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Sample records for adenosine receptor antagonism

  1. Antagonism by theophylline of respiratory inhibition induced by adenosine.

    PubMed

    Eldridge, F L; Millhorn, D E; Kiley, J P

    1985-11-01

    The effects on respiration of an analogue of adenosine, L-2-N6-(phenylisopropyl)adenosine (PIA), and of the methylxanthine, theophylline, were determined in 19 vagotomized glomectomized cats whose end-tidal PCO2 was kept constant by means of a servo-controlled ventilator. Integrated phrenic nerve activity was used to represent respiratory output. Our results show that PIA, whether given systemically or into the third cerebral ventricle, depressed respiration. Systemically administered theophylline stimulated respiration. Theophylline given intravenously, or into the third ventricle not only reversed the depressive effects of previously administered PIA but caused further increases of respiration above the control level. Prior systemic administration of theophylline blocked both respiratory and hypotensive effects of subsequently administered PIA. Effects of either agent on medullary extracellular fluid pH did not explain the results. We conclude that the adenosine analogue PIA, acts to inhibit neurons in the brain that are involved in the control of respiration and that its effects are blocked by theophylline. We suggest that adenosine acts as a tonic modulator of respiration and that theophylline stimulates breathing by competitive antagonism of adenosine at neuronal receptor sites. PMID:4066573

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

    PubMed Central

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

    2004-01-01

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

  3. Role of adenosine receptors in caffeine tolerance

    SciTech Connect

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

    1991-01-01

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

  4. Adenosine receptor interactions and anxiolytics.

    PubMed

    Bruns, R F; Katims, J J; Annau, Z; Snyder, S H; Daly, J W

    1983-12-01

    [3H]-N6-cyclohexyladenosine and [3H]-1,3-diethyl-8-phenylxanthine label the A1 subtype of adenosine receptor in brain membranes. The affinities of methylxanthines in competing for A1 adenosine receptors parallel their potencies as locomotor stimulants. The adenosine agonist N6-(phenylisopropyl) adenosine is a potent locomotor depressant. Both diazepam and N6-(L-phenylisopropyl)adenosine cause locomotor stimulation in a narrow range of subdepressant doses. Combined stimulant doses of the two agents depress motor activity, as do larger doses of either one, given separately. Evidence supporting and against the hypothesis that some of the actions of benzodiazepines are mediated via the adenosine system is reviewed. A number of compounds interact with both systems, probably because of physico-chemical similarities between adenosine and diazepam. It is concluded that of the four classic actions of benzodiazepines, the sedative and muscle relaxant (but not anxiolytic or anticonvulsant) actions could possibly be mediated by adenosine. PMID:6199685

  5. Adenosine receptor desensitization and trafficking.

    PubMed

    Mundell, Stuart; Kelly, Eamonn

    2011-05-01

    As with the majority of G-protein-coupled receptors, all four of the adenosine receptor subtypes are known to undergo agonist-induced regulation in the form of desensitization and trafficking. These processes can limit the ability of adenosine receptors to couple to intracellular signalling pathways and thus reduce the ability of adenosine receptor agonists as well as endogenous adenosine to produce cellular responses. In addition, since adenosine receptors couple to multiple signalling pathways, these pathways may desensitize differentially, while the desensitization of one pathway could even trigger signalling via another. Thus, the overall picture of adenosine receptor regulation can be complex. For all adenosine receptor subtypes, there is evidence to implicate arrestins in agonist-induced desensitization and trafficking, but there is also evidence for other possible forms of regulation, including second messenger-dependent kinase regulation, heterologous effects involving G proteins, and the involvement of non-clathrin trafficking pathways such as caveolae. In this review, the evidence implicating these mechanisms is summarized for each adenosine receptor subtype, and we also discuss those issues of adenosine receptor regulation that remain to be resolved as well as likely directions for future research in this field. PMID:20550943

  6. Antagonism of the adenosine A2A receptor attenuates akathisia-like behavior induced with MP-10 or aripiprazole in a novel non-human primate model.

    PubMed

    Bleickardt, Carina J; Kazdoba, Tatiana M; Jones, Nicholas T; Hunter, John C; Hodgson, Robert A

