results in a desensitization of adenosine receptor-stimulated cAMP production. Since adenosine is released by cells and is known to desensitize its own as well as other receptors, it may be involved in ethanol-induced desensitization of adenosine receptor function. Therefore, we have examine the acute and chronic effects of ethanol on the transport of adenosine via the nucleoside transport. Acute exposure to ethanol caused an inhibition of adenosine uptake in S49 lymphoma cells. This decrease in uptake resulted in accumulation of extracellular adenosine after ethanol exposure. The effect of ethanol was specific to nucleoside transport. Uptake of uridine, also transported by the nucleoside transporter, was inhibited by ethanol to the same degree as adenosine uptake, while neither isoleucine nor deoxyglucose uptake was altered by ethanol treatment. Inhibition of adenosine uptake by ethanol was non-competitive and dependent on the concentration of ethanol. After chronic exposure to ethanol, cells became tolerant to the acute effects of ethanol. There was no longer an acute inhibition of adenosine uptake, nor was these accumulation of extracellular adenosine. Chronic ethanol exposure also resulted in a decrease in the absolute rate of adenosine uptake. Binding studies using a high affinity lignad for the nucleoside transporter, nitrobenzylthioinosine (NBMPR), indicate that this decreased uptake was due to a decrease in the maximal number of binding sites. These ethanol-induced changes in adenosine transport may be important for the acute and chronic effects of ethanol.

  8. Separation of N2-ethyl-2'-deoxyguanosine-5'-monophosphate and four native deoxyribonucleoside monophosphates using capillary zone electrophoresis with polyethylene glycol as buffer additive.

    PubMed

    Esaka, Y; Inagaki, S; Goto, M; Sako, M

    2001-01-01

    We investigated the separation of five deoxyribonucleoside monophosphates: 2'-deoxyguanosine-5'-monophosphate (dGMP), 2'-deoxyadenosine-5'-monophosphate (dAMP), 2'-deoxycytosine-5'-monophosphate (dCMP), 2'-deoxythymidine-5'-monophosphate (dTMP) and a dGMP adduct possessing N2-ethyl-guanine, which has been noted in relation to mutagenesis of alcohol, using capillary zone electrophoresis (CZE). The concentration of polyethylene glycol (PEG) as a modifier and the pH of the running solutions can efficiently control the observed separation. Interaction of PEG with analytes was quantitatively evaluated. PEG worked effectively as a hydrophobic selector in these separations. The values of pKa of the acidic-NH-groups in the base moieties of dGMP, dTMP, and the dGMP adduct are close to that of boric acid used as buffer of the running solutions. The control of their charge was facilitated, enabling improved separations. A more sufficient and fast separation was achieved by both optimization of pH of the running solutions and PEG concentration compared with that obtained by pH control alone. On-line concentration using a stacking method followed by the PEG-assisted CZE was briefly studied.

  9. Identification of a novel phosphatase with high affinity for nucleotides monophosphate from common bean (Phaseolus vulgaris).

    PubMed

    Cabello-Díaz, Juan Miguel; Quiles, Francisco Antonio; Lambert, Rocío; Pineda, Manuel; Piedras, Pedro

    2012-04-01

    Common bean (Phaseolus vulgaris) seedlings accumulate ureides derived from purines after germination. The first step in the conversion of purines to ureides is the removal of the 5'-phosphate group by a phosphatase that has not been established yet. Two main phosphatase activities were detected in the embryonic axes of common bean using inosine monophosphate as substrate in an in-gel assay. Both activities differed in their sensitive to the common phosphatase inhibitor molybdate, with the molybdate-resistant as the first enzyme induced after radicle protrusion. The molybdate-resistant phosphatase has been purified to electrophoretic homogeneity and this is the first enzyme which shows this resistance purified and characterized from plant tissues. The native enzyme was a monomer of 55 kDa and it showed highest activity with nucleotides as substrates, with the K(m) values in the micromolar range. Among nucleotides, the highest specific constant (V(max)/K(m)) was observed for adenosine monophosphate. Furthermore, the enzyme was inhibited by nucleosides, the products of the enzymatic reaction, with maximum effect for adenosine. Common bean seedlings imbibed in the presence of adenosine monophosphate in vivo showed the highest molybdate-resistant phosphatase activity in the axes in addition to increased ureide content. The data presented suggests that purified phosphatase is involved in nucleotide metabolism in embryonic axes from common bean.

  10. The role of microorganisms in the degradation of adenosine triphosphate (ATP) in chill-stored common carp (Cyprinus carpio) fillets.

    PubMed

    Li, Dapeng; Zhang, Longteng; Song, Sijia; Wang, Zhiying; Kong, Chunli; Luo, Yongkang

    2017-06-01

    Biochemical and microbial changes after harvest strongly affect the final quality and shelf life of fish and fish products. In this study, the role of microbes in the degradation of adenosine triphosphate (ATP), and the origin of adenosine monophosphate deaminase (AMPD) and acid phosphatase (ACP) in common carp fillets during different stages of chilled storage (at 4°C) were investigated. The content of ATP, ADP, AMP, IMP, HxR, and Hx, the activity of AMPD and ACP, and the total count of viable, Aeromonas, Pseudomonas, H2S-producing bacteria, and lactic acid bacteria were examined. Results indicated that the population of microbial communities in control samples increased with storage time, and Pseudomonas peaked on the 10th day of storage. Changes in AMPD activity were less related to the abundance of microbes during the entire storage period. However, ACP was derived from both fish muscle and microbial secretion during the middle and late stages of storage. Degradation of ATP to IMP was not affected by spoilage bacteria, but the hydrolysis of IMP, and the transformation of HxR to Hx was affected considerably by the spoilage bacteria.

  11. Interactions of Calcium Fluctuations during Cardiomyocyte Contraction with Real-Time cAMP Dynamics Detected by FRET

    PubMed Central

    Sprenger, Julia U.; Bork, Nadja I.; Herting, Jonas; Fischer, Thomas H.; Nikolaev, Viacheslav O.

    2016-01-01

    Calcium (Ca2+) and 3’,5’-cyclic adenosine monophosphate (cAMP) play a critical role for cardiac excitation-contraction-coupling. Both second messengers are known to interact with each other, for example via Ca2+-dependent modulation of phosphodiesterase 1 (PDE1) and adenylyl cyclase 5/6 (AC 5/6) activities, which is supposed to occur especially at the local level in distinct subcellular microdomains. Currently, many studies analyze global and local cAMP signaling and its regulation in resting cardiomyocytes devoid of electrical stimulation. For example, Förster resonance energy transfer (FRET) microscopy is a popular approach for visualization of real time cAMP dynamics performed in resting cardiomyocytes to avoid potential contractility-related movement artifacts. However, it is unknown whether such data are comparable with the cell behavior under more physiologically relevant conditions during contraction. Here, we directly compare the cAMP-FRET responses to AC stimulation and PDE inhibition in resting vs. paced adult mouse ventricular cardiomyocytes for both cytosolic and subsarcolemmal microdomains. Interestingly, no significant differences in cAMP dynamics could be detected after β-adrenergic (isoproterenol) stimulation, suggesting low impact of rapidly changing contractile Ca2+ concentrations on cytosolic cAMP levels associated with AC activation. However, the contribution of the calcium-dependent PDE1, but not of the Ca2+-insensitive PDE4, to the regulation of cAMP levels after forskolin stimulation was significantly increased. This increase could be mimicked by pretreatment of resting cells with Ca2+ elevating agents. Ca2+ imaging demonstrated significantly higher amplitudes of Ca2+ transients in forskolin than in isoproterenol stimulated cells, suggesting that forskolin stimulation might lead to stronger activation of PDE1. In conclusion, changes in intracellular Ca2+ during cardiomyocyte contraction dynamically interact with cAMP levels, especially

  12. Evolutionary Adaptation of the Essential tRNA Methyltransferase TrmD to the Signaling Molecule 3′,5′-cAMP in Bacteria*

    PubMed Central

    Agrebi, Rym; Bellows, Lauren E.; Collet, Jean-François; Kaever, Volkhard

    2017-01-01

    The nucleotide signaling molecule 3′,5′-cyclic adenosine monophosphate (3′,5′-cAMP) plays important physiological roles, ranging from carbon catabolite repression in bacteria to mediating the action of hormones in higher eukaryotes, including human. However, it remains unclear whether 3′,5′-cAMP is universally present in the Firmicutes group of bacteria. We hypothesized that searching for proteins that bind 3′,5′-cAMP might provide new insight into this question. Accordingly, we performed a genome-wide screen and identified the essential Staphylococcus aureus tRNA m1G37 methyltransferase enzyme TrmD, which is conserved in all three domains of life as a tight 3′,5′-cAMP-binding protein. TrmD enzymes are known to use S-adenosyl-l-methionine (AdoMet) as substrate; we have shown that 3′,5′-cAMP binds competitively with AdoMet to the S. aureus TrmD protein, indicating an overlapping binding site. However, the physiological relevance of this discovery remained unclear, as we were unable to identify a functional adenylate cyclase in S. aureus and only detected 2′,3′-cAMP but not 3′,5′-cAMP in cellular extracts. Interestingly, TrmD proteins from Escherichia coli and Mycobacterium tuberculosis, organisms known to synthesize 3′,5′-cAMP, did not bind this signaling nucleotide. Comparative bioinformatics, mutagenesis, and biochemical analyses revealed that the highly conserved Tyr-86 residue in E. coli TrmD is essential to discriminate between 3′,5′-cAMP and the native substrate AdoMet. Combined with a phylogenetic analysis, these results suggest that amino acids in the substrate binding pocket of TrmD underwent an adaptive evolution to accommodate the emergence of adenylate cyclases and thus the signaling molecule 3′,5′-cAMP. Altogether this further indicates that S. aureus does not produce 3′,5′-cAMP, which would otherwise competitively inhibit an essential enzyme. PMID:27881678

  13. Protein Kinase A-independent Ras Protein Activation Cooperates with Rap1 Protein to Mediate Activation of the Extracellular Signal-regulated Kinases (ERK) by cAMP.

    PubMed

    Li, Yanping; Dillon, Tara J; Takahashi, Maho; Earley, Keith T; Stork, Philip J S

    2016-10-07

    Cyclic adenosine monophosphate (cAMP) is an important mediator of hormonal stimulation of cell growth and differentiation through its activation of the extracellular signal-regulated kinase (ERK) cascade. Two small G proteins, Ras and Rap1, have been proposed to mediate this activation, with either Ras or Rap1 acting in distinct cell types. Using Hek293 cells, we show that both Ras and Rap1 are required for cAMP signaling to ERKs. The roles of Ras and Rap1 were distinguished by their mechanism of activation, dependence on the cAMP-dependent protein kinase (PKA), and the magnitude and kinetics of their effects on ERKs. Ras was required for the early portion of ERK activation by cAMP and was activated independently of PKA. Ras activation required the Ras/Rap guanine nucleotide exchange factor (GEF) PDZ-GEF1. Importantly, this action of PDZ-GEF1 was disrupted by mutation within its putative cyclic nucleotide-binding domain within PDZ-GEF1. Compared with Ras, Rap1 activation of ERKs was of longer duration. Rap1 activation was dependent on PKA and required Src family kinases and the Rap1 exchanger C3G. This is the first report of a mechanism for the cooperative actions of Ras and Rap1 in cAMP activation of ERKs. One physiological role for the sustained activation of ERKs is the transcription and stabilization of a range of transcription factors, including c-FOS. We show that the induction of c-FOS by cAMP required both the early and sustained phases of ERK activation, requiring Ras and Rap1, as well as for each of the Raf isoforms, B-Raf and C-Raf.

  14. Cyclic AMP-elevating Agents Promote Cumulus Cell Survival and Hyaluronan Matrix Stability, Thereby Prolonging the Time of Mouse Oocyte Fertilizability.

    PubMed

    Di Giacomo, Monica; Camaioni, Antonella; Klinger, Francesca G; Bonfiglio, Rita; Salustri, Antonietta

    2016-02-19

    Cumulus cells sustain the development and fertilization of the mammalian oocyte. These cells are retained around the oocyte by a hyaluronan-rich extracellular matrix synthesized before ovulation, a process called cumulus cell-oocyte complex (COC) expansion. Hyaluronan release and dispersion of the cumulus cells progressively occur after ovulation, paralleling the decline of oocyte fertilization. We show here that, in mice, postovulatory changes of matrix are temporally correlated to cumulus cell death. Cumulus cell apoptosis and matrix disassembly also occurred in ovulated COCs cultured in vitro. COCs expanded in vitro with FSH or EGF underwent the same changes, whereas those expanded with 8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP) maintained integrity for a longer time. It is noteworthy that 8-Br-cAMP treatment was also effective on ovulated COCs cultured in vitro, prolonging the vitality of the cumulus cells and the stability of the matrix from a few hours to >2 days. Stimulation of endogenous adenylate cyclase with forskolin or inhibition of phosphodiesterase with rolipram produced similar effects. The treatment with selective cAMP analogues suggests that the effects of cAMP elevation are exerted through an EPAC-independent, PKA type II-dependent signaling pathway, probably acting at the post-transcriptional level. Finally, overnight culture of ovulated COCs with 8-Br-cAMP significantly counteracted the decrease of fertilization rate, doubling the number of fertilized oocytes compared with control conditions. In conclusion, these studies suggest that cAMP-elevating agents prevent cumulus cell senescence and allow them to continue to exert beneficial effects on oocyte and sperm, thereby extending in vitro the time frame of oocyte fertilizability.

  15. Cyclic AMP-elevating Agents Promote Cumulus Cell Survival and Hyaluronan Matrix Stability, Thereby Prolonging the Time of Mouse Oocyte Fertilizability*

    PubMed Central

    Di Giacomo, Monica; Camaioni, Antonella; Klinger, Francesca G.; Bonfiglio, Rita; Salustri, Antonietta

    2016-01-01

    Cumulus cells sustain the development and fertilization of the mammalian oocyte. These cells are retained around the oocyte by a hyaluronan-rich extracellular matrix synthesized before ovulation, a process called cumulus cell-oocyte complex (COC) expansion. Hyaluronan release and dispersion of the cumulus cells progressively occur after ovulation, paralleling the decline of oocyte fertilization. We show here that, in mice, postovulatory changes of matrix are temporally correlated to cumulus cell death. Cumulus cell apoptosis and matrix disassembly also occurred in ovulated COCs cultured in vitro. COCs expanded in vitro with FSH or EGF underwent the same changes, whereas those expanded with 8-bromo-adenosine-3′,5′-cyclic monophosphate (8-Br-cAMP) maintained integrity for a longer time. It is noteworthy that 8-Br-cAMP treatment was also effective on ovulated COCs cultured in vitro, prolonging the vitality of the cumulus cells and the stability of the matrix from a few hours to >2 days. Stimulation of endogenous adenylate cyclase with forskolin or inhibition of phosphodiesterase with rolipram produced similar effects. The treatment with selective cAMP analogues suggests that the effects of cAMP elevation are exerted through an EPAC-independent, PKA type II-dependent signaling pathway, probably acting at the post-transcriptional level. Finally, overnight culture of ovulated COCs with 8-Br-cAMP significantly counteracted the decrease of fertilization rate, doubling the number of fertilized oocytes compared with control conditions. In conclusion, these studies suggest that cAMP-elevating agents prevent cumulus cell senescence and allow them to continue to exert beneficial effects on oocyte and sperm, thereby extending in vitro the time frame of oocyte fertilizability. PMID:26694612

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-08-01

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

  18. Fluorescence detection of adenosine triphosphate in an aqueous solution using a combination of copper(II) complexes.

    PubMed

    Kataev, Evgeny; Arnold, René; Rüffer, Tobias; Lang, Heinrich

    2012-08-06

    Fluorescent ligands have been designed to form ternary complexes with a Cu(II) cation and phosphates in a buffer solution at physiological pH 7.4. It has been shown that a combination of two different ligands and CuCl(2) allows one to achieve high adenosine triphosphate/adenosine diphosphate, adenosine 5'-monophosphate selectivity, and ratiometric fluorescence sensing, while separately each ligand complex does not have such properties.

  19. Use of hypometabolic TRIS extenders and high cooling rate refrigeration for cryopreservation of stallion sperm: presence and sensitivity of 5' AMP-activated protein kinase (AMPK).

    PubMed

    Córdova, Alex; Strobel, Pablo; Vallejo, Andrés; Valenzuela, Pamela; Ulloa, Omar; Burgos, Rafael A; Menarim, Bruno; Rodríguez-Gil, Joan Enric; Ratto, Marcelo; Ramírez-Reveco, Alfredo

    2014-12-01

    This study evaluated the effect of the use of hypometabolic TRIS extenders in the presence or the absence of AMPK activators as well as the utilization of high cooling rates in the refrigeration step on the freezability of stallion sperm. Twelve ejaculates were cryopreserved using Botucrio® as a control extender and a basic TRIS extender (HM-0) separately supplemented with 10 mM metformin, 2mM 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), 2 mM Adenosine monophosphate (AMP), 40 μM compound C AMPK inhibitor or 2 mM AMP+40 μM compound C. Our results showed that the utilization of a hypometabolic TRIS extender supplemented or not with AMP or metformin significantly improves stallion sperm freezability when compared with a commercial extender. Additionally, high cooling rates do not affect stallion sperm quality after cooling and post-thawing. Finally, stallion spermatozoa present several putative AMPK sperm isoforms that do not seem to respond to classical activators, but do respond to the Compound C inhibitor.