    2014-03-01

    Akathisia is a subset of the larger antipsychotic side effect profile known as extrapyramidal syndrome (EPS). It is associated with antipsychotic treatment and is characterized as a feeling of inner restlessness that results in a compulsion to move. There are currently no primate models available to assess drug-induced akathisia; the present research was designed to address this shortcoming. We developed a novel rating scale based on both the Barnes Akathisia Rating Scale (BARS) and the Hillside Akathisia Scale (HAS) to measure the objective, observable incidence of antipsychotic-induced akathisia-like behavior in Cebus apella non-human primates (NHPs). To induce akathisia, we administered the atypical antipsychotic aripiprazole (1 mg/kg) or the selective phosphodiesterase 10A (PDE10A) inhibitor MP-10 (1-3 mg/kg). Treatment with both compounds produced significantly greater akathisia scores on the rating scale than vehicle treatment. Characteristic behaviors observed included vocalizations, stereotypies, teeth grinding, restless limb movements, and hyperlocomotion. Adenosine A2A receptor antagonists have previously been shown to be effective in blocking antipsychotic-induced EPS in primates. The selective A2A receptor antagonist, SCH 412348 (10-30 mg/kg), effectively reduced or reversed akathisia-like behavior induced by both aripiprazole and MP-10. This work represents the first NHP measurement scale of akathisia and demonstrates that NHPs are responsive to akathisia-inducing agents. As such, it provides a useful tool for the preclinical assessment of putative antipsychotics. In addition, these results provide further evidence of the utility of A2A receptor antagonists for the treatment of antipsychotic-induced movement disorders. PMID:24211858

  7. Adenosine Receptors and Membrane Microdomains

    PubMed Central

    Lasley, Robert D.

    2010-01-01

    Adenosine receptors are a member of the large family of seven transmembrane spanning G protein coupled receptors (GPCR). The four adenosine receptor subtypes – A1, A2a, A2b, A3 – exert their effects via the activation of one or more heterotrimeric G proteins resulting in the modulation of intracellular signaling. Numerous studies over the past decade have documented the complexity of GPCR signaling at the level of protein-protein interactions as well as through signaling crosstalk. With respect to adenosine receptors the activation of one receptor subtype can have profound direct effects in one cell type, but little or no effect in other cells. There is significant evidence that the compartmentation of subcellular signaling plays a physiological role in the fidelity of GPCR signaling. This compartmentation is evident at the level of the plasma membrane in the form of membrane microdomains such as caveolae and lipid rafts. This review will summarize and critically assess our current understanding of the role of membrane microdomains in regulating adenosine receptor signaling. PMID:20888790

  8. Molecular vibration-activity relationship in the agonism of adenosine receptors.

    PubMed

    Chee, Hyun Keun; Oh, S June

    2013-12-01

    The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibration patterns showed its potential for the functional classification of adenosine receptor ligands. PMID:24465242

  9. Molecular Vibration-Activity Relationship in the Agonism of Adenosine Receptors

    PubMed Central

    Chee, Hyun Keun

    2013-01-01

    The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibration patterns showed its potential for the functional classification of adenosine receptor ligands. PMID:24465242

  10. Xanthines as Adenosine Receptor Antagonists

    PubMed Central

    Jacobson, Kenneth A.

    2013-01-01

    The natural plant alkaloids caffeine and theophylline were the first adenosine receptor (AR) antagonists described in the literature. They exhibit micromolar affinities and are non-selective. A large number of derivatives and analogs have subsequently been synthesized and evaluated as AR antagonists. Very potent antagonists have thus been developed with selectivity for each of the four AR subtypes. PMID:20859796

  11. Adenosine kinase inhibitors attenuate opiate withdrawal via adenosine receptor activation.

    PubMed

    Kaplan, G B; Coyle, T S

    1998-11-27

    Previous studies have demonstrated a role for adenosine in mediating opiate effects. This study examines the effects of indirect activation of adenosine receptors, via treatment with adenosine kinase inhibitors, on the expression of opiate withdrawal in mice. Mice receive chronic morphine treatment via implantation of subcutaneous morphine pellets (75 mg) for 72 h. Mice then receive parenteral treatment with adenosine kinase inhibitors, either 5'-amino-5'-deoxyadenosine (2, 5, 20, 40 mg/kg, intraperitoneal or i.p.) or iodotubericidin (1, 2, 5 mg/kg, i.p.), followed by naloxone injection and opiate withdrawal signs are measured over 20 min. Both adenosine kinase inhibitors significantly reduce the following opiate withdrawal signs in a dose-dependent manner compared to vehicle: withdrawal jumps, teeth chattering, forepaw tremors, and forepaw treads. Additionally, 5'-amino-5'-deoxyadenosine significantly reduces withdrawal-induced diarrhea and weight loss. Effects of 5'-amino-5'-deoxyadenosine (40 mg/kg) on opiate withdrawal signs appear to be mediated via adenosine receptor activation as they are reversed by pretreatment by adenosine receptor antagonist caffeine (20 mg, i.p.) but not by selective phosphodiesterase inhibitor Ro 20-1724 (10 mg/kg, i.p.). Adenosine receptor activation via adenosine kinase inhibitor treatment attenuates opiate withdrawal and these agents may be generally useful in the treatment of drug withdrawal syndromes. PMID:9865523

  12. Fluorescent Ligands for Adenosine Receptors

    PubMed Central

    Kozma, Eszter; Jayasekara, P Suresh; Squarcialupi, Lucia; Paoletta, Silvia; Moro, Stefano; Federico, Stephanie; Spalluto, Giampiero; Jacobson, Kenneth A.

    2012-01-01

    Interest is increasing in developing fluorescent ligands for characterization of adenosine receptors (ARs), which hold a promise of usefulness in the drug discovery process. The size of a strategically labeled AR ligand can be greatly increased after the attachment of a fluorophore. The choice of dye moiety (e.g. Alexa Fluor 488), attachment point and linker length can alter the selectivity and potency of the parent molecule. Fluorescent derivatives of adenosine agonists and antagonists (e.g. XAC and other heterocyclic antagonist scaffolds) have been synthesized and characterized pharmacologically. Some are useful AR probes for flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer, and scanning confocal microscopy. Thus, the approach of fluorescent labeled GPCR ligands, including those for ARs, is a growing dynamic research field. PMID:23200243

  13. Identification and mechanism of ABA receptor antagonism

    SciTech Connect

    Melcher, Karsten; Xu, Yong; Ng, Ley-Moy; Zhou, X. Edward; Soon, Fen-Fen; Chinnusamy, Viswanathan; Suino-Powell, Kelly M; Kovach, Amanda; Tham, Fook S.; Cutler, Sean R.; Li, Jun; Yong, Eu-Leong; Zhu, Jian-Kang; Xu, H. Eric

    2010-11-11

    The phytohormone abscisic acid (ABA) functions through a family of fourteen PYR/PYL receptors, which were identified by resistance to pyrabactin, a synthetic inhibitor of seed germination. ABA activates these receptors to inhibit type 2C protein phosphatases, such as ABI1, yet it remains unclear whether these receptors can be antagonized. Here we demonstrate that pyrabactin is an agonist of PYR1 and PYL1 but is unexpectedly an antagonist of PYL2. Crystal structures of the PYL2-pyrabactin and PYL1-pyrabactin-ABI1 complexes reveal the mechanism responsible for receptor-selective activation and inhibition, which enables us to design mutations that convert PYL1 to a pyrabactin-inhibited receptor and PYL2 to a pyrabactin-activated receptor and to identify new pyrabactin-based ABA receptor agonists. Together, our results establish a new concept of ABA receptor antagonism, illustrate its underlying mechanisms and provide a rational framework for discovering novel ABA receptor ligands.

  14. Photoaffinity labeling of A1-adenosine receptors

    SciTech Connect

    Klotz, K.N.; Cristalli, G.; Grifantini, M.; Vittori, S.; Lohse, M.J.

    1985-11-25

    The ligand-binding subunit of the A1-adenosine receptor has been identified by photoaffinity labeling. A photolabile derivative of R-N6-phenylisopropyladenosine, R-2-azido-N6-p-hydroxyphenylisopropyladenosine (R-AHPIA), has been synthesized as a covalent specific ligand for A1-adenosine receptors. In adenylate cyclase studies with membranes of rat fat cells and human platelets, R-AHPIA has adenosine receptor agonist activity with a more than 60-fold selectivity for the A1-subtype. It competes for (TH)N6-phenylisopropyladenosine binding to A1-receptors of rat brain membranes with a Ki value of 1.6 nM. After UV irradiation, R-AHPIA binds irreversibly to the receptor, as indicated by a loss of (TH)N6-phenylisopropyladenosine binding after extensive washing; the Ki value for this photoinactivation is 1.3 nM. The p-hydroxyphenyl substituent of R-AHPIA can be directly radioiodinated to give a photoaffinity label of high specific radioactivity ( SVI-AHPIA). This compound has a KD value of about 1.5 nM as assessed from saturation and kinetic experiments. Adenosine analogues compete for SVI-AHPIA binding to rat brain membranes with an order of potency characteristic for A1-adenosine receptors. Dissociation curves following UV irradiation at equilibrium demonstrate 30-40% irreversible specific binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the probe is photoincorporated into a single peptide of Mr = 35,000. Labeling of this peptide can be blocked specifically and stereoselectively by adenosine receptor agonists and antagonists in a manner which is typical for the A1-subtype. The results indicate that SVI-AHPIA identifies the ligand-binding subunit of the A1-adenosine receptor, which is a peptide with Mr = 35,000.

  15. Improvement of Cold Tolerance by Selective A1 Adenosine Receptor Antagonists in Rats

    PubMed Central

    Lee, T. F.; Li, D. J.; Jacobson, K. A.; Wang, L. C. H.