  20. Induction of a Torpor-Like State by 5’-AMP Does Not Depend on H2S Production

    PubMed Central

    Dugbartey, George J.; Bouma, Hjalmar R.; Strijkstra, Arjen M.; Boerema, Ate S.; Henning, Robert H.

    2015-01-01

    Background Therapeutic hypothermia is used to reduce ischemia/reperfusion injury (IRI) during organ transplantation and major surgery, but does not fully prevent organ injury. Interestingly, hibernating animals undergo repetitive periods of low body temperature called ‘torpor’ without signs of organ injury. Recently, we identified an essential role of hydrogen sulfide (H2S) in entrance into torpor and preservation of kidney integrity during hibernation. A torpor-like state can be induced pharmacologically by injecting 5’-Adenosine monophosphate (5’-AMP). The mechanism by which 5’-AMP leads to the induction of a torpor-like state, and the role of H2S herein, remains to be unraveled. Therefore, we investigated whether induction of a torpor-like state by 5-AMP depends on H2S production. Methods To study the role of H2S on the induction of torpor, amino-oxyacetic acid (AOAA), a non-specific inhibitor of H2S, was administered before injection with 5'-AMP to block endogenous H2S production in Syrian hamster. To assess the role of H2S on maintenance of torpor induced by 5’-AMP, additional animals were injected with AOAA during torpor. Key Results During the torpor-like state induced by 5’-AMP, the expression of H2S- synthesizing enzymes in the kidneys and plasma levels of H2S were increased. Blockade of these enzymes inhibited the rise in the plasma level of H2S, but neither precluded torpor nor induced arousal. Remarkably, blockade of endogenous H2S production was associated with increased renal injury. Conclusions Induction of a torpor-like state by 5’-AMP does not depend on H2S, although production of H2S seems to attenuate renal injury. Unraveling the mechanisms by which 5’-AMP reduces the metabolism without organ injury may allow optimization of current strategies to limit (hypothermic) IRI and improve outcome following organ transplantation, major cardiac and brain surgery. PMID:26295351

  1. cAMP stimulates bicarbonate secretion across normal, but not cystic fibrosis airway epithelia.

    PubMed Central

    Smith, J J; Welsh, M J

    1992-01-01

    Adenosine 3',5'-cyclic monophosphate stimulates chloride (Cl-) secretion across airway epithelia. To determine whether cAMP also stimulates HCO3- secretion, we studied cultured canine and human airway epithelial cells bathed in a HCO3-/CO2-buffered, Cl(-)-free solution. Addition of forskolin stimulated an increase in short-circuit current that was likely a result of bicarbonate secretion because it was inhibited by a HCO3(-)-free solution, by addition of the carbonic anhydrase inhibitor, acetazolamide, or by mucosal addition of the anion channel blocker, diphenylamine 2-carboxylate. The current was dependent on Na+ because it was inhibited by removal of Na+ from the submucosal bathing solution, by addition of the Na+ pump inhibitor, ouabain, or by addition of amiloride (1 mM) to the submucosal solution. An increase in cytosolic Ca2+ produced by addition of a Ca2+ ionophore also stimulated short-circuit current. These data suggest that cAMP and Ca2+ stimulate HCO3- secretion across airway epithelium, and suggest that HCO3- leaves the cell across the apical membrane via conductive pathways. These results may explain previous observations that the short-circuit current across airway epithelia was not entirely accounted for by the sum of Na+ absorption and Cl- secretion. The cAMP-induced secretory response was absent in cystic fibrosis (CF) airway epithelial cells, although Ca(2+)-stimulated secretion was intact. This result suggests that HCO3- exist at the apical membrane is through the Cl- channel that is defectively regulated in CF epithelia. These results suggest the possibility that a defect in HCO3- secretion may contribute to the pathophysiology of CF pulmonary disease. PMID:1313448

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

    PubMed Central

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

    2007-01-01

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

  3. Phosphorylation of adenosine in renal brush-border membrane vesicles by an exchange reaction catalysed by adenosine kinase.

    PubMed Central

    Sayós, J; Solsona, C; Mallol, J; Lluis, C; Franco, R

    1994-01-01

    Uptake of [3H]adenosine in brush-border membrane (BBM) vesicles from either rat or pig kidney leads to an accumulation of intravesicular [3H]AMP. The lack of significant levels of ATP and the presence of AMP in BBM indicated that a phosphotransfer between [3H]adenosine and AMP occurs. The phosphotransfer activity is inhibited by iodotubercidin, which suggests that it is performed by adenosine kinase acting in an ATP-independent manner. The existence of a similar phosphotransferase activity was demonstrated in membrane-free extracts from pig kidney. From the compounds tested it was shown that a variety of mononucleotides could act as phosphate donors. The results suggest that phosphotransfer reactions may be physiologically relevant in kidney. PMID:8110185

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

    PubMed

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

    1989-08-01

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

  5. Effects of 5-hydroxytryptamine, dopamine, and acetylcholine on accumulation of cyclic AMP and cyclic GMP in the anterior byssus retractor muscle of Mytilus edulis L. (Mollusca).

    PubMed

    Köhler, G; Lindl, T

    1980-02-01

    We investigated in vitro accumulation of adenosine 3',5'-monophosphate (induced by 5-hydroxytryptamine and dopamine) and of guanosine 3',5'-monophosphate (induced by acetylcholine) in the anterior byssus retractor muscle of Mytilus. The response to 5-hydroxytryptamine exceeded that induced by equimolar concentrations of dopamine. 1-methyl lysergic acid, a 5-hydroxytryptamine-blocking agent, diminished the 5-hydroxytryptamine-induced increase of cyclic AMP level. This parallels the effect of this amine on the contracted muscle. Acetylcholine, which causes a tonic contraction of the muscle, increased intracellular levels of cyclic GMP in a dose-dependent (max. 45-fold at 10(-4) M ACh) manner. The time course of the rise in cyclic GMP level was rapid and transient (peak concentration of cyclic GMP at 2 min). Mytolon was the most effective of all cholinergic blockers tested. It was concluded that cyclic nucleotides may play a role in the modulatory process of the transmitters. A direct relation to the relaxation-contraction process could not be established.

  6. Role of adenosine receptors in caffeine tolerance

    SciTech Connect

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

    1991-01-01

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

  7. The Janus face of adenosine: antiarrhythmic and proarrhythmic actions.

    PubMed

    Szentmiklosi, A József; Galajda, Zoltán; Cseppento, Ágnes; Gesztelyi, Rudolf; Susán, Zsolt; Hegyi, Bence; Nánási, Péter P

    2015-01-01

    Adenosine is a ubiquitous, endogenous purine involved in a variety of physiological and pathophysiological regulatory mechanisms. Adenosine has been proposed as an endogenous antiarrhythmic substance to prevent hypoxia/ischemia-induced arrhythmias. Adenosine (and its precursor, ATP) has been used in the therapy of various cardiac arrhythmias over the past six decades. Its primary indication is treatment of paroxysmal supraventricular tachycardia, but it can be effective in other forms of supraventricular and ventricular arrhythmias, like sinus node reentry based tachycardia, triggered atrial tachycardia, atrioventricular nodal reentry tachycardia, or ventricular tachycardia based on a cAMP-mediated triggered activity. The main advantage is the rapid onset and the short half life (1- 10 sec). Adenosine exerts its antiarrhythmic actions by activation of A1 adenosine receptors located in the sinoatrial and atrioventricular nodes, as well as in activated ventricular myocardium. However, adenosine can also elicit A2A, A2B and A3 adenosine receptor-mediated global side reactions (flushing, dyspnea, chest discomfort), but it may display also proarrhythmic actions mediated by primarily A1 adenosine receptors (e.g. bradyarrhythmia or atrial fibrillation). To avoid the non-specific global adverse reactions, A1 adenosine receptor- selective full agonists (tecadenoson, selodenoson, trabodenoson) have been developed, which agents are currently under clinical trial. During long-term administration with orthosteric agonists, adenosine receptors can be internalized and desensitized. To avoid desensitization, proarrhythmic actions, or global adverse reactions, partial A1 adenosine receptor agonists, like CVT-2759, were developed. In addition, the pharmacologically "silent" site- and event specific adenosinergic drugs, such as adenosine regulating agents and allosteric modulators, might provide attractive opportunity to increase the effectiveness of beneficial actions of adenosine

  8. Pleiotropic effects of cAMP on germination, antibiotic biosynthesis and morphological development in Streptomyces coelicolor.

    PubMed

    Süsstrunk, U; Pidoux, J; Taubert, S; Ullmann, A; Thompson, C J

    1998-10-01

    In wild-type Streptomyces coelicolor MT1110 cultures, cyclic adenosine 3',5' monophosphate (cAMP) was synthesized throughout the developmental programme with peaks of accumulation both during germination and later when aerial mycelium and actinorhodin were being produced. Construction and characterization of an adenylate cyclase disruption mutant (BZ1) demonstrated that cAMP facilitated these developmental processes. Although pulse-labelling experiments showed that a similar germination process was initiated in BZ1 and MT1110, germ-tube emergence was severely delayed in BZ1 and never occurred in more than 85% of the spores. Studies of growth and development on solid glucose minimal medium (SMMS, buffered or unbuffered) showed that MT1110 and BZ1 produced acid during the first rapid growth phase, which generated substrate mycelium. Thereafter, on unbuffered SMMS, only MT1110 resumed growth and produced aerial mycelium by switching to an alternative metabolism that neutralized its medium, probably by reincorporating and metabolizing extracellular acids. BZ1 was not able to neutralize its medium or produce aerial mycelium on unbuffered SMMS; these defects were suppressed by high concentrations (>1 mM) of cAMP during early growth or on buffered medium. Other developmental mutants (bldA, bldB, bldC, bldD, bldG) also irreversibly acidified this medium. However, these bald mutants were not suppressed by exogenous cAMP or neutralizing buffer. BZ1 also differentiated when it was cultured in close proximity to MT1110, a property observed in cross-feeding experiments between bald mutants and commonly thought to reflect diffusion of a discrete positively acting signalling molecule. In this case, MT1110 generated a more neutral pH environment that allowed BZ1 to reinitiate growth and form aerial mycelium. The fact that actinorhodin synthesis could be induced by concentrations of cAMP (< 20 microM) found in the medium of MT1110 cultures, suggested that it may serve as a

  9. Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalysing the formation of adenosine 5' -phosphoramidate from adenosine 5' -phosphosulphate and ammonia.

    PubMed

    Fankhauser, H; Schiff, J A; Garber, L J

    1981-06-01

    Extracts of Chlorella pyrenoidosa, Euglena gracilis var. bacillaris, spinach, barley, Dictyostelium discoideum and Escherichia coli form an unknown compound enzymically from adenosine 5'-phosphosulphate in the presence of ammonia. This unknown compound shares the following properties with adenosine 5'-phosphoramidate: molar proportions of constituent parts (1 adenine:1 ribose:1 phosphate:1 ammonia released at low pH), co-electrophoresis in all buffers tested including borate, formation of AMP at low pH through release of ammonia, mass and i.r. spectra and conversion into 5'-AMP by phosphodiesterase. This unknown compound therefore appears to be identical with adenosine 5'-phosphoramidate. The enzyme that catalyses the formation of adenosine 5'-phosphoramidate from ammonia and adenosine 5'-phosphosulphate was purified 1800-fold (to homogeneity) from Chlorella by using (NH(4))(2)SO(4) precipitation and DEAE-cellulose, Sephadex and Reactive Blue 2-agarose chromatography. The purified enzyme shows one band of protein, coincident with activity, at a position corresponding to 60000-65000 molecular weight, on polyacrylamide-gel electrophoresis, and yields three subunits on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 26000, 21000 and 17000 molecular weight, consistent with a molecular weight of 64000 for the native enzyme. Isoelectrofocusing yields one band of pI4.2. The pH optimum of the enzyme-catalysed reaction is 8.8. ATP, ADP or adenosine 3'-phosphate 5'-phosphosulphate will not replace adenosine 5'-phosphosulphate, and the apparent K(m) for the last-mentioned compound is 0.82mm. The apparent K(m) for ammonia (assuming NH(3) to be the active species) is about 10mm. A large variety of primary, secondary and tertiary amines or amides will not replace ammonia. One mol.prop. of adenosine 5'-phosphosulphate reacts with 1 mol.prop. of ammonia to yield 1 mol.prop. each of adenosine 5'-phosphoramidate and sulphate; no AMP is found. The highly purified enzyme

  10. 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.

  11. Sustained antagonism of acute ethanol-induced ataxia following microinfusion of cyclic AMP and cpt-cAMP in the mouse cerebellum.

    PubMed

    Dar, M Saeed

    2011-05-01

    Ataxia is a conspicuous physical manifestation of alcohol consumption in humans and laboratory animals. Previously we reported possible involvement of cAMP in ethanol-induced ataxia. We now report a sustained antagonism of ataxia due to multiple ethanol injections following intracerebellar (ICB) cAMP or cpt-cAMP microinfusion. Adenylyl cyclase drugs cAMP, cpt-cAMP, Sp-cAMP, Rp-cAMP, adenosine A₁ agonist, N⁶-cyclohexyladenosine (CHA) and GABA(A) agonist muscimol were directly microinfused into the cerebellum of CD-1 male mice to evaluate their effect on ethanol (2 g/kg; i.p.) ataxia. Drug microinfusions were made via stereotaxically implanted stainless steel guide cannulas. Rotorod was used to evaluate the ethanol's ataxic response. Intracerebellar cAMP (0.1, 1, 10 fmol) or cpt-cAMP (0.5, 1, 2 fmol) 60 min before ethanol treatment, dose-dependently attenuated ethanol-induced ataxia in general agreement with previous observations. Intracerebellar microinfusion of cAMP (100 fmol) or cpt-cAMP (2 fmol) produced a sustained attenuation of ataxia following ethanol administration at 1, 4, 7 and 25 h or 31 h post-cAMP/cpt-cAMP microinfusion. At 31 h post-cAMP, the ataxic response of ethanol reappeared. Additionally, marked antagonism to the accentuation of ethanol-induced ataxia by adenosine A₁ and GABA(A) agonists, CHA (34 pmol) and muscimol (88 pmol), respectively, was noted 24h after cAMP and cpt-cAMP treatment. This indicated possible participation of AC/cAMP/PKA signaling in the co-modulation of ethanol-induced ataxia by A₁ adenosinergic and GABAergic systems. No change in normal motor coordination was noted when cAMP or cpt-cAMP microinfusion was followed by saline. Finally, Rp-cAMP (PKA inhibitor, 22 pmol) accentuated ethanol-induced ataxia and antagonized its attenuation by cAMP whereas Sp-cAMP (PKA activator, 22 pmol) produced just the opposite effects, further indicating participation of cAMP-dependent PKA downstream. Overall, the results support a role of

  12. cAMP generation inhibits inositol 1,4,5-trisphosphate binding in rabbit tracheal smooth muscle.

    PubMed

    Schramm, C M; Chuang, S T; Grunstein, M M

    1995-11-01

    Agonist-induced airway contraction involves the generation and subsequent binding of the phosphoinositide-derived second messenger, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], to its Ca(2+)-mobilizing intracellular receptor. To the extent that regulatory cross-talk is known to exist between different signal transduction pathways, the present study examined whether activation of the adenosine 3',5'-cyclic monophosphate (cAMP)/protein kinase A (PKA) pathway induces altered binding of Ins(1,4,5)P3 to its receptor in membrane homogenates of rabbit tracheal smooth muscle (TSM). In control TSM, monophasic binding curves provided mean +/- SE values for Ins(1,4,5)P3 receptor density (Bmax) and binding affinity (Kd) amounting to 940 +/- 43 fmol/mg protein and 10.7 +/- 1.2 nM, respectively. Relative to control, binding of [3H]Ins(1,4,5)P3 was significantly reduced in paired TSM separately treated with isoproterenol, forskolin, or dibutyryl-cAMP. Ins(1,4,5)P3 binding was inhibited to a level averaging 60% of control binding by maximal concentrations of each agonist, an effect attributed to a reduction in Ins(1,4,5)P3 binding sites rather than altered ligand affinity. Collectively, these findings demonstrate that activation of the cAMP-dependent signaling pathway is associated with inhibition of Ins(1,4,5)P3 receptor binding and implicate a novel mechanism of action of beta-adrenergic agents in preventing and/or reversing airway contraction.

  13. Helicobacter pylori Induces miR-155 in T Cells in a cAMP-Foxp3-Dependent Manner

    PubMed Central

    Fassi Fehri, Lina; Koch, Manuel; Belogolova, Elena; Khalil, Hany; Bolz, Christian; Kalali, Behnam; Mollenkopf, Hans J.; Beigier-Bompadre, Macarena; Karlas, Alexander; Schneider, Thomas; Churin, Yuri; Gerhard, Markus; Meyer, Thomas F.