    2015-01-01

    Previously we have shown that the improvement of cold tolerance by theophylline is due to antagonism at adenosine receptors rather than inhibition of phosphodiesterase. Since theophylline is a nonselective adenosine receptor antagonist for both A1 and A2 receptors, the present study investigated the adenosine receptor subtype involved in theophylline’s action. Acute systemic injection of selective A1 receptor antagonists (1,3-dialkyl-8-aryl or 1,3-dialkyl-8-cyclopentyl xanthine derivatives) significantly increased both the total and maximal heat production as well as cold tolerance. In contrast, injection of a relatively selective A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (compound No. 19), failed to significantly alter the thermogenic response of the rat under cold exposure. Further, the relative effectiveness of these compounds in increasing total thermogenesis was positively correlated with their potency in blocking the A1 adenosine receptor (r= .52, p<0.01), but not in A2 adenosine receptor (r= .20, p<0.2). It is likely that the thermally beneficial effects of adenosine A1 antagonists are due to their attenuation of the inhibitory effects of endogenously released adenosine on lipolysis and glucose utilization, resulting in increased substrate mobilization and utilization for enhanced thermogenesis. PMID:2263650

  16. Internalization and desensitization of adenosine receptors

    PubMed Central

    Klaasse, Elisabeth C.; de Grip, Willem J.; Beukers, Margot W.

    2007-01-01

    Until now, more than 800 distinct G protein-coupled receptors (GPCRs) have been identified in the human genome. The four subtypes of the adenosine receptor (A1, A2A, A2B and A3 receptor) belong to this large family of GPCRs that represent the most widely targeted pharmacological protein class. Since adenosine receptors are widespread throughout the body and involved in a variety of physiological processes and diseases, there is great interest in understanding how the different subtypes are regulated, as a basis for designing therapeutic drugs that either avoid or make use of this regulation. The major GPCR regulatory pathway involves phosphorylation of activated receptors by G protein-coupled receptor kinases (GRKs), a process that is followed by binding of arrestin proteins. This prevents receptors from activating downstream heterotrimeric G protein pathways, but at the same time allows activation of arrestin-dependent signalling pathways. Upon agonist treatment, adenosine receptor subtypes are differently regulated. For instance, the A1Rs are not (readily) phosphorylated and internalize slowly, showing a typical half-life of several hours, whereas the A2AR and A2BR undergo much faster downregulation, usually shorter than 1 h. The A3R is subject to even faster downregulation, often a matter of minutes. The fast desensitization of the A3R after agonist exposure may be therapeutically equivalent to antagonist occupancy of the receptor. This review describes the process of desensitization and internalization of the different adenosine subtypes in cell systems, tissues and in vivo studies. In addition, molecular mechanisms involved in adenosine receptor desensitization are discussed. PMID:18368531

  17. Adenosine receptor agonists attenuate and adenosine receptor antagonists exacerbate opiate withdrawal signs.

    PubMed

    Kaplan, G B; Sears, M T

    1996-01-01

    Previous studies have demonstrated a role for adenosine in mediating opiate effects. Adenosine receptors and their functions have been shown to be regulated by chronic opiate treatment. This study examines the role of adenosine receptors in the expression of opiate withdrawal behaviors. The effects of single doses of parenterally administered adenosine receptor subtype-selective agonists and antagonists on opiate withdrawal signs in morphine-dependent mice were measured. Mice received subcutaneous morphine pellet treatment for 72 h and then underwent naloxone-precipitated withdrawal after pretreatment with adenosinergic agents. Adenosine agonists attenuated different opiate withdrawal signs. The A1 agonist R-N6(phenylisopropyl)adenosine (0, 0.01, 0.02 mg/kg, IP) significantly reduced wet dog shakes and withdrawal diarrhea, while the A2a-selective agonist 2-p-(2-carboxethyl)phenylethylamino-5'-N-ethylcarboxamido adenosine or CGS 21680 (0, 0.01, 0.05 mg/kg, IP) significantly inhibited teeth chattering and forepaw treads. Adenosine receptor antagonists enhanced different opiate withdrawal signs. The adenosine A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (0, 1, 10 mg/kg, IP) significantly increased weight loss and the A2 antagonist, 3,7-dimethyl-1-propargylxanthine (0, 1 and 10 mg/kg, IP) enhanced wet dog shakes and withdrawal diarrhea. Treatment effects of adenosinergic agents were not due to nonspecific motor effects, as demonstrated by activity monitoring studies. These results support a role for adenosine receptors in the expression of opiate withdrawal and suggest the potential utility of adenosine agonists in its treatment. PMID:8741956

  18. Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein

    SciTech Connect

    Zolnierowicz, S.; Work, C.; Hutchison, K.; Fox, I.H. )

    1990-04-01

    The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-({sup 3}H)ethylcarboxamidoadenosine (({sup 3}H)NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the ({sup 3}H)NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors.

  19. Novel adenosine receptors in rat hippocampus identification and characterization

    SciTech Connect

    Chin, J.H.; Mashman, W.E.; DeLorenzo, R.J.

    1985-05-06

    2-chloro(/sup 3/H)adenosine, a stable analog of adenosine, was used to investigate the presence of adenosine receptors in rat hippocampal membranes that may mediate the depressant effects of adenosine on synaptic transmission in this tissue. Equilibrium binding studies reveal the presence of a previously undescribed class of receptors with a K/sub D/ of 4.7 ..mu..M and a Bmax of 130 pmol/mg of protein. Binding is sensitive to alkylxanthines and to a number of adenosine-related compounds. The pharmacological properties of this binding site are distinct from those of the A1 and A2 adenosine receptors associated with adenylate cyclase. The results suggest that this adenosine binding site is a novel central purinergic receptor through which adenosine may regulate hippocampal excitability. 50 references, 2 figures, 1 table.

  20. Characteristic molecular vibrations of adenosine receptor ligands.

    PubMed

    Chee, Hyun Keun; Yang, Jin-San; Joung, Je-Gun; Zhang, Byoung-Tak; Oh, S June

    2015-02-13

    Although the regulation of membrane receptor activation is known to be crucial for molecular signal transduction, the molecular mechanism underlying receptor activation is not fully elucidated. Here we study the physicochemical nature of membrane receptor behavior by investigating the characteristic molecular vibrations of receptor ligands using computational chemistry and informatics methods. By using information gain, t-tests, and support vector machines, we have identified highly informative features of adenosine receptor (AdoR) ligand and corresponding functional amino acid residues such as Asn (6.55) of AdoR that has informative significance and is indispensable for ligand recognition of AdoRs. These findings may provide new perspectives and insights into the fundamental mechanism of class A G protein-coupled receptor activation. PMID:25622891

  1. Agonism and Antagonism at the Insulin Receptor

    PubMed Central

    Knudsen, Louise; Hansen, Bo Falck; Jensen, Pia; Pedersen, Thomas Åskov; Vestergaard, Kirsten; Schäffer, Lauge; Blagoev, Blagoy; Oleksiewicz, Martin B.; Kiselyov, Vladislav V.; De Meyts, Pierre

    2012-01-01

    Insulin can trigger metabolic as well as mitogenic effects, the latter being pharmaceutically undesirable. An understanding of the structure/function relationships between insulin receptor (IR) binding and mitogenic/metabolic signalling would greatly facilitate the preclinical development of new insulin analogues. The occurrence of ligand agonism and antagonism is well described for G protein-coupled receptors (GPCRs) and other receptors but in general, with the exception of antibodies, not for receptor tyrosine kinases (RTKs). In the case of the IR, no natural ligand or insulin analogue has been shown to exhibit antagonistic properties, with the exception of a crosslinked insulin dimer (B29-B’29). However, synthetic monomeric or dimeric peptides targeting sites 1 or 2 of the IR were shown to be either agonists or antagonists. We found here that the S961 peptide, previously described to be an IR antagonist, exhibited partial agonistic effects in the 1–10 nM range, showing altogether a bell-shaped dose-response curve. Intriguingly, the agonistic effects of S961 were seen only on mitogenic endpoints (3H-thymidine incorporation), and not on metabolic endpoints (14C-glucose incorporation in adipocytes and muscle cells). The agonistic effects of S961 were observed in 3 independent cell lines, with complete concordance between mitogenicity (3H-thymidine incorporation) and phosphorylation of the IR and Akt. Together with the B29-B’29 crosslinked dimer, S961 is a rare example of a mixed agonist/antagonist for the human IR. A plausible mechanistic explanation based on the bivalent crosslinking model of IR activation is proposed. PMID:23300584

  2. A(3) adenosine receptor ligands: history and perspectives.

    PubMed

    Baraldi, P G; Cacciari, B; Romagnoli, R; Merighi, S; Varani, K; Borea, P A; Spalluto, G

    2000-03-01

    Adenosine regulates many physiological functions through specific cell membrane receptors. On the basis of pharmacological studies and molecular cloning, four different adenosine receptors have been identified and classified as A(1), A(2A), A(2B), and A(3). These adenosine receptors are members of the G-protein-coupled receptor family. While adenosine A(1) and A(2A) receptor subtypes have been pharmacologically characterized through the use of selective ligands, the A(3) adenosine receptor subtype is presently under study in order to better understand its physio-pathological functions. Activation of adenosine A(3) receptors has been shown to stimulate phospholipase C and D and to inhibit adenylate cyclase. Activation of A(3) adenosine receptors also causes the release of inflammatory mediators such as histamine from mast cells. These mediators are responsible for processes such as inflammation and hypotension. It has also been suggested that the A(3) receptor plays an important role in brain ischemia, immunosuppression, and bronchospasm in several animal models. Based on these results, highly selective A(3) adenosine receptor agonists and/or antagonists have been indicated as potential drugs for the treatment of asthma and inflammation, while highly selective agonists have been shown to possess cardioprotective effects. The updated material related to this field of research has been rationalized and arranged in order to offer an overview of the topic. PMID:10723024

  3. Targeting of Adenosine Receptors in Ischemia-Reperfusion Injury

    PubMed Central

    Laubach, Victor E.; French, Brent A.; Okusa, Mark D.