    2010-01-01

    Amongst the most severe clinical outcomes of life-long infections with Helicobacter pylori is the development of peptic ulcers and gastric adenocarcinoma - diseases often associated with an increase of regulatory T cells. Understanding H. pylori-driven regulation of T cells is therefore of crucial clinical importance. Several studies have defined mammalian microRNAs as key regulators of the immune system and of carcinogenic processes. Hence, we aimed here to identify H. pylori-regulated miRNAs, mainly in human T cells. MicroRNA profiling of non-infected and infected human T cells revealed H. pylori infection triggers miR-155 expression in vitro and in vivo. By using single and double H. pylori mutants and the corresponding purified enzymes, the bacterial vacuolating toxin A (VacA) and γ-glutamyl transpeptidase (GGT) plus lipopolysaccharide (LPS) tested positive for their ability to regulate miR-155 and Foxp3 expression in human lymphocytes; the latter being considered as the master regulator and marker of regulatory T cells. RNAi-mediated knockdown (KD) of the Foxp3 transcription factor in T cells abolished miR-155 expression. Using adenylate cyclase inhibitors, the miR-155 induction cascade was shown to be dependent on the second messenger cyclic adenosine monophosphate (cAMP). Furthermore, we found that miR-155 directly targets the protein kinase A inhibitor α (PKIα) mRNA in its 3′UTR, indicative of a positive feedback mechanism on the cAMP pathway. Taken together, our study describes, in the context of an H. pylori infection, a direct link between Foxp3 and miR-155 in human T cells and highlights the significance of cAMP in this miR-155 induction cascade. PMID:20209161

  14. Extracellular adenosine triphosphate and adenosine in cancer.

    PubMed

    Stagg, J; Smyth, M J

    2010-09-30

    Adenosine triphosphate (ATP) is actively released in the extracellular environment in response to tissue damage and cellular stress. Through the activation of P2X and P2Y receptors, extracellular ATP enhances tissue repair, promotes the recruitment of immune phagocytes and dendritic cells, and acts as a co-activator of NLR family, pyrin domain-containing 3 (NLRP3) inflammasomes. The conversion of extracellular ATP to adenosine, in contrast, essentially through the enzymatic activity of the ecto-nucleotidases CD39 and CD73, acts as a negative-feedback mechanism to prevent excessive immune responses. Here we review the effects of extracellular ATP and adenosine on tumorigenesis. First, we summarize the functions of extracellular ATP and adenosine in the context of tumor immunity. Second, we present an overview of the immunosuppressive and pro-angiogenic effects of extracellular adenosine. Third, we present experimental evidence that extracellular ATP and adenosine receptors are expressed by tumor cells and enhance tumor growth. Finally, we discuss recent studies, including our own work, which suggest that therapeutic approaches that promote ATP-mediated activation of inflammasomes, or inhibit the accumulation of tumor-derived extracellular adenosine, may constitute effective new means to induce anticancer activity.

  15. From totipotent embryonic stem cells to spontaneously contracting smooth muscle cells: a retinoic acid and db-cAMP in vitro differentiation model.

    PubMed

    Drab, M; Haller, H; Bychkov, R; Erdmann, B; Lindschau, C; Haase, H; Morano, I; Luft, F C; Wobus, A M

    1997-09-01

    Vascular smooth muscle cell (VSMC) differentiation is important in understanding vascular disease; however, no in vitro model is available. Totipotent mouse embryonic stem (ES) cells were used to establish such a model. To test whether the ES cell-derived smooth muscle cells expressed VSMC-specific properties, the differentiated cells were characterized by 1) morphological analysis, 2) gene expression, 3) immunostaining for VSMC-specific proteins, 4) expression of characteristic VSMC ion channels, and 5) formation of [Ca2+]i transients in response to VSMC-specific agonists. Treatment of embryonic stem cell-derived embryoid bodies with retinoic acid and dibutyryl-cyclic adenosine monophosphate (db-cAMP) induced differentiation of spontaneously contracting cell clusters in 67% of embryoid bodies compared with 10% of untreated controls. The highest differentiation rate was observed when retinoic acid and db-cAMP were applied to the embryoid bodies between days 7 and 11 in combination with frequent changes of culture medium. Other protocols with retinoic acid and db-cAMP, as well as single or combined treatment with VEGF, ECGF, bFGF, aFGF, fibronectin, matrigel, or hypoxia did not influence the differentiation rate. Single-cell RT-PCR and sequencing of the PCR products identified myosin heavy chain (MHC) splice variants distinguishing between gut and VSMC isoforms. RT-PCR with VSMC-specific MHC primers and immunostaining confirmed the presence of VSMC transcripts and MHC protein. Furthermore, VSMC expressing MHC had typical ion channels and responded to specific agonists with an increased [Ca2+]i. Here we present a retinoic acid + db-cAMP-inducible embryonic stem cell model of in vitro vasculogenesis. ES cell-derived cells expressing VSMC-specific MHC and functional VSMC properties may be a suitable system to study mechanisms of VSMC differentiation.

  16. G-protein coupled receptor 6 deficiency alters striatal dopamine and cAMP concentrations and reduces dyskinesia in a mouse model of Parkinson's disease.

    PubMed

    Oeckl, Patrick; Hengerer, Bastian; Ferger, Boris

    2014-07-01

    The orphan G-protein coupled receptor 6 (GPR6) is a constitutively active receptor which is positively coupled to the formation of cyclic adenosine-3',5'-monophosphate (cAMP). GPR6 is predominantly expressed in striatopallidal neurons. Here, we investigated neurochemical and behavioural effects of Gpr6 deficiency in mice. Gpr6 depletion decreased in vivo cAMP tissue concentrations (20%) in the striatum. An increase of striatal tissue dopamine concentrations (10%) was found in Gpr6(-/-) mice, whereas basal extracellular dopamine levels were not changed compared with Gpr6(+/+) mice, as shown by in vivo microdialysis. Western blot analyses revealed no alteration in the expression and subcellular localisation of the dopamine D2 receptor in the striatum of Gpr6(-/-) mice, and the number of tyrosine hydroxylase positive neurons in the substantia nigra was unchanged. DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32kDa) expression in the striatum of Gpr6(-/-) mice was not altered, however, a twofold increase in the phosphorylation of DARPP-32 at Thr34 was detected in Gpr6(-/-) compared with Gpr6(+/+) mice. Gpr6(-/-) mice showed higher locomotor activity in the open field, which persisted after treatment with the dopamine D2 receptor antagonist haloperidol. They also displayed reduced abnormal involuntary movements after apomorphine and quinpirole treatment in the mouse dyskinesia model of Parkinson's disease. In conclusion, the depletion of Gpr6 reduces cAMP concentrations in the striatum and alters the striatal dopaminergic system. Gpr6 deficiency causes an interesting behavioural phenotype in the form of enhanced motor activity combined with reduced abnormal involuntary movements. These findings could offer an opportunity for the treatment of Parkinson's disease beyond dopamine replacement.

  17. cAMP Signaling in Brain is Decreased in Unmedicated Depressed Patients and Increased by Treatment with a Selective Serotonin Reuptake Inhibitor

    PubMed Central

    Fujita, Masahiro; Richards, Erica M.; Niciu, Mark J.; Ionescu, Dawn F.; Zoghbi, Sami S.; Hong, Jinsoo; Telu, Sanjay; Hines, Christina S.; Pike, Victor W.; Zarate, Carlos A.; Innis, Robert B.

    2016-01-01

    Basic studies exploring the importance of the cyclic adenosine monophosphate (cAMP) cascade in major depressive disorder (MDD) have noted that the cAMP cascade is downregulated in MDD and upregulated by antidepressant treatment. We investigated cAMP cascade activity by using 11C-(R)-rolipram to image phosphodiesterase-4 (PDE4) in unmedicated MDD patients and after approximately eight weeks of treatment with a selective serotonin reuptake inhibitor (SSRI). 11C-(R)-rolipram PET scans were performed in 44 unmedicated patients during a major depressive episode and 35 healthy controls. Twenty-three of the 44 patients had a follow-up 11C-(R)-rolipram PET scan approximately eight weeks after treatment with an SSRI. Patients were moderately depressed (Montgomery-Åsberg Depression Rating Scale=30±6) and about half were treatment-naïve. 11C-(R)-Rolipram binding was measured using arterial sampling to correct for individual differences in radioligand metabolism. We found in unmedicated MDD patients widespread, ~20% reductions in 11C-(R)-rolipram binding compared to controls (P=0.001). SSRI treatment significantly increased rolipram binding (12%, P<0.001) with significantly greater increases observed in older patients (P<0.001). Rolipram binding did not correlate with severity of baseline symptoms, and increased rolipram binding during treatment did not correlate with symptom improvement. In brief, consistent with the results of basic studies, PDE4 was decreased in unmedicated MDD patients and increased after SSRI treatment. The lack of correlation between PDE4 binding and depressive symptoms could reflect the heterogeneity of the disease and/or the heterogeneity of the target, given that PDE4 has four subtypes. These results suggest that PDE4 inhibitors, which increase cAMP cascade activity, may have antidepressant effects. PMID:27725657

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

    PubMed Central

    Flitney, F W; Singh, J

    1980-01-01

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

  19. Lack of the presynaptic RhoGAP protein oligophrenin1 leads to cognitive disabilities through dysregulation of the cAMP/PKA signalling pathway

    PubMed Central

    Khelfaoui, Malik; Gambino, Frédéric; Houbaert, Xander; Ragazzon, Bruno; Müller, Christian; Carta, Mario; Lanore, Frédéric; Srikumar, Bettadapura N.; Gastrein, Philippe; Lepleux, Marilyn; Zhang, Chun-Lei; Kneib, Marie; Poulain, Bernard; Reibel-Foisset, Sophie; Vitale, Nicolas; Chelly, Jamel; Billuart, Pierre; Lüthi, Andreas; Humeau, Yann

    2014-01-01

    Loss-of-function mutations in the gene encoding for the RhoGAP protein of oligophrenin-1 (OPHN1) lead to cognitive disabilities (CDs) in humans, yet the underlying mechanisms are not known. Here, we show that in mice constitutive lack of Ophn1 is associated with dysregulation of the cyclic adenosine monophosphate/phosphate kinase A (cAMP/PKA) signalling pathway in a brain-area-specific manner. Consistent with a key role of cAMP/PKA signalling in regulating presynaptic function and plasticity, we found that PKA-dependent presynaptic plasticity was completely abolished in affected brain regions, including hippocampus and amygdala. At the behavioural level, lack of OPHN1 resulted in hippocampus- and amygdala-related learning disabilities which could be fully rescued by the ROCK/PKA kinase inhibitor fasudil. Together, our data identify OPHN1 as a key regulator of presynaptic function and suggest that, in addition to reported postsynaptic deficits, loss of presynaptic plasticity contributes to the pathophysiology of CDs. PMID:24298161

  20. The gene transcription factor cyclic AMP-responsive element binding protein: role in positive and negative affective states of alcohol addiction.

    PubMed

    Pandey, Subhash C

    2004-10-01

    The gene transcription factor cyclic adenosine monophosphate (cAMP)-responsive element binding (CREB) protein is a nuclear protein that regulates synaptic plasticity via modulating the expression of several (cAMP)-inducible genes. Alcohol addiction is a complex psychiatric disorder and is characterized by a compulsive and uncontrolled pattern of alcohol drinking by an individual in spite of the adverse consequences of its abuse. Ethanol produces both euphoric (reward and reinforcing) and dysphoric (negative withdrawal reactions) effects and these are most likely involved in the initiation and maintenance of alcohol use and abuse. Several neurotransmitter systems in the brain might be involved in the effects of alcohol but the exact molecular mechanisms of both the positive and negative affective states of alcohol abuse are still unclear. Recent research in molecular neurosciences using animal models have identified the role of extended amygdaloid (shell structures of nucleus accumbens [NAc] and central and medial amygdaloid nuclei) CREB signaling in positive and negative affective states of alcohol drinking behaviors. This review article highlights the current findings on the role of nucleus accumbal and amygdaloid CREB signaling in behavioral consequences of alcohol use and abuse.

  1. Efficient and robust differentiation of endothelial cells from human induced pluripotent stem cells via lineage control with VEGF and cyclic AMP

    PubMed Central

    Ikuno, Takeshi; Masumoto, Hidetoshi; Yamamizu, Kohei; Yoshioka, Miki; Minakata, Kenji; Ikeda, Tadashi; Sakata, Ryuzo; Yamashita, Jun K.

    2017-01-01

    Blood vessels are essential components for many tissues and organs. Thus, efficient induction of endothelial cells (ECs) from human pluripotent stem cells is a key method for generating higher tissue structures entirely from stem cells. We previously established an EC differentiation system with mouse pluripotent stem cells to show that vascular endothelial growth factor (VEGF) is essential to induce ECs and that cyclic adenosine monophosphate (cAMP) synergistically enhances VEGF effects. Here we report an efficient and robust EC differentiation method from human pluripotent stem cell lines based on a 2D monolayer, serum-free culture. We controlled the direction of differentiation from mesoderm to ECs using stage-specific stimulation with VEGF and cAMP combined with the elimination of non-responder cells at early EC stage. This “stimulation-elimination” method robustly achieved very high efficiency (>99%) and yield (>10 ECs from 1 hiPSC input) of EC differentiation, with no purification of ECs after differentiation. We believe this method will be a valuable technological basis broadly for regenerative medicine and 3D tissue engineering. PMID:28288160

  2. Adenosine signaling promotes hematopoietic stem and progenitor cell emergence.

    PubMed

    Jing, Lili; Tamplin, Owen J; Chen, Michael J; Deng, Qing; Patterson, Shenia; Kim, Peter G; Durand, Ellen M; McNeil, Ashley; Green, Julie M; Matsuura, Shinobu; Ablain, Julien; Brandt, Margot K; Schlaeger, Thorsten M; Huttenlocher, Anna; Daley, George Q; Ravid, Katya; Zon, Leonard I

    2015-05-04

    Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1(+)/cmyb(+) HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl(+) hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP-protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates.

  3. A possible mechanism for improvement by a cognition-enhancer nefiracetam of spatial memory function and cAMP-mediated signal transduction system in sustained cerebral ischaemia in rats.

    PubMed

    Takeo, Satoshi; Niimura, Makiko; Miyake-Takagi, Keiko; Nagakura, Akira; Fukatsu, Tomoko; Ando, Tsuyoshi; Takagi, Norio; Tanonaka, Kouichi; Hara, Junko

    2003-02-01

    1. Accumulated evidence indicates that the adenylyl cyclase (AC)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP-responsive element binding protein (CREB) signal transduction system may be linked to learning and memory function. 2. The effects of nefiracetam, which has been developed as a cognition enhancer, on spatial memory function and the AC/cAMP/PKA/CREB signal transduction system in rats with sustained cerebral ischaemia were examined. 3. Microsphere embolism (ME)-induced sustained cerebral ischaemia was produced by injection of 700 microspheres (48 micro m in diameter) into the right hemisphere of rats. Daily oral administration of nefiracetam (10 mg kg(-1) day(-1)) was started from 15 h after the operation. 4. The delayed treatment with nefiracetam attenuated the ME-induced prolongation of the escape latency in the water maze task that was examined on day 7 to 9 after ME, but it did not reduce the infarct size. 5. ME decreased Ca(2+)/calmodulin (CaM)-stimulated AC (AC-I) activity, cAMP content, cytosolic PKA Cbeta level, nuclear PKA Calpha and Cbeta levels, and reduced the phosphorylation and DNA-binding activity of CREB in the nucleus in the right parietal cortex and hippocampus on day 3 after ME. The ME-induced changes in these variables did not occur by the delayed treatment with nefiracetam. 6. These results suggest that nefiracetam preserved cognitive function, or prevented cognitive dysfunction, after sustained cerebral ischaemia and that the effect is, in part, attributable to the prevention of the ischaemia-induced impairment of the AC/cAMP/PKA/CREB signal transduction pathway.

  4. Role of yessotoxin in calcium and cAMP-crosstalks in primary and K-562 human lymphocytes: the effect is mediated by anchor kinase A mitochondrial proteins.

    PubMed

    Tobío, Araceli; Fernández-Araujo, Andrea; Alfonso, Amparo; Botana, Luis M

    2012-12-01

    Yessotoxin (YTX) is a marine polyether toxin previously described as a phosphodiesterase (PDE) activator in fresh human lymphocytes. This toxin induces a decrease of adenosine 3',5'-cyclic monophosphate (cAMP) levels in fresh human lymphocytes in a medium with calcium (Ca(2+) ), whereas the contrary effect has been observed in a Ca(2+) -free medium. In the present article, the effect of YTX in K-562 lymphocytes cell line has been analysed. Surprisingly, results obtained in K-562 cell line are completely opposite than in fresh human lymphocytes, since in K-562 cells YTX induces an increase of cAMP levels. YTX cytotoxicity was also studied in both K-562 cell line and fresh human lymphocytes. Results demonstrate that YTX does not modify fresh human lymphocytes viability, whereas in K-562 cells, YTX has a highly cytotoxic effect. It has been described in a previous study that YTX induces a small cytosolic Ca(2+) increase in fresh human lymphocytes but no effect was observed on Ca(2+) pools depletion in these cells. However, our results show that, in K-562 cells, YTX has no effect on cytosolic Ca(2+) levels in a medium with Ca(2+) and induces an increase on Ca(2+) pools depletion followed by a Ca(2+) influx. As far as Ca(2+) modulation is concerned these results demonstrate that YTX has a clear opposite effect in tumoural and fresh human lymphocytes. In addition, intracellular Ca(2+) reservoirs affected by YTX are different than thapsigargin-sensible pools. Furthermore, YTX-dependent Ca(2+) pools depletion was abolished by cAMP analogue (dibutyryl cAMP), phosphodiesterase-4 (PDE4) inhibitor (rolipram), protein kinase A inhibitor (H89) and oxidative phosphorylation uncoupler carbonyl cyanide p-(trifluoromethoxy) (FCCP) treatments. This evidences the crosstalks between Ca(2+) , YTX and cAMP pathways. Also, results obtain demonstrate that YTX-dependent Ca(2+) influx was only abolished by FCCP pre-treatment, which indicates a link between YTX and mitochondria in K-562 cell

  5. Mitotic activation of the DISC1-inducible cyclic AMP phosphodiesterase-4D9 (PDE4D9), through multi-site phosphorylation, influences cell cycle progression.