    2010-01-01

    Importance of the field Ischemia-reperfusion (IR) injury is a common clinical problem after transplantation as well as myocardial infarction and stroke. IR initiates an inflammatory response leading to rapid tissue damage. Adenosine, produced in response to IR, is generally considered as a protective signaling molecule and elicits its physiological responses through four distinct adenosine receptors. The short half-life, lack of specificity, and rapid metabolism limits the use of adenosine as a therapeutic agent. Thus intense research efforts have focused on the synthesis and implementation of specific adenosine receptor agonists and antagonists as potential therapeutic agents for a variety of inflammatory conditions including IR injury. Areas covered by this review This review summarizes current knowledge on IR injury with a focus on lung, heart, and kidney, and examines studies that have advanced our understanding of the role of adenosine receptors and the therapeutic potential of adenosine receptor agonists and antagonists for the prevention of IR injury. What the reader will gain The reader will gain insight into the role of adenosine receptor signaling in IR injury. Take home message No clinical therapies are currently available that specifically target IR injury; however, targeting of specific adenosine receptors may offer therapeutic strategies in this regard. PMID:21110787

  4. Photo-antagonism of the GABAA receptor.

    PubMed

    Mortensen, Martin; Iqbal, Favaad; Pandurangan, Arun P; Hannan, Saad; Huckvale, Rosemary; Topf, Maya; Baker, James R; Smart, Trevor G

    2014-01-01

    Neurotransmitter receptor trafficking is fundamentally important for synaptic transmission and neural network activity. GABAA receptors and inhibitory synapses are vital components of brain function, yet much of our knowledge regarding receptor mobility and function at inhibitory synapses is derived indirectly from using recombinant receptors, antibody-tagged native receptors and pharmacological treatments. Here we describe the use of a set of research tools that can irreversibly bind to and affect the function of recombinant and neuronal GABAA receptors following ultraviolet photoactivation. These compounds are based on the competitive antagonist gabazine and incorporate a variety of photoactive groups. By using site-directed mutagenesis and ligand-docking studies, they reveal new areas of the GABA binding site at the interface between receptor β and α subunits. These compounds enable the selected inactivation of native GABAA receptor populations providing new insight into the function of inhibitory synapses and extrasynaptic receptors in controlling neuronal excitation. PMID:25072879

  5. ANDROGEN RECEPTOR ANTAGONISM BY THE ORGANOPHOSPHATE INSECTICIDE FENITROTHION

    EPA Science Inventory

    Androgen receptor antagonism by the organophosphate insecticide fenitrothion. Tamura, H., Maness, S.C., Reischmann, K. Dorman, D.C., Gray, L.E., and Gaido, K.W. (2000). Toxicol. Sci.

    Organophosphate insecticides represent one of the most widely used classes of pesticide...

  6. Equilibrium and kinetic selectivity profiling on the human adenosine receptors.

    PubMed

    Guo, Dong; Dijksteel, Gabrielle S; van Duijl, Tirsa; Heezen, Maxime; Heitman, Laura H; IJzerman, Adriaan P

    2016-04-01

    Classical evaluation of target selectivity is usually undertaken by measuring the binding affinity of lead compounds against a number of potential targets under equilibrium conditions, without considering the kinetics of the ligand-receptor interaction. In the present study we propose a combined strategy including both equilibrium- and kinetics-based selectivity profiling. The adenosine receptor (AR) was chosen as a prototypical drug target. Six in-house AR antagonists were evaluated in a radioligand displacement assay for their affinity and in a competition association assay for their binding kinetics on three AR subtypes. One of the compounds with a promising kinetic selectivity profile was also examined in a [(35)S]-GTPγS binding assay for functional activity. We found that XAC and LUF5964 were kinetically more selective for the A1R and A3R, respectively, although they are non-selective in terms of their affinity. In comparison, LUF5967 displayed a strong equilibrium-based selectivity for the A1R over the A2AR, yet its kinetic selectivity thereon was less pronounced. In a GTPγS assay, LUF5964 exhibited insurmountable antagonism on the A3R while having a surmountable effect on the A1R, consistent with its kinetic selectivity profile. This study provides evidence that equilibrium and kinetic selectivity profiling can both be important in the early phases of the drug discovery process. Our proposed combinational strategy could be considered for future medicinal chemistry efforts and aid the design and discovery of different or even better leads for clinical applications. PMID:26930564

  7. Alterations of adenosine A1 receptors in morphine dependence.

    PubMed

    Kaplan, G B; Leite-Morris, K A; Sears, M T

    1994-09-19

    The possibility that central adenosine A1 and A2a receptors mediate opiate dependence was examined in morphine-treated mice using radioligand binding methods. Mice treated with morphine for 72 h demonstrated significant increases in naloxone precipitated abstinence behaviors of jumping, wet-dog shakes, teeth chattering, forepaw trends, forepaw tremors and diarrhea compared to vehicle-treated mice. Increased concentrations of cortical adenosine A1 receptor sites, but not striatal adenosine A2a sites, were found in saturation binding studies from morphine-dependent mice. Decreases in cortical A1 agonist binding affinity values along with increases in agonist binding sites were demonstrated in competition binding studies. These results suggest that adaptive changes of upregulation and sensitization of adenosine A1 receptors play a role in mediating the opiate abstinence syndrome. PMID:7820640

  8. Noncompetitive, Voltage-Dependent NMDA Receptor Antagonism by Hydrophobic Anions

    PubMed Central

    Linsenbardt, Andrew J.; Chisari, Mariangela; Yu, Andrew; Shu, Hong-Jin; Zorumski, Charles F.

    2013-01-01

    NMDA receptor (NMDAR) antagonists are dissociative anesthetics, drugs of abuse, and are of therapeutic interest in neurodegeneration and neuropsychiatric disease. Many well-known NMDAR antagonists are positively charged, voltage-dependent channel blockers. We recently showed that the hydrophobic anion dipicrylamine (DPA) negatively regulates GABAA receptor function by a mechanism indistinguishable from that of sulfated neurosteroids. Because sulfated neurosteroids also modulate NMDARs, here we examined the effects of DPA on NMDAR function. In rat hippocampal neurons DPA inhibited currents gated by 300 µM NMDA with an IC50 of 2.3 µM. Neither onset nor offset of antagonism exhibited dependence on channel activation but exhibited a noncompetitive profile. DPA antagonism was independent of NMDAR subunit composition and was similar at extrasynaptic and total receptor populations. Surprisingly, similar to cationic channel blockers but unlike sulfated neurosteroids, DPA antagonism was voltage dependent. Onset and offset of DPA antagonism were nearly 10-fold faster than DPA-induced increases in membrane capacitance, suggesting that membrane interactions do not directly explain antagonism. Furthermore, voltage dependence did not derive from association of DPA with a site on NMDARs directly accessible to the outer membrane leaflet, assessed by DPA translocation experiments. Consistent with the expected lack of channel block, DPA antagonism did not interact with permeant ions. Therefore, we speculate that voltage dependence may arise from interactions of DPA with the inherent voltage dependence of channel gating. Overall, we conclude that DPA noncompetitively inhibits NMDA-induced current by a novel voltage-dependent mechanism and represents a new class of anionic NMDAR antagonists. PMID:23144238

  9. Characterization of adenosine receptors involved in adenosine-induced bronchoconstriction in allergic rabbits.

    PubMed Central

    el-Hashim, A.; D'Agostino, B.; Matera, M. G.; Page, C.

    1996-01-01

    1. Recent work has suggested that adenosine may be involved in asthma via the activation of A1 receptors. However, the role of the recently cloned A3 receptor in airways is largely unknown. In the present study, we have investigated the role of the A3 receptor in adenosine-induced bronchoconstriction in allergic rabbits. 2. Aerosol challenge of antigen (Ag) immunized rabbits with the adenosine precursor, adenosine 5'-monophosphate (AMP), resulted in a dose-dependent fall in dynamic compliance (Cdyn). The maximum fall in Cdyn in these rabbits was significantly greater than that in litter matched, sham immunized animals (P < 0.05). However, there was no significant difference in the maximum increase in airways resistance (Rt) between Ag and sham immunized rabbits (P > 0.05). 3. Aerosol challenge of Ag immunized rabbits with cyclopentyl-adenosine (CPA) (A1-receptor agonist) elicited a dose-dependent fall in Cdyn in Ag immunized rabbits and the maximum fall in Cdyn in these rabbits was significantly greater than that observed in sham immunized rabbits (P < 0.05). Similarly, CPA induced dose-dependent increases in R1 in Ag immunized rabbits whereas sham immunized rabbits failed to respond to CPA within the same dose range. The maximum increase in RL in Ag immunized rabbits was significantly greater than that of sham immunized rabbits (P < 0.05). 4. Aerosol challenge of either Ag or sham immunized rabbits with the A3 agonist aminophenylethyladenosine (APNEA) did not elicit dose-dependent changes in either RL or Cdyn. Moreover, there was no significant difference in the maximum response, measured by either parameter, between the two animal groups (P > 0.05). 5. These data provide further evidence for a role of the A1 receptor in the airways, but do not support a role for the A3 receptor in adenosine-induced bronchoconstriction in the allergic rabbit. PMID:8937732

  10. Impact on monoclonal antibody production in murine hybridoma cell cultures of adenosine receptor antagonists and phosphodiesterase inhibitors.