    PubMed

    Sheppard, Catherine L; Lee, Louisa C Y; Hill, Elaine V; Henderson, David J P; Anthony, Diana F; Houslay, Daniel M; Yalla, Krishna C; Cairns, Lynne S; Dunlop, Allan J; Baillie, George S; Huston, Elaine; Houslay, Miles D

    2014-09-01

    In Rat-1 cells, the dramatic decrease in the levels of both intracellular cyclic 3'5' adenosine monophosphate (cyclic AMP; cAMP) and in the activity of cAMP-activated protein kinase A (PKA) observed in mitosis was paralleled by a profound increase in cAMP hydrolyzing phosphodiesterase-4 (PDE4) activity. The decrease in PKA activity, which occurs during mitosis, was attributable to PDE4 activation as the PDE4 selective inhibitor, rolipram, but not the phosphodiesterase-3 (PDE3) inhibitor, cilostamide, specifically ablated this cell cycle-dependent effect. PDE4 inhibition caused Rat-1 cells to move from S phase into G2/M more rapidly, to transit through G2/M more quickly and to remain in G1 for a longer period. Inhibition of PDE3 elicited no observable effects on cell cycle dynamics. Selective immunopurification of each of the four PDE4 sub-families identified PDE4D as being selectively activated in mitosis. Subsequent analysis uncovered PDE4D9, an isoform whose expression can be regulated by Disrupted-In-Schizophrenia 1 (DISC1)/activating transcription factor 4 (ATF4) complex, as the sole PDE4 species activated during mitosis in Rat-1 cells. PDE4D9 becomes activated in mitosis through dual phosphorylation at Ser585 and Ser245, involving the combined action of ERK and an unidentified 'switch' kinase that has previously been shown to be activated by H2O2. Additionally, in mitosis, PDE4D9 also becomes phosphorylated at Ser67 and Ser81, through the action of MK2 (MAPKAPK2) and AMP kinase (AMPK), respectively. The multisite phosphorylation of PDE4D9 by all four of these protein kinases leads to decreased mobility (band-shift) of PDE4D9 on SDS-PAGE. PDE4D9 is predominantly concentrated in the perinuclear region of Rat-1 cells but with a fraction distributed asymmetrically at the cell margins. Our investigations demonstrate that the diminished levels of cAMP and PKA activity that characterise mitosis are due to enhanced cAMP degradation by PDE4D9. PDE4D9, was found to

  6. The cAMP analogs have potent anti-proliferative effects on medullary thyroid cancer cell lines.

    PubMed

    Dicitore, Alessandra; Grassi, Elisa Stellaria; Caraglia, Michele; Borghi, Maria Orietta; Gaudenzi, Germano; Hofland, Leo J; Persani, Luca; Vitale, Giovanni

    2016-01-01

    The oncogenic activation of the rearranged during transfection (RET) proto-oncogene has a main role in the pathogenesis of medullary thyroid cancer (MTC). Several lines of evidence suggest that RET function could be influenced by cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity. We evaluated the in vitro anti-tumor activity of 8-chloroadenosine-3',5'-cyclic monophosphate (8-Cl-cAMP) and PKA type I-selective cAMP analogs [equimolar combination of the 8-piperidinoadenosine-3',5'-cyclic monophosphate (8-PIP-cAMP) and 8-hexylaminoadenosine-3',5'-cyclic monophosphate (8-HA-cAMP) in MTC cell lines (TT and MZ-CRC-1)]. 8-Cl-cAMP and the PKA I-selective cAMP analogs showed a potent anti-proliferative effect in both cell lines. In detail, 8-Cl-cAMP blocked significantly the transition of TT cell population from G2/M to G0/G1 phase and from G0/G1 to S phase and of MZ-CRC-1 cells from G0/G1 to S phase. Moreover, 8-Cl-cAMP induced apoptosis in both cell lines, as demonstrated by FACS analysis for annexin V-FITC/propidium iodide, the activation of caspase-3 and PARP cleavage. On the other hand, the only effect induced by PKA I-selective cAMP analogs was a delay in G0/G1-S and S-G2/M progression in TT and MZ-CRC-1 cells, respectively. In conclusion, these data demonstrate that cAMP analogs, particularly 8-Cl-cAMP, significantly suppress in vitro MTC proliferation and provide rationale for a potential clinical use of cAMP analogs in the treatment of advanced MTC.

  7. A High-Affinity Adenosine Kinase from Anopheles Gambiae

    SciTech Connect

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

    2011-12-31

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

  8. Temporal variations of adenosine metabolism in human blood.

    PubMed

    Chagoya de Sánchez, V; Hernández-Muñoz, R; Suárez, J; Vidrio, S; Yáñez, L; Aguilar-Roblero, R; Oksenberg, A; Vega-González, A; Villalobos, L; Rosenthal, L; Fernández-Cancino, F; Drucker-Colín, R; Díaz-Muñoz, M

    1996-08-01

    Eight diurnally active (06:00-23:00 h) subjects were adapted for 2 days to the room conditions where the experiments were performed. Blood sampling for adenosine metabolites and metabolizing enzymes was done hourly during the activity span and every 30 min during sleep. The results showed that adenosine and its catabolites (inosine, hypoxanthine, and uric acid), adenosine synthesizing (S-adenosylhomocysteine hydrolase and 5'-nucleotidase), degrading (adenosine deaminase) and nucleotide-forming (adenosine kinase) enzymes as well as adenine nucleotides (AMP, ADP, and ATP) undergo statistically significant fluctuations (ANOVA) during the 24 h. However, energy charge was invariable. Glucose and lactate chronograms were determined as metabolic indicators. The same data analyzed by the chi-square periodogram and Fourier series indicated ultradian oscillatory periods for all the metabolites and enzymatic activities determined, and 24-h oscillatory components for inosine, hypoxanthine, adenine nucleotides, glucose, and the activities of SAH-hydrolase, 5'-nucleotidase, and adenosine kinase. The single cosinor method showed significant oscillatory components exclusively for lactate. As a whole, these results suggest that adenosine metabolism may play a role as a biological oscillator coordinating and/or modulating the energy homeostasis and physiological status of erythrocytes in vivo and could be an important factor in the distribution of purine rings for the rest of the organism.

  9. Anticancer activity of 7,8-dihydroxyflavone in melanoma cells via downregulation of α-MSH/cAMP/MITF pathway.

    PubMed

    Sim, Deok Yong; Sohng, Jae Kyung; Jung, Hye Jin

    2016-07-01

    Malignant melanoma is one of the most aggressive skin cancer and highly resistant to most conventional treatment. In the present study, we aimed to investigate the anticancer effects and mechanisms of action of 7,8-dihydroxyflavone (7,8-DHF), a monophenolic flavone, in melanoma cells. At concentrations not exhibiting cytotoxicity, 7,8-DHF potently inhibited growth and clonogenic survival of alpha-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 melanoma cells. Furthermore, it significantly blocked migration and invasion of the metastatic melanoma cells. We also observed that 7,8-DHF exhibits anti-melanogenic activity through inhibition of tyrosinase activity in α-MSH-stimulating condition. Notably, the suppressive activities of 7,8-DHF on melanoma progression were associated with the downregulation of microphthalmia-associated transcription factor (MITF) and its main downstream transcription targets, including hypoxia-inducible factor 1α (HIF1α) and c-MET, by a decrease in cyclic adenosine monophosphate (cAMP) level. In addition, combination treatment with 7,8-DHF and resveratrol, a known therapeutic agent against melanoma, had greater anticancer activities and MITF inhibition than treatment with each single agent in α-MSH-treated B16F10 cells. Collectively, these findings may contribute to the potential application of 7,8-DHF in the prevention and treatment of malignant melanoma.

  10. Spatial variation in sediment-water exchange of phosphorus in Florida Bay: AMP as a model organic compound.

    PubMed

    Huang, Xiao-Lan; Zhang, Jia-Zhong

    2010-10-15

    Dissolved organic phosphorus (DOP) has been recognized as dominant components in total dissolved phosphorus (TDP) pools in many coastal waters, and its exchange between sediment and water is an important process in biogeochemical cycle of phosphorus. Adenosine monophosphate (AMP) was employed as a model DOP compound to simulate phosphorus exchange across sediment-water interface in Florida Bay. The sorption data from 40 stations were fitted to a modified Freundlich equation and provided a detailed spatial distribution both of the sediment's zero equilibrium phosphorus concentration (EPC(0-T)) and of the distribution coefficient (K(d-T)) with respect to TDP. The K(d-T) was found to be a function of the index of phosphorus saturation (IPS), a molar ratio of the surface reactive phosphorus to the surface reactive iron oxide content in the sediment, across the entire bay. However, the EPC(0-T) was found to correlate to the contents of phosphorus in the eastern bay only. Sediment in the western bay might act as a source of the phosphorus in the exchange process due to their high EPC(0-T) and low K(d-T), whereas sediments in the eastern bay might act as a sink because of their low EPC(0-T) and high K(d-T). These results strongly support the hypothesis that both phosphorus and iron species in calcareous marine sediments play a critical role in governing the sediment-water exchange of both phosphate and DOP in the coastal and estuarine ecosystems.

  11. Ameliorative effect of adenosine on hypoxia-reoxygenation injury in LLC-PK1, a porcine kidney cell line.

    PubMed

    Yonehana, T; Gemba, M

    1999-06-01

    We studied the effects of adenosine on injury caused by hypoxia and reoxygenation in LLC-PK1 cells. Lactate dehydrogenase and gamma-glutamyltranspeptidase were released from cells exposed to hypoxia for 6 hr and then reoxygenation for 1 hr. The addition of adenosine at 100 microM to the medium before hypoxia began significantly decreased enzyme leakage into medium during both hypoxia and reoxygenation. The adenosine A1-receptor agonist, R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA), at the concentration of 100 microM, did not affect enzyme release, but the adenosine A2-receptor agonist 2-p-[2-car-boxyethyl]phenethyl-amino-5'-N-ethylcarboxamido-adenosi ne hydrochloride (CGS 21680) at the concentration of 100 nM, suppressed the injury caused by hypoxia and reoxygenation. There were decreases in cAMP contents and ATP levels in LLC-PK1 cells injured by hypoxia and reoxygenation. Adenosine (100 microM) restored ATP levels in the cells during reoxygenation. With adenosine, the intracellular cAMP level was increased prominently during reoxygenation. These results suggest that adenosine protects LLC-PK1 cells from injury caused by hypoxia and reoxygenation by increasing the intracellular cAMP level via adenosine A2 receptor.

  12. Temporal changes in the calcium-dependence of the histamine H1-receptor-stimulation of cyclic AMP accumulation in guinea-pig cerebral cortex.

    PubMed Central

    Donaldson, J.; Brown, A. M.; Hill, S. J.

    1989-01-01

    1. 2-Chloroadenosine (2CA) causes a maintained rise in adenosine 3':5'-cyclic monophosphate (cyclic AMP) content of guinea-pig cerebral cortical slices which is augmented by addition of histamine. We have investigated the temporal profile of the sensitivity of this response to calcium. 2. Rapid removal of extracellular calcium with EGTA (5 mM) at 2CA (30 microM)-induced steady state caused a slight increase in the cyclic AMP response to 2CA alone and completely abolished the augmentation produced by histamine (0.1 mM) added 20 min later. When EGTA was added only 2 min before histamine, the augmentation was reduced by 72%. 3. The calcium sensitivity of the histamine response was also indicated in studies in which EGTA was added 1 or 3 min after histamine at 2CA-induced steady state. Following addition of EGTA at either of these times, the augmentation was not maintained. 4. When calcium was rapidly removed with EGTA once a steady state level of cyclic AMP had been achieved with histamine, the augmentation response was maintained. This was despite the fact that EGTA had a similar effect on both extracellular free calcium and tissue calcium content when it was applied before or after histamine. 5. The 2CA response was augmented by phorbol esters (which mimic the actions of diacylglycerol) in a calcium-independent manner. 6. These results suggest that calcium is important for the initiation and early stages of the histamine-induced augmentation response. The apparent lack of calcium sensitivity of the response at later stages could mean that calcium is not involved in the maintenance of the response or that the intracellular machinery involved in the augmentation process becomes more sensitive to calcium as the response progresses, such that it becomes able to operate at a much lower level of intracellular calcium. A possible role for diacylglycerol in the maintenance of the response is discussed. PMID:2558762

  13. Glucagon and cAMP inhibit cholesterol 7alpha-hydroxylase (CYP7A1) gene expression in human hepatocytes: discordant regulation of bile acid synthesis and gluconeogenesis.

    PubMed

    Song, Kwang-Hoon; Chiang, John Y L

    2006-01-01

    The gene encoding cholesterol 7alpha-hydroxylase (CYP7A1) is tightly regulated to control bile acid synthesis and maintain lipid homeostasis. Recent studies in mice suggest that bile acid synthesis is regulated by the fasted-to-fed cycle, and fasting induces CYP7A1 gene expression in parallel to the induction of peroxisome proliferators-activated receptor gamma co-activator 1alpha (PGC-1alpha) and phosphoenolpyruvate carboxykinase (PEPCK). How glucagon regulates CYP7A1 gene expression in the human liver is not clear. Here we show that glucagon and cyclic adenosine monophosphate (cAMP) strongly repressed CYP7A1 mRNA expression in human primary hepatocytes. Reporter assays confirmed that cAMP and protein kinase A (PKA) inhibited human CYP7A1 gene transcription, in contrast to their stimulation of the PEPCK gene. Mutagenesis analysis identified a PKA-responsive region located within the previously identified HNF4alpha binding site in the human CYP7A1 promoter. Glucagon and cAMP increased HNF4alpha phosphorylation and reduced the amount of HNF4alpha present in CYP7A1 chromatin. Our findings suggest that glucagon inhibited CYP7A1 gene expression via PKA phosphorylation of HNF4alpha, which lost its ability to bind the CYP7A1 gene and resulted in inhibition of human CYP7A1 gene transcription. In conclusion, this study unveils a species difference in nutrient regulation of the human and mouse CYP7A1 gene and suggests a discordant regulation of bile acid synthesis and gluconeogenesis by glucagon in human livers during fasting.

  14. Vasopressin induces dopamine release and cyclic AMP efflux from the brain of water-deprived rats: inhibitory effect of vasopressin V2 receptor-mediated phosphorylation.

    PubMed

    Tyagi, M G; Handa, R K; Stephen, P M; Bapna, J S

    1998-01-01

    The neurohypophyseal hormone vasopressin (AVP) is widely distributed throughout the central nervous system. It acts as an excitatory transmitter in the CNS and plays an important physiological role in water and electrolyte homeostasis. However, water deprivation has been shown to induce changes in the levels of monoamines, but there is little knowledge about the influence of AVP on monoamine levels after water deprivation. In this study, we investigated the effect of AVP and its receptor antagonists on alterations in dopamine (DA) release and cyclic adenosine 3',5' monophosphate (cAMP) efflux from rat brain slices following water deprivation. Striatal brain slices (500 microm thick) were incubated in a medium with or without AVP (0. 1-1.0 microM) for 30 min. After 2 h of washout in normal medium, high KCl (40 mM)-evoked DA release and cAMP efflux from the rat brain slices were examined. In the brain slices of euhydrated animals, treatment with AVP slightly altered DA release and cAMP efflux from the brain. This increase in DA release and cAMP efflux was not significantly affected by the addition of a calcium/calmodulin-dependent protein phosphatase, calcineurin (20 microM), to the incubation medium or either by a V1 or V2 AVP receptor antagonist. In contrast, AVP significantly increased the DA release and enhanced the cAMP efflux from the brain slices of water-deprived animals. The AVP-induced increase of brain response in the water-deprived animals was significantly attenuated by a V2 receptor antagonist, partially by calcineurin, but not by a V1 receptor antagonist. The present results suggest that AVP may play a role in water-deprivation-induced DA release and cAMP efflux, which is possibly mediated through the activation of the V2 receptor. The V2 receptor action is attenuated by calcium/calmodulin-dependent dephosphorlyation of some cellular proteins critical for signal transduction.

  15. Role of intragenic binding of cAMP responsive protein (CRP) in regulation of the succinate dehydrogenase genes Rv0249c-Rv0247c in TB complex mycobacteria.

    PubMed

    Knapp, Gwendowlyn S; Lyubetskaya, Anna; Peterson, Matthew W; Gomes, Antonio L C; Ma, Zhuo; Galagan, James E; McDonough, Kathleen A

    2015-06-23

    Bacterial pathogens adapt to changing environments within their hosts, and the signaling molecule adenosine 3', 5'-cyclic monophosphate (cAMP) facilitates this process. In this study, we characterized in vivo DNA binding and gene regulation by the cAMP-responsive protein CRP in M. bovis BCG as a model for tuberculosis (TB)-complex bacteria. Chromatin immunoprecipitation followed by deep-sequencing (ChIP-seq) showed that CRP associates with ∼900 DNA binding regions, most of which occur within genes. The most highly enriched binding region was upstream of a putative copper transporter gene (ctpB), and crp-deleted bacteria showed increased sensitivity to copper toxicity. Detailed mutational analysis of four CRP binding sites upstream of the virulence-associated Rv0249c-Rv0247c succinate dehydrogenase genes demonstrated that CRP directly regulates Rv0249c-Rv0247c expression from two promoters, one of which requires sequences intragenic to Rv0250c for maximum expression. The high percentage of intragenic CRP binding sites and our demonstration that these intragenic DNA sequences significantly contribute to biologically relevant gene expression greatly expand the genome space that must be considered for gene regulatory analyses in mycobacteria. These findings also have practical implications for an important bacterial pathogen in which identification of mutations that affect expression of drug target-related genes is widely used for rapid drug resistance screening.

  16. Cyanidin-3-O-Glucoside Protects against 1,3-Dichloro-2-Propanol-Induced Reduction of Progesterone by Up-regulation of Steroidogenic Enzymes and cAMP Level in Leydig Cells.

    PubMed

    Sun, Jianxia; Xu, Wei; Zhu, Cuijuan; Hu, Yunfeng; Jiang, Xinwei; Ou, Shiyi; Su, Zhijian; Huang, Yadong; Jiao, Rui; Bai, Weibin

    2016-01-01

    1,3-Dichloro-2-propanol (1,3-DCP) is a food processing contaminant and has been shown to perturb male reproductive function. Cyanidin-3-O-glucoside (C3G), an anthocyanin antioxidant, is reported to have protective effects on many organs. However, it remains unclear whether C3G protects against chemical-induced reproductive toxicity. The present study was therefore to investigate the intervention of C3G on 1,3-DCP-induced reproductive toxicity in R2C Leydig cells. Results demonstrated that C3G inhibited the 1,3-DCP-induced cytotoxicity and cell shape damage with the effective doses being ranging from 10 to 40 μmol/L. In addition, 1,3-DCP (2 mmol/L) exposure significantly increased the ROS level and mitochondrial membrane potential damage ratio, leading to a decrease in progesterone production, while C3G intervention reduced the ROS level, and increased the progesterone production after 24 h treatment. Most importantly, C3G intervention could up-regulate the cyclic adenosine monophosphate (cAMP) level and protein expression of steroidogenic acute regulatory protein and 3β-hydroxysteroid dehydrogenase. It was concluded that C3G is effective in reducing 1,3-DCP-induced reproductive toxicity via activating steroidogenic enzymes and cAMP level.

  17. Cyanidin-3-O-Glucoside Protects against 1,3-Dichloro-2-Propanol-Induced Reduction of Progesterone by Up-regulation of Steroidogenic Enzymes and cAMP Level in Leydig Cells

    PubMed Central

    Sun, Jianxia; Xu, Wei; Zhu, Cuijuan; Hu, Yunfeng; Jiang, Xinwei; Ou, Shiyi; Su, Zhijian; Huang, Yadong; Jiao, Rui; Bai, Weibin

    2016-01-01

    1,3-Dichloro-2-propanol (1,3-DCP) is a food processing contaminant and has been shown to perturb male reproductive function. Cyanidin-3-O-glucoside (C3G), an anthocyanin antioxidant, is reported to have protective effects on many organs. However, it remains unclear whether C3G protects against chemical-induced reproductive toxicity. The present study was therefore to investigate the intervention of C3G on 1,3-DCP-induced reproductive toxicity in R2C Leydig cells. Results demonstrated that C3G inhibited the 1,3-DCP-induced cytotoxicity and cell shape damage with the effective doses being ranging from 10 to 40 μmol/L. In addition, 1,3-DCP (2 mmol/L) exposure significantly increased the ROS level and mitochondrial membrane potential damage ratio, leading to a decrease in progesterone production, while C3G intervention reduced the ROS level, and increased the progesterone production after 24 h treatment. Most importantly, C3G intervention could up-regulate the cyclic adenosine monophosphate (cAMP) level and protein expression of steroidogenic acute regulatory protein and 3β-hydroxysteroid dehydrogenase. It was concluded that C3G is effective in reducing 1,3-DCP-induced reproductive toxicity via activating steroidogenic enzymes and cAMP level. PMID:27867356

  18. Kinetic and mechanistic analysis of dinucleotide and oligonucleotide formation from the 5'-phosphorimidazolide of adenosine on Na(+)-montmorillonite

    NASA Technical Reports Server (NTRS)

    Kawamura, K.; Ferris, J. P.

    1994-01-01

    The rate constants for the condensation reaction of the 5'-phosphorimidazolide of adenosine (ImpA) to form dinucleotides and oligonucleotides have been measured in the presence of Na(+)-volclay (a Na(+)-montmorillonite) in pH 8 aqueous solution at 25 degrees C. The rates of the reaction of ImpA with an excess of adenosine 5'-monophosphoramidate (NH2pA), P1,P2-diadenosine 5',5'-pyrophosphate (A5'ppA), or adenosine 5'-monophosphate (5'-AMP or pA) in the presence of the montmorillonite to form NH2pA3'pA, A5'ppA3'pA, and pA3'pA, respectively, were measured. Only 3',5'-linked products were observed. The magnitude of the rate constants decrease in the order NH2pA3'pA > A5'-ppA3'pA > pA3'pA. The binding of ImpA to montmorillonite was measured, and the adsorption isotherm was determined. The binding of ImpA to montmorillonite and the formation of higher oligonucleotides is not observed in the absence of salts. Mg2+ enhances binding and oligonucleotide formation more than Ca2+ and Na+. The rate constants for the oligonucleotide formation were determined from the reaction products formed from 10 to 40 mM ImpA in the presence of Na(+)-montmorillonite using the computer program SIMFIT. The magnitudes of the rate constants for the formation of oligonucleotides increased in the order 2-mer < 3-mer < 4-mer ... 7-mer. The rate constants for dinucleotide and trinucleotide formation are more than 1000 times larger than those measured in the absence of montmorillonite. The rate constants for the formation of dinucleotide, trinucleotide, and tetranucleotide are 41,2.6, and 3.7 times larger than those for the formation of oligo(G)s with a poly(C) template. The hydrolysis of ImpA was accelerated 35 times in the presence of the montmorillonite. The catalytic ability of montmorillonite to form dinucleotides and oligonucleotides is quantitatively evaluated and possible pathways for oligo(A) formation are proposed.

  19. Modafinil inhibits K(Ca)3.1 currents and muscle contraction via a cAMP-dependent mechanism.

    PubMed

    Choi, Shinkyu; Kim, Moon Young; Joo, Ka Young; Park, Seonghee; Kim, Ji Aee; Jung, Jae-Chul; Oh, Seikwan; Suh, Suk Hyo

    2012-07-01

    Modafinil has been used as a psychostimulant for the treatment of narcolepsy. However, its primary mechanism of action remains elusive. Therefore, we examined the effects of modafinil on K(Ca)3.1 channels and vascular smooth muscle contraction. K(Ca)3.1 currents and channel activity were measured using a voltage-clamp technique and inside-out patches in mouse embryonic fibroblast cell line, NIH-3T3 fibroblasts. Intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration was measured, and the phosphorylation of K(Ca)3.1 channel protein was examined using western blotting in NIH-3T3 fibroblasts and/or primary cultured mouse aortic smooth muscle cells (SMCs). Muscle contractions were recorded from mouse aorta and rat pulmonary artery by using a myograph developed in-house. Modafinil was found to inhibit K(Ca)3.1 currents in a concentration-dependent manner, and the half-maximal inhibition (IC(50)) of modafinil for the current inhibition was 6.8 ± 0.7 nM. The protein kinase A (PKA) activator forskolin also inhibited K(Ca)3.1 currents. The inhibitory effects of modafinil and forskolin on K(Ca)3.1 currents were blocked by the PKA inhibitors PKI(14-22) or H-89. In addition, modafinil relaxed blood vessels (mouse aorta and rat pulmonary artery) in a concentration-dependent manner. Modafinil increased cAMP concentrations in NIH-3T3 fibroblasts or primary cultured mouse aortic SMCs and phosphorylated K(Ca)3.1 channel protein in NIH-3T3 fibroblasts. However, open probability and single-channel current amplitudes of K(Ca)3.1 channels were not changed by modafinil. From these results, we conclude that modafinil inhibits K(Ca)3.1 channels and vascular smooth muscle contraction by cAMP-dependent phosphorylation, suggesting that modafinil can be used as a cAMP-dependent K(Ca)3.1 channel blocker and vasodilator.

  20. 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

  1. The ability of denbufylline to inhibit cyclic nucleotide phosphodiesterase and its affinity for adenosine receptors and the adenosine re-uptake site.

    PubMed Central

    Nicholson, C. D.; Jackman, S. A.; Wilke, R.

    1989-01-01

    1. Denbufylline has been examined for its ability to inhibit cyclic nucleotide phosphodiesterase isoenzymes from rat cardiac ventricle and cerebrum, as well as for its affinity for adenosine A1 and A2 receptors and the re-uptake site. For comparison, SK&F 94120, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) were examined as phosphodiesterase inhibitors whilst N6-cyclohexyladenosine, R(-)-N6-(2-phenylisopropyl)-adenosine, 5'-N-ethylcarboxamido-adenosine, 2-nitrobenzylthioinosine, theophylline and IBMX were examined for their affinity for adenosine binding sites. 2. This investigation confirmed the presence of four phosphodiesterase activities in rat cardiac ventricle; in rat cerebrum only three were present. 3. Denbufylline selective inhibited one form of Ca2+-independent, low Km cyclic AMP phosphodiesterase. The form inhibited was one of two present in cardiac ventricle and the sole one in cerebrum. This form was not inhibited by cyclic GMP. The inotropic agent SK&F 94120 selectively inhibited the form of cyclic AMP phosphodiesterase which was inhibited by cyclic GMP present in cardiac ventricle. Theophylline and IBMX were relatively non-selective phosphodiesterase inhibitors. 4. Denbufylline was a less potent inhibitor of ligand binding to adenosine receptors than of cyclic AMP phosphodiesterase. This contrasted with theophylline, which had a higher affinity for adenosine receptors, and IBMX which showed no marked selectivity. Denbufylline, theophylline and IBMX all had a low affinity for the adenosine re-uptake site. 5. Denbufylline is being developed as an agent for the therapy of multi-infarct dementia. The selective inhibition of a particular low Km cyclic AMP phosphodiesterase may account for the activity of this compound. PMID:2474352

  2. Evidence for the primary role for 4-aminopyridine-sensitive K(v) channels in beta(3)-adrenoceptor-mediated, cyclic AMP-independent relaxations of guinea-pig gastrointestinal smooth muscles.

    PubMed

    Horinouchi, Takahiro; Tanaka, Yoshio; Koike, Katsuo

    2003-02-01

    Gastrointestinal smooth muscles exhibit relaxation in response to the stimulation of beta-adrenoceptors with catecholamines. Subtypes of beta-adrenoceptors which mediate catecholamine-elicited relaxations in gastrointestinal smooth muscles are predominantly atypical beta-adrenoceptors including beta(3)-adrenoceptors. Gastrointestinal smooth muscle relaxations mediated via beta(3)-adrenoceptors can occur independently of intracellular cyclic adenosine monophosphate (AMP) elevation. One of the mechanisms responsible for cyclic AMP-independent smooth muscle relaxation following activation of G(s) protein-coupled receptors could be activation of voltage-gated K(+) channels. In the present study, possible contribution of two types of K(+) (large-conductance, Ca(2+)-sensitive and voltage-gated K(+), BK(Ca); voltage-gated, K(v)) channels to beta(3)-adrenoceptor-mediated, cyclic AMP-independent relaxations was compared in gastric fundus and duodenum smooth muscles isolated from the guinea-pig. In these gastrointestinal smooth muscles, three catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline) and two beta(3)-adrenoceptor agonists ((R(*), R(*))-(+/-)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium (BRL37344) and (+/-)-[4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy] -1,3-dihydro-2H-benzimidazol-2-one] hydrochloride ((+/-)-CGP12177A)) elicited a concentration-dependent relaxation in the presence of beta(1)- and beta(2)-adrenoceptor antagonists. The relaxations were unaffected by an adenylyl cyclase inhibitor, SQ-22536 (100 microM), which indicates their characteristic of cyclic AMP-independency. On the other hand, the SQ-22536-resistant, beta(3)-adrenoceptor-mediated relaxant components were potently attenuated when the tone was raised using high-KCl (80 mM) or in the presence of a K(v) channel blocker, 4-aminopyridine (4-AP, 1-3 mM). Iberiotoxin (100 nM), a selective blocker of BK(Ca) channels which significantly

  3. Ionizing Radiation Activates AMP-Activated Kinase (AMPK): A Target for Radiosensitization of Human Cancer Cells

    SciTech Connect

    Sanli, Toran; Rashid, Ayesha; Liu Caiqiong

    2010-09-01

    Purpose: Adenosine monophosphate (AMP)-activated kinase (AMPK) is a molecular energy sensor regulated by the tumor suppressor LKB1. Starvation and growth factors activate AMPK through the DNA damage sensor ataxia-telangiectasia mutated (ATM). We explored the regulation of AMPK by ionizing radiation (IR) and its role as a target for radiosensitization of human cancer cells. Methods and Materials: Lung, prostate, and breast cancer cells were treated with IR (2-8 Gy) after incubation with either ATM or AMPK inhibitors or the AMPK activator metformin. Then, cells were subjected to either lysis and immunoblotting, immunofluorescence microscopy, clonogenic survival assays, or cell cycle analysis. Results: IR induced a robust phosphorylation and activation of AMPK in all tumor cells, independent of LKB1. IR activated AMPK first in the nucleus, and this extended later into cytoplasm. The ATM inhibitor KU-55933 blocked IR activation of AMPK. AMPK inhibition with Compound C or anti-AMPK {alpha} subunit small interfering RNA (siRNA) blocked IR induction of the cell cycle regulators p53 and p21{sup waf/cip} as well as the IR-induced G2/M arrest. Compound C caused resistance to IR, increasing the surviving fraction after 2 Gy, but the anti-diabetic drug metformin enhanced IR activation of AMPK and lowered the surviving fraction after 2 Gy further. Conclusions: We provide evidence that IR activates AMPK in human cancer cells in an LKB1-independent manner, leading to induction of p21{sup waf/cip} and regulation of the cell cycle and survival. AMPK appears to (1) participate in an ATM-AMPK-p21{sup waf/cip} pathway, (2) be involved in regulation of the IR-induced G2/M checkpoint, and (3) may be targeted by metformin to enhance IR responses.

  4. Release of Adenosine and ATP During Ischemia and Epilepsy

    PubMed Central

    Dale, Nicholas; Frenguelli, Bruno G

    2009-01-01

    Eighty years ago Drury & Szent-Györgyi described the actions of adenosine, AMP (adenylic acid) and ATP (pyrophosphoric or diphosphoric ester of adenylic acid) on the mammalian cardiovascular system, skeletal muscle, intestinal and urinary systems. Since then considerable insight has been gleaned on the means by which these compounds act, not least of which in the distinction between the two broad classes of their respective receptors, with their many subtypes, and the ensuing diversity in cellular consequences their activation invokes. These myriad actions are of course predicated on the release of the purines into the extracellular milieu, but, surprisingly, there is still considerable ambiguity as to how this occurs in various physiological and pathophysiological conditions. In this review we summarise the release of ATP and adenosine during seizures and cerebral ischemia and discuss mechanisms by which the purines adenosine and ATP may be released from cells in the CNS under these conditions. PMID:20190959

  5. Correlation between blood adenosine metabolism and sleep in humans.

    PubMed

    Díaz-Muñoz, M; Hernández-Muñoz, R; Suárez, J; Vidrio, S; Yááñez, L; Aguilar-Roblero, R; Rosenthal, L; Villalobos, L; Fernández-Cancino, F; Drucker-Colín, R; Chagoya De Sanchez, V

    1999-01-01

    Blood adenosine metabolism, including metabolites and metabolizing enzymes, was studied during the sleep period in human volunteers. Searching for significant correlations among biochemical parameters found: adenosine with state 1 of slow-wave sleep (SWS); activity of 5'-nucleotidase with state 2 of SWS; inosine and AMP with state 3-4 of SWS; and activity of 5'-nucleotidase and lactate with REM sleep. The correlations were detected in all of the subjects that presented normal hypnograms, but not in those who had fragmented sleep the night of the experiment. The data demonstrate that it is possible to obtain information of complex brain operations such as sleep by measuring biochemical parameters in blood. The results strengthen the notion of a role played by adenosine, its metabolites and metabolizing enzymes, during each of the stages that constitute the sleep process in humans.

  6. Inhibitory action of certain cyclophosphate derivatives of cAMP on cAMP-dependent protein kinases.

    PubMed

    de Wit, R J; Hekstra, D; Jastorff, B; Stec, W J; Baraniak, J; Van Driel, R; Van Haastert, P J

    1984-07-16

    A series cAMP derivatives with modifications in the adenine, ribose and cyclophosphate moiety were screened for their binding affinity for the two types of cAMP-binding sites in mammalian protein kinase type 1. In addition, the activation of the kinase by these analogs was monitored. The binding data indicate that cAMP is bound to both sites in a comparable manner: the adenine appears to have no hydrogen-bond interactions with the binding sites, whereas the ribose may be bound by three hydrogen bonds involving the 2', 3' and 5' positions of cAMP. The binding data are not conclusive about the nature of the interaction with the exocyclic oxygen atoms on phosphorus, though a charge interaction seems to be absent. The cAMP molecule seems to be bound in the syn conformation. The results of activation experiments show that modifications in the adenine and ribose moiety do not affect the maximal activation level, while alteration of the two exocyclic oxygen atoms may result in a reduced maximal activation level and in one case, (Rp)-adenosine 3', 5'-monophosphorothioate [Rp-cAMPS], in total absence of activation even at concentrations at which the analog saturates both binding sites. Since occupancy of the cAMP-binding sites by this derivative apparently did not lead to activation of the enzyme, we examined whether this compound could antagonize the activation by cAMP. Indeed (Rp)-cAMPS was found to inhibit cAMP stimulated kinase activity at concentrations compatible to its binding affinity. Also with mammalian protein kinase type II (Rp)-cAMPS showed antagonistic activity, while with a cAMP-dependent protein kinase from Dictyostelium discoideum partial agonistic activity was observed. Previously a mechanism for activation of protein kinase type I was proposed involving a charge interaction between the equatorial exocyclic oxygen atom and the binding site [De Wit et. al. (1982) Eur. J. Biochem 122, 95-99]. This was based on measurements with impure preparations of (Rp)-cAMPS

  7. Effects of adenosine metabolism in astrocytes on central nervous system oxygen toxicity.

    PubMed

    Chen, Yu-liang; Zhang, Ya-nan; Wang, Zhong-zhuang; Xu, Wei-gang; Li, Run-ping; Zhang, Jun-dong

    2016-03-15

    Hyperbaric oxygen (HBO) is widely used in military operations, especially underwater missions. However, prolonged and continuous inhalation of HBO can cause central nervous system oxygen toxicity (CNS-OT), which greatly limits HBO's application. The regulation of astrocytes to the metabolism of adenosine is involved in epilepsy. In our study, we aimed to observe the effects of HBO exposure on the metabolism of adenosine in the brain. Furthermore, we aimed to confirm the possible mechanism underlying adenosine's mediation of the CNS-OT. Firstly, anesthetized rats exposed to 5 atm absolute HBO for 80 min. The concentrations of extracellular adenosine, ATP, ADP, and AMP were detected. Secondly, free-moving rats were exposed to HBO at the same pressure for 20 min, and the activities of 5'-nucleotidase and ADK in brain tissues were measured. For the mechanism studies, we observed the effects of a series of different doses of drugs related to adenosine metabolism on the latency of CNS-OT. Results showed HBO exposure could increase adenosine content by inhibiting ADK activity and improving 5'-nucleotidase activity. And adenosine metabolism during HBO exposure may be a protective response against HBO-induced CNS-OT. Moreover, the improvement of adenosine concentration, activation of adenosine A1R, or suppression of ADK and adenosine A2AR, which are involved in the prevention of HBO-induced CNS-OT. This is the first study to demonstrate HBO exposure regulated adenosine metabolism in the brain. Adenosine metabolism and adenosine receptors are related to HBO-induced CNS-OT development. These results will provide new potential targets for the termination or the attenuation of CNS-OT.

  8. H3 histamine receptor agonist inhibits biliary growth of BDL rats by downregulation of the cAMP-dependent PKA/ERK1/2/ELK-1 pathway.

    PubMed

    Francis, Heather; Franchitto, Antonio; Ueno, Yoshiyuki; Glaser, Shannon; DeMorrow, Sharon; Venter, Julie; Gaudio, Eugenio; Alvaro, Domenico; Fava, Giammarco; Marzioni, Marco; Vaculin, Bradley; Alpini, Gianfranco

    2007-05-01

    Histamine regulates many functions by binding to four histamine G-coupled receptor proteins (H1R, H2R, H3R and H4R). As H3R exerts their effects by coupling to Galpha(i/o) proteins reducing adenosine 3', 5'-monophosphate (cAMP) levels (a key player in the modulation of cholangiocyte hyperplasia/damage), we evaluated the role of H3R in the regulation of biliary growth. We posed the following questions: (1) Do cholangiocytes express H3R? (2) Does in vivo administration of (R)-(alpha)-(-)-methylhistamine dihydrobromide (RAMH) (H3R agonist), thioperamide maleate (H3R antagonist) or histamine, in the absence/presence of thioperamide maleate, to bile duct ligated (BDL) rats regulate cholangiocyte proliferation? and (3) Does RAMH inhibit cholangiocyte proliferation by downregulation of cAMP-dependent phosphorylation of protein kinase A (PKA)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ets-like gene-1 (Elk-1)? The expression of H3R was evaluated in liver sections by immunohistochemistry and immunofluorescence, and by real-time PCR in cholangiocyte RNA from normal and BDL rats. BDL rats (immediately after BDL) were treated daily with RAMH, thioperamide maleate or histamine in the absence/presence of thioperamide maleate for 1 week. Following in vivo treatment of BDL rats with RAMH for 1 week, and in vitro stimulation of BDL cholangiocytes with RAMH, we evaluated cholangiocyte proliferation, cAMP levels and PKA, ERK1/2 and Elk-1 phosphorylation. Cholangiocytes from normal and BDL rats express H3R. The expression of H3R mRNA increased in BDL compared to normal cholangiocytes. Histamine decreased cholangiocyte growth of BDL rats to a lower extent than that observed in BDL RAMH-treated rats; histamine-induced inhibition of cholangiocyte growth was partly blocked by thioperamide maleate. In BDL rats treated with thioperamide maleate, cholangiocyte hyperplasia was slightly higher than that of BDL rats. In vitro, RAMH inhibited the proliferation of BDL cholangiocytes. RAMH

  9. Adenosine receptor neurobiology: overview.

    PubMed

    Chen, Jiang-Fan; Lee, Chien-fei; Chern, Yijuang

    2014-01-01

    Adenosine is a naturally occurring nucleoside that is distributed ubiquitously throughout the body as a metabolic intermediary. In the brain, adenosine functions as an important upstream neuromodulator of a broad spectrum of neurotransmitters, receptors, and signaling pathways. By acting through four G-protein-coupled receptors, adenosine contributes critically to homeostasis and neuromodulatory control of a variety of normal and abnormal brain functions, ranging from synaptic plasticity, to cognition, to sleep, to motor activity to neuroinflammation, and cell death. This review begun with an overview of the gene and genome structure and the expression pattern of adenosine receptors (ARs). We feature several new developments over the past decade in our understanding of AR functions in the brain, with special focus on the identification and characterization of canonical and noncanonical signaling pathways of ARs. We provide an update on functional insights from complementary genetic-knockout and pharmacological studies on the AR control of various brain functions. We also highlight several novel and recent developments of AR neurobiology, including (i) recent breakthrough in high resolution of three-dimension structure of adenosine A2A receptors (A2ARs) in several functional status, (ii) receptor-receptor heterodimerization, (iii) AR function in glial cells, and (iv) the druggability of AR. We concluded the review with the contention that these new developments extend and strengthen the support for A1 and A2ARs in brain as therapeutic targets for neurologic and psychiatric diseases.

  10. Galangin inhibits proliferation of HepG2 cells by activating AMPK via increasing the AMP/TAN ratio in a LKB1-independent manner.

    PubMed

    Zhang, Haitao; Li, Ning; Wu, Jun; Su, Lijuan; Chen, Xiaoyi; Lin, Biyun; Luo, Hui

    2013-10-15

    Galangin, a flavonol derived from Alpinia officinarum Hance and used as food additives in southern China, induces apoptosis and autophagy to suppress the proliferation of HepG2 cells. In this study, we demonstrated that galangin induced autophagy by increasing the ratio of AMP/TAN in HepG2 cells. It stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and LKB1, but inhibited the phosphorylation of AKT and mTOR. Inhibition of AMPK activation suppressed the dephosphorylation of mTOR to block galangin-induced autophagy. AMPK activation by galangin appeared to be independent of the LKB1 signaling pathway because the down-regulation of LKB1 by its siRNA failed to affect galangin-induced autophagy. Collectively, the findings demonstrated a novel mechanism of how galangin induces autophagy via activating AMPK in a LKB1- independent manner. The induction of autophagy can thus reflect the anti-proliferation effect of galangin in HCC cells.

  11. Sesamol decreases melanin biosynthesis in melanocyte cells and zebrafish: Possible involvement of MITF via the intracellular cAMP and p38/JNK signalling pathways.

    PubMed

    Baek, Seung-hwa; Lee, Sang-Han

    2015-10-01

    The development of antimelanogenic agents is important for the prevention of serious aesthetic problems such as melasma, freckles, age spots and chloasma. The aim of this study was to investigate the antimelanogenic effect of sesamol, an active lignan isolated from Sesamum indicum, in melan-a cells. Sesamol strongly inhibited melanin biosynthesis and the activity of intracellular tyrosinase by decreasing cyclic adenosine monophosphate (cAMP) accumulation. Sesamol significantly decreased the expression of melanogenesis-related genes, such as tyrosinase, tyrosinase-related protein-1,2 (TRP-1,2), microphthalmia-associated transcription factor (MITF) and melanocortin 1 receptor (MC1R). In addition, sesamol also induces phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK). Moreover, sesamol dose-dependently decreased zebrafish pigment formation, tyrosinase activity and expression of melanogenesis-related genes. These findings indicate that sesamol inhibited melanin biosynthesis by down-regulating tyrosinase activity and melanin production via regulation of gene expression of melanogenesis-related proteins through modulation of MITF activity, which promoted phosphorylation of p38 and JNK in melan-a cells. Together, these results suggest that sesamol strongly inhibits melanin biosynthesis, and therefore, sesamol represents a new skin-whitening agent for use in cosmetics.

  12. Overexpression, purification and crystallographic analysis of a unique adenosine kinase from Mycobacterium tuberculosis

    SciTech Connect

    Wang, Yimin; Long, Mary C.; Ranganathan, Senthil; Escuyer, Vincent; Parker, William B.; Li, Rongbao

    2005-06-01

    Adenosine kinase from M. tuberculosis has been overexpressed, purified and crystallized in the presence of adenosine. Structure determination using molecular replacement with diffraction data collected at 2.2 Å reveals a dimeric structure. Adenosine kinase from Mycobacterium tuberculosis is the only prokaryotic adenosine kinase that has been isolated and characterized. The enzyme catalyzes the phosphorylation of adenosine to adenosine monophosphate and is involved in the activation of 2-methyladenosine, a compound that has demonstrated selective activity against M. tuberculosis. The mechanism of action of 2-methyladenosine is likely to be different from those of current tuberculosis treatments and this compound (or other adenosine analogs) may prove to be a novel therapeutic intervention for this disease. The M. tuberculosis adenosine kinase was overexpressed in Escherichia coli and the enzyme was purified with activity comparable to that reported previously. The protein was crystallized in the presence of adenosine using the vapour-diffusion method. The crystals diffracted X-rays to high resolution and a complete data set was collected to 2.2 Å using synchrotron radiation. The crystal belonged to space group P3{sub 1}21, with unit-cell parameters a = 70.2, c = 111.6 Å, and contained a single protein molecule in the asymmetric unit. An initial structural model of the protein was obtained by the molecular-replacement method, which revealed a dimeric structure. The monomers of the dimer were related by twofold crystallographic symmetry. An understanding of how the M. tuberculosis adenosine kinase differs from the human homolog should aid in the design of more potent and selective antimycobacterial agents that are selectively activated by this enzyme.

  13. Quantitative effect and regulatory function of cyclic adenosine 5'-phosphate in Escherichia coli.

    PubMed

    Narang, Atul

    2009-09-01

    Cyclic adenosine 5'-phosphate (cAMP) is a global regulator of gene expression in Escherichia coli. Despite decades of intensive study, the quantitative effect and regulatory function of cAMP remain the subjects of considerable debate. Here, we analyse the data in the literature to show that: (a) In carbon-limited cultures (including cultures limited by glucose), cAMP is at near-saturation levels with respect to expression of several catabolic promoters (including lac, ara and gal). It follows that cAMP receptor protein (CRP) cAMP-mediated regulation cannot account for the strong repression of these operons in the presence of glucose. (b) The cAMP levels in carbon-excess cultures are substantially lower than those observed in carbon-limited cultures under these conditions, the expression of catabolic promoters is very sensitive to variation of cAMP levels. (c)=CRPcAMP invariably activates the expression of catabolic promoters, but it appears to inhibit the expression of anabolic promoters. (d) These results suggest that the physiological function of cAMP is to maintain homeostatic energy levels. In carbon-limited cultures, growth is limited by the supply of energy; the cAMP levels therefore increase to enhance energy accumulation by activating the catabolic promoters and inhibiting the anabolic promoters. Conversely, in carbonexcess cultures, characterized by the availability of excess energy, the cAMP levels decrease in order to depress energy accumulation by inhibiting the catabolic promoters and activating the anabolic promoters.

  14. Recognition of Nucleoside Monophosphate Substrates by Haemophilus influenzae Class C Acid Phosphatase

    SciTech Connect

    Singh, Harkewal; Schuermann, Jonathan P.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2010-12-08

    The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD{sup +} utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5{prime},3{prime}-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5{prime}-AMP, 3{prime}-AMP, and 2{prime}-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5{prime}-nucleotides and 3{prime}-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5{prime} substrates in an anti conformation and 3{prime} substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.

  15. Recognition of nucleoside monophosphate substrates by Haemophilus influenzae class C acid phosphatase.

    PubMed

    Singh, Harkewal; Schuermann, Jonathan P; Reilly, Thomas J; Calcutt, Michael J; Tanner, John J

    2010-12-10

    The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD(+) utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5',3'-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5'-AMP, 3'-AMP, and 2'-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5'-nucleotides and 3'-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5' substrates in an anti conformation and 3' substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.

  16. Adenosine and sleep

    SciTech Connect

    Yanik, G.M. Jr.

    1987-01-01

    Behavioral and biochemical approaches have been used to determine the relative contribution of endogenous adenosine and adenosine receptors to the sleep-wake cycle in the rat. Adenosine concentrations in specific areas of the rat brain were not affected by 24 hours of total sleep deprivation, or by 24 or 48 hours of REM sleep deprivation. In order to assess the effect of REM sleep deprivation on adenosine A/sub 1/ receptors, /sup 3/H-L-PIA binding was measured. The Bmax values for /sup 3/H-L-PIA binding to membrane preparations of the cortices and corpus striata from 48 hour REM sleep-deprived animals were increased 14.8% and 23%, respectively. These increases were not maintained following the cessation of sleep deprivation and recovered within 2 hours. The results of a 96 hour REM deprivation experiment were similar to those of the 48 hour REM sleep deprivation experiment. However, these increases were not evident in similar structures taken from stress control animals, and conclusively demonstrated that the changes in /sup 3/H-L-PIA binding resulted from REM sleep deprivation and not from stress.

  17. Adenosine deaminase from Streptomyces coelicolor: recombinant expression, purification and characterization.

    PubMed

    Pornbanlualap, Somchai; Chalopagorn, Pornchanok

    2011-08-01

    The sequencing of the genome of Streptomyces coelicolor A3(2) identified seven putative adenine/adenosine deaminases and adenosine deaminase-like proteins, none of which have been biochemically characterized. This report describes recombinant expression, purification and characterization of SCO4901 which had been annotated in data bases as a putative adenosine deaminase. The purified putative adenosine deaminase gives a subunit Mr=48,400 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 182,000 by comparative gel filtration. These values are consistent with the active enzyme being composed of four subunits with identical molecular weights. The turnover rate of adenosine is 11.5 s⁻¹ at 30 °C. Since adenine is deaminated ∼10³ slower by the enzyme when compared to that of adenosine, these data strongly show that the purified enzyme is an adenosine deaminase (ADA) and not an adenine deaminase (ADE). Other adenine nucleosides/nucleotides, including 9-β-D-arabinofuranosyl-adenine (ara-A), 5'-AMP, 5'-ADP and 5'-ATP, are not substrates for the enzyme. Coformycin and 2'-deoxycoformycin are potent competitive inhibitors of the enzyme with inhibition constants of 0.25 and 3.4 nM, respectively. Amino acid sequence alignment of ScADA with ADAs from other organisms reveals that eight of the nine highly conserved catalytic site residues in other ADAs are also conserved in ScADA. The only non-conserved residue is Asn317, which replaces Asp296 in the murine enzyme. Based on these data, it is suggested here that ADA and ADE proteins are divergently related enzymes that have evolved from a common α/β barrel scaffold to catalyze the deamination of different substrates, using a similar catalytic mechanism.

  18. Identification and partial characterization of an adenosine(5')tetraphospho(5')adenosine hydrolase on intact bovine aortic endothelial cells.

    PubMed Central

    Ogilvie, A; Lüthje, J; Pohl, U; Busse, R

    1989-01-01

    The biologically active dinucleotides adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')-triphospho(5')adenosine (Ap3A), which are both releasable into the circulation from storage pools in thrombocytes, are catabolized by intact bovine aortic endothelial cells. 1. Compared with extracellular ATP and ADP, which are very rapidly hydrolysed, the degradation of Ap4A and Ap3A by endothelial ectohydrolases is relatively slow, resulting in a much longer half-life on the endothelial surface of the blood vessel. The products of hydrolysis are further degraded and finally taken up as adenosine. 2. Ap4A hydrolase has high affinity for its substrate (Km 10 microM). 3. ATP as well as AMP transiently accumulates in the extracellular fluid, suggesting an asymmetric split of Ap4A by the ectoenzyme. 4. Mg2+ or Mn2+ at millimolar concentration are needed for maximal activity; Zn2+ and Ca2+ are inhibitory. 5. The hydrolysis of Ap4A is retarded by other nucleotides, such as ATP and Ap3A, which are released from platelets simultaneously with Ap4A. PMID:2541689

  19. 1,3-Dichloro-2-propanol inhibits progesterone production through the expression of steroidogenic enzymes and cAMP concentration in Leydig cells.

    PubMed

    Sun, Jianxia; Bai, Shun; Bai, Weibin; Zou, Feiyan; Zhang, Lei; Li, Guoqiang; Hu, Yunfeng; Li, Mingwei; Yan, Rian; Su, Zhijian; Huang, Yadong

    2014-07-01

    1,3-Dichloro-2-propanol (1,3-DCP) is a well-known food processing contaminant that has been shown to impede male reproductive function. However, its mechanism of action remains elusive. In this study, the effects of 1,3-DCP on progesterone production were investigated using the R2C Leydig cell model. 1,3-DCP significantly reduced cell viability from 7.48% to 97.4% at doses comprised between 0.5 and 6mM. Single cell gel/comet assays and atomic force microscopy assays showed that 1,3-DCP induced early phase cell apoptosis. In addition, 1,3-DCP significantly reduced progesterone production detected by radioimmunoassay (RIA). The results from quantitative polymerase chain reaction and western blotting demonstrated that the mRNA expression levels of steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase were significantly down-regulated in R2C cells. Particularly, the change rhythm of Star expression was highly consistent with progesterone production. Furthermore, the cyclic adenosine monophosphate (cAMP) and the mitochondrial membrane potential mediated by ROS, which are involved in regulating progesterone synthesis were also decreased in response to the 1,3-DCP treatment. Overall, the data presented here suggested that 1,3-DCP interferes with the male steroidogenic capacity mainly by down-regulating the level of cAMP and the key enzymes involved in the androgen synthesis pathway.

  20. Beta-Adrenoceptor Activation Reduces Both Dermal Microvascular Endothelial Cell Migration via a cAMP-Dependent Mechanism and Wound Angiogenesis

    PubMed Central

    O'Leary, Andrew P; Fox, James M; Pullar, Christine E

    2015-01-01

    Angiogenesis is an essential process during tissue regeneration; however, the amount of angiogenesis directly correlates with the level of wound scarring. Angiogenesis is lower in scar-free foetal wounds while angiogenesis is raised and abnormal in pathophysiological scarring such as hypertrophic scars and keloids. Delineating the mechanisms that modulate angiogenesis and could reduce scarring would be clinically useful. Beta-adrenoceptors (β-AR) are G protein-coupled receptors (GPCRs) expressed on all skin cell-types. They play a role in wound repair but their specific role in angiogenesis is unknown. In this study, a range of in vitro assays (single cell migration, scratch wound healing, ELISAs for angiogenic growth factors and tubule formation) were performed with human dermal microvascular endothelial cells (HDMEC) to investigate and dissect mechanisms underpinning β-AR-mediated modulation of angiogenesis in chick chorioallantoic membranes (CAM) and murine excisional skin wounds. β-AR activation reduced HDMEC migration via cyclic adenosine monophosphate (cAMP)-dependent and protein kinase A (PKA)-independent mechanisms as demonstrated through use of an EPAC agonist that auto-inhibited the cAMP-mediated β-AR transduced reduction in HDMEC motility; a PKA inhibitor was, conversely, ineffective. ELISA studies demonstrated that β-AR activation reduced pro-angiogenic growth factor secretion from HDMECs (fibroblast growth factor 2) and keratinocytes (vascular endothelial growth factor A) revealing possible β-AR-mediated autocrine and paracrine anti-angiogenic mechanisms. In more complex environments, β-AR activation delayed HDMEC tubule formation and decreased angiogenesis both in the CAM assay and in murine excisional skin wounds in vivo. β-AR activation reduced HDMEC function in vitro and angiogenesis in vivo; therefore, β-AR agonists could be promising anti-angiogenic modulators in skin. J. Cell. Physiol. 230: 356–365, 2015. © 2014 The Authors. Journal

  1. Concentration dependence of sodium permeation and sodium ion interactions in the cyclic AMP-gated channels of mammalian olfactory receptor neurons.

    PubMed

    Balasubramanian, S; Lynch, J W; Barry, P H

    1997-09-01

    The dependence of currents through the cyclic nucleotide-gated (CNG) channels of mammalian olfactory receptor neurons (ORNs) on the concentration of NaCl was studied in excised inside-out patches from their dendritic knobs using the patch-clamp technique. With a saturating concentration (100 microM) of adenosine 3',5'-cyclic monophosphate (cAMP), the changes in the reversal potential of macroscopic currents were studied at NaCl concentrations from 25 to 300 mM. In symmetrical NaCl solutions without the addition of divalent cations, the current-voltage relations were almost linear, reversing close to 0 mV. When the external NaCl concentration was maintained at 150 mM and the internal concentrations were varied, the reversal potentials of the cAMP-activated currents closely followed the Na+ equilibrium potential indicating that PCl/PNa approximately 0. However, at low external NaCl concentrations (< or = 100 mM) there was some significant chloride permeability. Our results further indicated that Na+ currents through these channels: (i) did not obey the independence principle; (ii) showed saturation kinetics with K(m)s in the range of 100-150 mM and (iii) displayed a lack of voltage dependence of conductance in asymmetric solutions that suggested that ion-binding sites were situated midway along the channel. Together, these characteristics indicate that the permeation properties of the olfactory CNG channels are significantly different from those of photoreceptor CNG channels.

  2. Adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase.

    PubMed Central

    Guranowski, A; Günther Sillero, M A; Sillero, A

    1994-01-01

    Yeast (Saccharomyces cerevisiae) acetyl coenzyme A (CoA) synthetase (EC 6.2.1.1) catalyzes the synthesis of adenosine 5'-tetraphosphate (P4A) and adenosine 5'-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate (P3 or P4), with relative velocities of 7:1, respectively. Of 12 nucleotides tested as potential donors of nucleotidyl moiety, only ATP, adenosine-5'-O-[3-thiotriphosphate], and acetyl-AMP were substrates, with relative velocities of 100, 62, and 80, respectively. The Km values for ATP, P3, and acetyl-AMP were 0.16, 4.7, and 1.8 mM, respectively. The synthesis of p4A could proceed in the absence of exogenous acetate but was stimulated twofold by acetate, with an apparent Km value of 0.065 mM. CoA did not participate in the synthesis of p4A (p5A) and inhibited the reaction (50% inhibitory concentration of 0.015 mM). At pH 6.3, which was optimum for formation of p4A (p5A), the rate of acetyl-CoA synthesis (1.84 mumol mg-1 min-1) was 245 times faster than the rate of synthesis of p4A measured in the presence of acetate. The known formation of p4A (p5A) in yeast sporulation and the role of acetate may therefore be related to acetyl-CoA synthetase. Images PMID:7910605

  3. Structural Studies of Thiamin Monophosphate Kinase in Complex with Substrates and Products.

    SciTech Connect

    McCulloch, K.M.; Kinsland, C.; Begley, T.P.; Ealick, S E.

    2008-06-03

    Thiamin monophosphate kinase (ThiL) catalyzes the ATP-dependent phosphorylation of thiamin monophosphate (TMP) to form thiamin pyrophosphate (TPP), the active form of vitamin B1. ThiL is a member of a small ATP binding superfamily that also includes the purine biosynthetic enzymes, PurM and PurL, NiFe hydrogenase maturation protein, HypE, and selenophosphate synthase, SelD. The latter four enzymes are believed to utilize phosphorylated intermediates during catalysis. To understand the mechanism of ThiL and its relationship to the other superfamily members, we determined the structure of Aquifex aeolicus ThiL (AaThiL) with nonhydrolyzable AMP-PCP and TMP, and also with the products of the reaction, ADP and TPP. The results suggest that AaThiL utilizes a direct, inline transfer of the {gamma}-phosphate of ATP to TMP rather than a phosphorylated enzyme intermediate. The structure of ThiL is compared to those of PurM, PurL, and HypE, and the ATP binding site is compared to that of PurL, for which nucleotide complexes are available.

  4. 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.

  5. A cell wall-bound adenosine nucleosidase is involved in the salvage of extracellular ATP in Solanum tuberosum.

    PubMed

    Riewe, David; Grosman, Lukasz; Fernie, Alisdair R; Zauber, Henrik; Wucke, Cornelia; Geigenberger, Peter

    2008-10-01

    Extracellular ATP (eATP) has recently been demonstrated to play a crucial role in plant development and growth. To investigate the fate of eATP within the apoplast, we used intact potato (Solanum tuberosum) tuber slices as an experimental system enabling access to the apoplast without interference of cytosolic contamination. (i) Incubation of intact tuber slices with ATP led to the formation of ADP, AMP, adenosine, adenine and ribose, indicating operation of apyrase, 5'-nucleotidase and nucleosidase. (ii) Measurement of apyrase, 5'-nucleotidase and nucleosidase activities in fractionated tuber tissue confirmed the apoplastic localization for apyrase and phosphatase in potato and led to the identification of a novel cell wall-bound adenosine nucleosidase activity. (iii) When intact tuber slices were incubated with saturating concentrations of adenosine, the conversion of adenosine into adenine was much higher than adenosine import into the cell, suggesting a potential bypass of adenosine import. Consistent with this, import of radiolabeled adenine into tuber slices was inhibited when ATP, ADP or AMP were added to the slices. (iv) In wild-type plants, apyrase and adenosine nucleosidase activities were found to be co-regulated, indicating functional linkage of these enzymes in a shared pathway. (v) Moreover, adenosine nucleosidase activity was reduced in transgenic lines with strongly reduced apoplastic apyrase activity. When taken together, these results suggest that a complete ATP salvage pathway is present in the apoplast of plant cells.

  6. Preferential activation of excitatory adenosine receptors at rat hippocampal and neuromuscular synapses by adenosine formed from released adenine nucleotides.

    PubMed Central

    Cunha, R. A.; Correia-de-Sá, P.; Sebastião, A. M.; Ribeiro, J. A.

    1996-01-01

    1. In the present work, we investigated the action of adenosine originating from extracellular catabolism of adenine nucleotides, in two preparations where synaptic transmission is modulated by both inhibitory A1 and excitatory A(2a)-adenosine receptors, the rat hippocampal Schaffer fibres/CA1 pyramid synapses and the rat innervated hemidiaphragm. 2. Endogenous adenosine tonically inhibited synaptic transmission, since 0.5-2 u ml-1 of adenosine deaminase increased both the population spike amplitude (30 +/- 4%) and field excitatory post-synaptic potential (f.e.p.s.p.) slope (27 +/- 4%) recorded from hippocampal slices and the evoked [3H]-acetylcholine ([3H]-ACh) release from the motor nerve terminals (25 +/- 2%). 3. alpha, beta-Methylene adenosine diphosphate (AOPCP) in concentrations (100-200 microM) that almost completely inhibited the formation of adenosine from the extracellular catabolism of AMP, decreased population spike amplitude by 39 +/- 5% and f.e.p.s.p. slope by 32 +/- 3% in hippocampal slices and [3H]-ACh release from motor nerve terminals by 27 +/- 3%. 4. Addition of exogenous 5'-nucleotidase (5 u ml-1) prevented the inhibitory effect of AOPCP on population spike amplitude and f.e.p.s.p. slope by 43-57%, whereas the P2 antagonist, suramin (100 microM), did not modify the effect of AOPCP. 5. In both preparations, the effect of AOPCP resulted from prevention of adenosine formation since it was no longer evident when accumulation of extracellular adenosine was hindered by adenosine deaminase (0.5-2 u ml-1). The inhibitory effect of AOPCP was still evident when A1 receptors were blocked by 1,3-dipropyl-8-cyclopentylxanthine (2.5-5 nM), but was abolished by the A2 antagonist, 3,7-dimethyl-1-propargylxanthine (10 microM). 6. These results suggest that adenosine originating from catabolism of released adenine nucleotides preferentially activates excitatory A2 receptors in hippocampal CAI pyramid synapses and in phrenic motor nerve endings. PMID:8886406

  7. Genetic and Functional Studies Implicate Synaptic Overgrowth and Ring Gland cAMP/PKA Signaling Defects in the Drosophila melanogaster Neurofibromatosis-1 Growth Deficiency

    PubMed Central

    Walker, James A.; Gouzi, Jean Y.; Long, Jennifer B.; Huang, Sidong; Maher, Robert C.; Xia, Hongjing; Khalil, Kheyal; Ray, Arjun; Van Vactor, David; Bernards, René; Bernards, André

    2013-01-01

    Neurofibromatosis type 1 (NF1), a genetic disease that affects 1 in 3,000, is caused by loss of a large evolutionary conserved protein that serves as a GTPase Activating Protein (GAP) for Ras. Among Drosophila melanogaster Nf1 (dNf1) null mutant phenotypes, learning/memory deficits and reduced overall growth resemble human NF1 symptoms. These and other dNf1 defects are relatively insensitive to manipulations that reduce Ras signaling strength but are suppressed by increasing signaling through the 3′-5′ cyclic adenosine monophosphate (cAMP) dependent Protein Kinase A (PKA) pathway, or phenocopied by inhibiting this pathway. However, whether dNf1 affects cAMP/PKA signaling directly or indirectly remains controversial. To shed light on this issue we screened 486 1st and 2nd chromosome deficiencies that uncover >80% of annotated genes for dominant modifiers of the dNf1 pupal size defect, identifying responsible genes in crosses with mutant alleles or by tissue-specific RNA interference (RNAi) knockdown. Validating the screen, identified suppressors include the previously implicated dAlk tyrosine kinase, its activating ligand jelly belly (jeb), two other genes involved in Ras/ERK signal transduction and several involved in cAMP/PKA signaling. Novel modifiers that implicate synaptic defects in the dNf1 growth deficiency include the intersectin-related synaptic scaffold protein Dap160 and the cholecystokinin receptor-related CCKLR-17D1 drosulfakinin receptor. Providing mechanistic clues, we show that dAlk, jeb and CCKLR-17D1 are among mutants that also suppress a recently identified dNf1 neuromuscular junction (NMJ) overgrowth phenotype and that manipulations that increase cAMP/PKA signaling in adipokinetic hormone (AKH)-producing cells at the base of the neuroendocrine ring gland restore the dNf1 growth deficiency. Finally, supporting our previous contention that ALK might be a therapeutic target in NF1, we report that human ALK is expressed in cells that give rise

  8. Effects of fucoidan on insulin stimulation and pancreatic protection via the cAMP signaling pathway in vivo and in vitro.

    PubMed

    Jiang, Xiaoming; Yu, Jinfeng; Ma, Zhi; Zhang, Hong; Xie, Fengjie

    2015-09-01

    Diabetes is a global disease, in which pancreatic dysfunction is an important pathological process. In previous years, interest in the biological activities of seaweed has increased. Fucoidan is an extract of the seaweed Fucus vesiculosus, which has been widely investigated. The present study aimed to determine the effects of fucoidan on insulin stimulation and pancreatic protection in vivo and in vitro. Goto‑Kakizaki (GK) rats were provided with free access to standard food, with or without fucoidan, for 13 weeks, following which the body weights, and blood glucose and serum insulin levels of the rats were measured. Wistar rats were used as a control. In addition, the RIN‑5F rat insulin‑secreting cell line was treated with fucoidan in high glucose conditions, following which the dose‑dependent and time‑dependent effects of fucoidan were determined, and the concentration of insulin was measured. Glybenclamide was used as a positive control. In vivo, the body weight and serum insulin levels decreased, whereas blood glucose levels increased significantly in the GK rats, compared with the Wistar control rats. Although, fucoidan did not improve changes in body weight, the increased blood glucose levels were reduced and the decreased serum insulin levels were increased in the GK rats following oral administration of fucoidan. In vitro, fucoidan did not exhibit significant cytotoxicity towards the RIN‑5F cells, and the insulin secretion increased significantly in a dose‑ and time‑dependent manner. Treatment with amylin, an islet amyloid polypeptide and glybenclamide inhibitor, did not inhibit the stimulatory activity of fucoidan. The results of the present study also demonstrated that the concentration of cyclic adenosine monophosphate (cAMP) was significantly increased in the fucoidan‑treated RIN‑5F cells, and this increase was dose‑ and time‑dependent. In addition, treatment with a phosphodiesterase inhibitor, which decreases the degradation of

  9. Rat cardiac myocyte adenosine transport and metabolism

    SciTech Connect

    Ford, D.A.; Rovetto, M.J.

    1987-01-01

    Based on the importance of myocardial adenosine and adenine nucleotide metabolism, the adenosine salvage pathway in ventricular myocytes was studied. Accurate estimates of transport rates, separate from metabolic fllux, were determined. Adenosine influx was constant between 3 and 60 s. Adenosine metabolism maintained intracellular adenosine concentrations < 10% of the extracellular adenosine concentrations and thus unidirectional influx could be measured. Myocytes transported adenosine via saturable and nonsaturable processes. A minimum estimate of the V/sub max/ of myocytic adenosine kinase indicated the saturable component of adenosine influx was independent of adenosine kinase activity. Saturable transport was inhibited by nitrobenzylthioinosine and verapamil. Extracellular adenosine taken up myocytes was rapidly phosphorylated to adenine taken up by myocytes was rapidly phosphorylated to adenine nucleotides. Not all extracellular adenosine, though, was phosphorylated on entering myocytes, since free, as opposed to protein-bound, intracellular adenosine was detected after digitonin extraction of cells in the presence of 1 mM ethylene-diaminetetraacetic acid.

  10. Central or peripheral delivery of an adenosine A1 receptor agonist improves mechanical allodynia in a mouse model of painful diabetic neuropathy.

    PubMed

    Katz, N K; Ryals, J M; Wright, D E

    2015-01-29

    Diabetic peripheral neuropathy is a common complication of diabetes mellitus, and a significant proportion of individuals suffer debilitating pain that significantly affects their quality of life. Unfortunately, symptomatic treatment options have limited efficacy, and often carry significant risk of systemic adverse effects. Activation of the adenosine A1 receptor (A1R) by the analgesic small molecule adenosine has been shown to have antinociceptive benefits in models of inflammatory and neuropathic pain. The current study used a mouse model of painful diabetic neuropathy to determine the effect of diabetes on endogenous adenosine production, and if central or peripheral delivery of adenosine receptor agonists could alleviate signs of mechanical allodynia in diabetic mice. Diabetes was induced using streptozocin in male A/J mice. Mechanical withdrawal thresholds were measured weekly to characterize neuropathy phenotype. Hydrolysis of AMP into adenosine by ectonucleotidases was determined in the dorsal root ganglia (DRG) and spinal cord at 8 weeks post-induction of diabetes. AMP, adenosine and the specific A1R agonist, N(6)-cyclopentyladenosine (CPA), were administered both centrally (intrathecal) and peripherally (intraplantar) to determine the effect of activation of adenosine receptors on mechanical allodynia in diabetic mice. Eight weeks post-induction, diabetic mice displayed significantly decreased hydrolysis of extracellular AMP in the DRG; at this same time, diabetic mice displayed significantly decreased mechanical withdrawal thresholds compared to nondiabetic controls. Central delivery AMP, adenosine and CPA significantly improved mechanical withdrawal thresholds in diabetic mice. Surprisingly, peripheral delivery of CPA also improved mechanical allodynia in diabetic mice. This study provides new evidence that diabetes significantly affects endogenous AMP hydrolysis, suggesting that altered adenosine production could contribute to the development of

  11. Structure of Staphylococcus aureus cytidine monophosphate kinase in complex with cytidine 5'-monophosphate.

    PubMed

    Dhaliwal, Balvinder; Ren, Jingshan; Lockyer, Michael; Charles, Ian; Hawkins, Alastair R; Stammers, David K

    2006-08-01

    The crystal structure of Staphylococcus aureus cytidine monophosphate kinase (CMK) in complex with cytidine 5'-monophosphate (CMP) has been determined at 2.3 angstroms resolution. The active site reveals novel features when compared with two orthologues of known structure. Compared with the Streptococcus pneumoniae CMK solution structure of the enzyme alone, S. aureus CMK adopts a more closed conformation, with the NMP-binding domain rotating by approximately 16 degrees towards the central pocket of the molecule, thereby assembling the active site. Comparing Escherichia coli and S. aureus CMK-CMP complex structures reveals differences within the active site, including a previously unreported indirect interaction of CMP with Asp33, the replacement of a serine residue involved in the binding of CDP by Ala12 in S. aureus CMK and an additional sulfate ion in the E. coli CMK active site. The detailed understanding of the stereochemistry of CMP binding to CMK will assist in the design of novel inhibitors of the enzyme. Inhibitors are required to treat the widespread hospital infection methicillin-resistant S. aureus (MRSA), currently a major public health concern.

  12. Inhibition of Platelet Activation and Thrombus Formation by Adenosine and Inosine: Studies on Their Relative Contribution and Molecular Modeling

    PubMed Central

    Fuentes, Eduardo; Pereira, Jaime; Mezzano, Diego; Alarcón, Marcelo; Caballero, Julio; Palomo, Iván

    2014-01-01

    Background The inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine. Objectives The mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated. Results Adenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [n = 6, p<0.01] and 72±1.9% occlusion at 60 min, [n = 6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, n = 6). A2A is the adenosine receptor present in platelets; it is known that inosine is not an A2A ligand. Docking of adenosine and inosine inside A2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH2 of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A2A receptor. Conclusion Therefore

  13. 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.

  14. 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.

  15. Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay.

    PubMed

    Pacini, Stefania; Morucci, Gabriele; Punzi, Tiziana; Gulisano, Massimo; Ruggiero, Marco

    2011-04-01

    The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory effect of GcMAF on PBMCs. In addition, we demonstrate that GcMAF (1 ng/ml) inhibited prostaglandin E(1)- and human breast cancer cell-stimulated angiogenesis in chick embryo chorionallantoic membrane (CAM) assay. Finally, we tested different GcMAF preparations on CAM, and the assay proved to be a reliable, reproducible and inexpensive method to determine the relative potencies of different preparations and their stability; we observed that storage at room temperature for 15 days decreased GcMAF potency by about 50%. These data could prove useful for upcoming clinical trials on GcMAF.

  16. Kinetic mechanism of Toxoplasma gondii adenosine kinase and the highly efficient utilization of adenosine.

    PubMed

    Naguib, Fardos N M; Rais, Reem H; Al Safarjalani, Omar N; el Kouni, Mahmoud H

    2015-10-01

    Initial velocity and product inhibition studies of Toxoplasma gondii adenosine kinase (TgAK, EC 2.7.1.20) demonstrated that the basic mechanism of this enzyme is a hybrid random bi-uni ping-pong uni-bi. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation and a binding pattern indicating that binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KAdo=0.002±0.0002 mM, KATP=0.05±0.008 mM, and Vmax=920±35 μmol/min/mg protein. Ado exhibited substrate inhibition suggesting the presence of more than one binding site for Ado on the enzyme. ATP relieved substrate inhibition by Ado. Thus, Ado also binds to the ATP binding site. AMP was competitive with ATP, inferring that AMP binds to the same site as ATP. AMP, ADP and ATP were non-competitive with Ado, therefore, none of these nucleotides binds to the Ado binding site. Combining ATP with ADP was additive. Therefore, the binding of either ATP or ADP does not interfere with the binding of the other. It is concluded that for every ATP consumed, TgAK generates three new AMPs. These findings along with the fact that a wide range of nucleoside 5'-mono, di, and triphosphates could substitute for ATP as phosphate donors in this reaction may explain the efficient and central role played by TgAK in the utilization of Ado as the major source from which all other purines can be synthesized in T. gondii.

  17. The A2B adenosine receptor promotes Th17 differentiation via stimulation of dendritic cell IL-6.

    PubMed

    Wilson, Jeffrey M; Kurtz, Courtney C; Black, Steven G; Ross, William G; Alam, Mohammed S; Linden, Joel; Ernst, Peter B

    2011-06-15

    Adenosine is an endogenous metabolite produced during hypoxia or inflammation. Previously implicated as an anti-inflammatory mediator in CD4(+) T cell regulation, we report that adenosine acts via dendritic cell (DC) A(2B) adenosine receptor (A(2B)AR) to promote the development of Th17 cells. Mouse naive CD4(+) T cells cocultured with DCs in the presence of adenosine or the stable adenosine mimetic 5'-(N-ethylcarboximado) adenosine resulted in the differentiation of IL-17- and IL-22-secreting cells and elevation of mRNA that encode signature Th17-associated molecules, such as IL-23R and RORγt. The observed response was similar when DCs were generated from bone marrow or isolated from small intestine lamina propria. Experiments using adenosine receptor antagonists and cells from A(2B)AR(-/-) or A(2A)AR(-/-)/A(2B)AR(-/-) mice indicated that the DC A(2B)AR promoted the effect. IL-6, stimulated in a cAMP-independent manner, is an important mediator in this pathway. Hence, in addition to previously noted direct effects of adenosine receptors on regulatory T cell development and function, these data indicated that adenosine also acts indirectly to modulate CD4(+) T cell differentiation and suggested a mechanism for putative proinflammatory effects of A(2B)AR.

  18. Thyroid expression of an A2 adenosine receptor transgene induces thyroid hyperplasia and hyperthyroidism.

    PubMed Central

    Ledent, C; Dumont, J E; Vassart, G; Parmentier, M

    1992-01-01

    Cyclic AMP (cAMP) is the major intracellular second messenger of thyrotropin (TSH) action on thyroid cells. It stimulates growth as well as the function and differentiation of cultured thyrocytes. The adenosine A2 receptor, which activates adenylyl cyclase via coupling to the stimulating G protein (Gs), has been shown to promote constitutive activation of the cAMP cascade when transfected into various cell types. In order to test whether the A2 receptor was able to function similarly in vivo and to investigate the possible consequences of permanent adenylyl cyclase activation in thyroid cells, lines of transgenic mice were generated expressing the canine A2 adenosine receptor under control of the bovine thyroglobulin gene promoter. Thyroid-specific expression of the A2 adenosine receptor transgene promoted gland hyperplasia and severe hyperthyroidism causing premature death of the animals. The resulting goitre represents a model of hyperfunctioning adenomas: it demonstrates that constitutive activation of the cAMP cascade in such differentiated epithelial cells is sufficient to stimulate autonomous and uncontrolled function and growth. Images PMID:1371462

  19. Cocaine exposure modulates dopamine and adenosine signaling in the fetal brain

    PubMed Central

    Kubrusly, Regina C. C.; Bhide, Pradeep G.

    2009-01-01

    Exposure to cocaine during the fetal period can produce significant lasting changes in the structure and function of the brain. Cocaine exerts its effects on the developing brain by blocking monoamine transporters and impairing monoamine receptor signaling. Dopamine is a major central target of cocaine. In a mouse model, we show that cocaine exposure from embryonic day 8 (E8) to E14 produces significant reduction in dopamine transporter activity, attenuation of dopamine D1-receptor function and upregulation of dopamine D2-receptor function. Cocaine’s effects on the D1-receptor are at the level of protein expression as well as activity. The cocaine exposure also produces significant increases in basal cAMP levels in the striatum and cerebral cortex. The increase in the basal cAMP levels was independent of dopamine receptor activity. In contrast, blocking the adenosine A2a receptor downregulated of the basal cAMP levels in the cocaine-exposed brain to physiological levels, suggesting the involvement of adenosine receptors in mediating cocaine’s effects on the embryonic brain. In support of this suggestion, we found that the cocaine exposure downregulated adenosine transporter function. We also found that dopamine D2- and adenosine A2a-receptors antagonize each other’s function in the embryonic brain in a manner consistent with their interactions in the mature brain. Thus, our data show that prenatal cocaine exposure produces direct effects on both the dopamine and adenosine systems. Furthermore, the dopamine D2 and adenosine A2a receptor interactions in the embryonic brain discovered in this study unveil a novel substrate for cocaine’s effects on the developing brain. PMID:19765599

  20. [Interaction of adenosin-3',5'-cyclosulfate with adenosine-3'5'-cyclophosphate dependent protein kinase and phosphodiesterase].

    PubMed

    Severin, E S; Tkachuk, V A; Guliaev, N N

    1976-02-01

    Interaction of adenosine-3',5'-cyclosulphate (cAMS) cAMP analogue, having sulphur atom instead of phosphorus in a six-term cyclic system with pig brain proteinkinase and rabbit skeletal muscle phosphodiesterase is studied. The affinity of proteinkinase to cAMS was found to be in 25000 times lower than the affinity of cAMP, the affinity of cAMS to the active site of phosphodiesterase being high enough. It is suggested that in the regulatory subunit of proteinkinase positive kationic group participates in nucleotide binding by interacting with negative oxygen atom of six-term cyclophosphate system. There is no such a group in the active site of phospodiesterase, because the absence of negative charge in case of cAMS only slightly affects the constant of cAMS binding by phosphodiesterase.

  1. Antioxidant and Anti-tyrosinase Activities of Phenolic Extracts from Rape Bee Pollen and Inhibitory Melanogenesis by cAMP/MITF/TYR Pathway in B16 Mouse Melanoma Cells

    PubMed Central

    Sun, Liping; Guo, Yan; Zhang, Yanxin; Zhuang, Yongliang

    2017-01-01

    Rape bee pollen possesses many nutritional and therapeutic properties because of its abundant nutrimental and bioactive components. In this study, free (FPE) and bound (BPE) phenolic extracts of rape bee pollen were obtained, phenolic and flavonoid contents were determined, and composition of phenolic acids was analyzed. In vitro antioxidant and anti-tyrosinase (TYR) activities of FPE and BPE were compared, and inhibitory melanogenesis of FPE was further evaluated. Results showed FPE and BPE contain total phenolic contents of 11.76 and 0.81 mg gallic acid equivalents/g dry weight (DW) and total flavonoid contents of 19.24 and 3.65 mg rutin equivalents/g DW, respectively. Phenolic profiling showed FPE and BPE fractions contained 12 and 9 phenolic acids, respectively. FPE contained the highest rutin content of 774.87 μg/g. FPE and BPE showed the high antioxidant properties in vitro and high inhibitory activities for mushroom TYR. Higher activities of FPE than those of BPE can be attributed to difference in their phenolic compositions. Inhibitory melanogenesis activities of FPE against B16 were further evaluated. Results showed suppressed intracellular TYR activity, reduced melanin content, and promoted glutathione synthesis (p < 0.05) in FPE-treated cells. FPE reduced mRNA expression of TYR, TYR-related protein (TRP)-1 and TRP-2, and significantly suppressed cyclic adenosine monophosphate (cAMP) levels through down-regulation of melanocortin 1 receptor gene expression (p < 0.05). FPE reduced mRNA expression of microphthalmia-associated transcription factor (MITF), significantly inhibiting intracellular melanin synthesis (p < 0.05). Hence, FPE regulates melanogenesis of B16 cells involved in cAMP/MITF/TYR pathway. These results revealed that FPE can be used as pharmaceutical agents and cosmetics to protect cells from abnormal melanogenesis. PMID:28337140

  2. Lysosomal alkalization and dysfunction in human fibroblasts with the Alzheimer's disease-linked presenilin 1 A246E mutation can be reversed with cAMP.

    PubMed

    Coffey, E E; Beckel, J M; Laties, A M; Mitchell, C H

    2014-03-28

    Mutation in presenilin 1 (PS1) is one of the leading causes of familial Alzheimer's disease (fAD). PS1 mutation exacerbates the autophagic and lysosomal pathology in AD patients, leading to accumulation of partially degraded material in bloated lysosomes and autophagosomes - a pathology that bears some resemblance to other diseases characterized by elevated lysosomal pH, like age-related macular degeneration. In this study, we examined the effect of the PS1-fAD mutation A246E on lysosomal pH and lysosomal function, and asked whether restoration of lysosomal pH could reverse some of these changes. Lysosomal pH was elevated by 0.2-0.3 pH units in human fibroblasts with the PS1-fAD mutation. The lysosomal alkalization in PS1-fAD fibroblasts was supported by a reduction in the pH-dependent cleavage of cathepsin D and by a reduction in binding of boron-dipyrromethene (BODIPY) FL-pepstatin A to the cathepsin D active site. PS1-fAD cells had increased LC3B-II/-I ratios and p62 levels, consistent with impaired lysosomal degradation and analogous to changes induced by lysosomal alkalinization with chloroquine. PS1-fAD fibroblasts had increased expression of ATP6V1B2, ATG5, BECN1 TFEB mRNA, and of ATP6V1B2, ATG5 and beclin at the protein level, consistent with chronic impairment of autophagic and lysosomal functions in the mutant cells. Critically, cyclic adenosine monophosphate (cAMP) treatment reacidified lysosomal pH in mutant PS1-fAD; cAMP also increased the availability of active cathepsin D and lowered the LC3B-II/-I ratio. These results confirm a small elevation in the lysosomal pH of human PS1-fAD fibroblasts, demonstrate that this lysosomal alkalization is associated with chronic changes in autophagy and degradation, and suggest that treatment to reacidify the lysosomes with cAMP can reverse these changes.

  3. Uptake of intact nucleoside monophosphates by Bdellovibrio bacteriovorus 109J.

    PubMed Central

    Ruby, E G; McCabe, J B; Barke, J I

    1985-01-01

    The degraded nucleic acids and ribosomes of its prey cell provide Bdellovibrio bacteriovorus 109J with a source of ribonucleoside monophosphates and deoxyribonucleoside monophosphates for biosynthesis and respiration. We demonstrate that bdellovibrios, in contrast to almost all other bacteria, take up these nucleoside monophosphates into the cell in an intact, phosphorylated form. In this way they are able to assimilate more effectively the cellular contents of their prey. Studies with UMP and dTMP demonstrate that they are transported and accumulated against a concentration gradient, achieving internal levels at least 10 times the external levels. Treatment of the bdellovibrios with azide or carbonyl cyanide m-chlorophenylhydrazone eliminates their ability to either transport or maintain accumulated UMP and suggests the presence of a freely reversible exchange mechanism. There are at least two separate classes of transport systems for nucleoside monophosphates, each exhibiting partial specificity for either ribonucleoside monophosphates or deoxyribonucleoside monophosphates. Kinetic analyses of UMP transport in different developmental stages of strain 109J indicate that each stage expresses a single, saturable uptake system with a distinct apparent substrate affinity constant (Kt) of 104 microM in attack phase cells and 35 microM in prematurely released growth phase filaments. The capacity for transport of UMP by the growth phase filaments was 2.4 times that of the attack phase cells. These data, in addition to the apparent lack of environmental control of UMP transport capacity in attack phase cells, suggest that there are two transport systems for UMP in bdellovibrios and that the high-affinity, high-capacity growth phase system is developmentally regulated. PMID:4030692

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

    PubMed Central

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

    2014-01-01

    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

  5. Regulation of histamine- and UTP-induced increases in Ins(1,4,5)P3, Ins (1,3,4,5)P4 and Ca2+ by cyclic AMP in DDT1 MF-2 cells.