    PubMed

    Kelso, Geoffrey F; Kazi, Shahid A; Harris, Simon J; Boysen, Reinhard I; Chowdhury, Jamil; Hearn, Milton T W

    2016-01-15

    The effects of different adenosine receptor antagonists and cyclic nucleotide phosphodiesterase (PDE) inhibitors on monoclonal antibody (mAb) titer and cell viability of murine hybridoma cells in culture were measured as part of our investigations to discover additives that enhance mAb production. Specific adenosine receptor antagonists and PDE inhibitors were found to enhance or decrease the titer of immunoglobulin G1 (IgG1) mAbs relative to negative controls, depending on the specific compound and cell line employed. The observed enhancements or decreases in IgG1 mAb titer appeared to be mainly due to an increase or decrease in specific productivity rates (ngmAb/cell), respectively. The different effects of the selective adenosine antagonists suggest that antagonism at the level of the adenosine A2A and A1 or the adenosine A3 receptors result in either enhancement or suppression of IgG1 mAb production by hybridoma cells. Overall, these studies have identified hitherto unknown activities of specific adenosine antagonists and PDE inhibitors which indicate they may have valuable roles as cell culture additives in industrial biomanufacturing processes designed to enhance the yields of mAbs or other recombinant proteins produced by mammalian cell culture procedures. PMID:26646217

  11. Role of A3 adenosine receptor in diabetic neuropathy.

    PubMed

    Yan, Heng; Zhang, Enshui; Feng, Chang; Zhao, Xin

    2016-10-01

    Neuropathy is the most common diabetic complication. Although the A1 and A2A adenosine receptors are important pharmacological targets in alleviating diabetic neuropathy, the role of the A3 adenosine receptor remains unknown. Because the A3 adenosine receptor regulates pain induced by chronic constriction injury or chemotherapy, its stimulation might also attenuate diabetic neuropathy. This study examines the effects of systemic treatment with the A3 adenosine receptor agonist 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-β-d-ribofuranuronamide (IB-MECA) on diabetic neuropathy and explores the putative mechanisms underlying its pharmacological effects. We show that IB-MECA alleviated mechanical hyperalgesia and thermal hypoalgesia in mice 2 weeks but not 4 weeks after streptozocin (STZ) treatment. Furthermore, IB-MECA prevented the reduction in sciatic motor nerve conduction velocity and sensory nerve conduction velocity in diabetic mice 2 weeks but not 4 weeks after STZ treatment. Similarly, IB-MECA inhibited the activation of nuclear factor-κB and decreased the generation of tumor necrosis factor-α in the spinal cord of mice 2 weeks but not 4 weeks after STZ treatment. These phenomena were associated with reduction of A3 adenosine receptor expression in the spinal cord after long-term diabetes. Our results suggest that the A3 adenosine receptor plays a critical role in regulating diabetic neuropathy and that reduction in A3 adenosine receptor expression/function might contribute to the progression of diabetic neuropathy. © 2016 Wiley Periodicals, Inc. PMID:27319979

  12. Striatal adenosine-cannabinoid receptor interactions in rats over-expressing adenosine A2A receptors.

    PubMed

    Chiodi, Valentina; Ferrante, Antonella; Ferraro, Luca; Potenza, Rosa Luisa; Armida, Monica; Beggiato, Sarah; Pèzzola, Antonella; Bader, Michael; Fuxe, Kjell; Popoli, Patrizia; Domenici, Maria Rosaria

    2016-03-01

    Adenosine A2A receptors (A2 A Rs) and cannabinoid CB1 receptors (CB1 Rs) are highly expressed in the striatum, where they functionally interact and form A2A /CB1 heteroreceptor complexes. We investigated the effects of CB1 R stimulation in a transgenic rat strain over-expressing A2 A Rs under the control of the neural-specific enolase promoter (NSEA2A rats) and in age-matched wild-type (WT) animals. The effects of the CB1 R agonist WIN 55,212-2 (WIN) were significantly lower in NSEA2A rats than in WT animals, as demonstrated by i) electrophysiological recordings of synaptic transmission in corticostriatal slices; ii) the measurement of glutamate outflow from striatal synaptosomes and iii) in vivo experiments on locomotor activity. Moreover, while the effects of WIN were modulated by both A2 A R agonist (CGS 21680) and antagonists (ZM 241385, KW-6002 and SCH-442416) in WT animals, the A2 A R antagonists failed to influence WIN-mediated effects in NSEA2A rats. The present results demonstrate that in rats with genetic neuronal over-expression of A2 A Rs, the effects mediated by CB1 R activation in the striatum are significantly reduced, suggesting a change in the stoichiometry of A2A and CB1 receptors and providing a strategy to dissect the involvement of A2 A R forming or not forming heteromers in the modulation of striatal functions. These findings add additional evidence for the existence of an interaction between striatal A2 A Rs and CB1 Rs, playing a fundamental role in the regulation of striatal functions. We studied A2A -CB1 receptor interaction in transgenic rats over-expressing adenosine A2A receptors under the control of the neuron-specific enolase promoter (NSEA2A ). In these rats, we demonstrated a reduced effect of the CB1 receptor agonist WIN 55,212-2 in the modulation of corticostriatal synaptic transmission and locomotor activity, while CB1 receptor expression level did not change with respect to WT rats. A reduction in the expression of A2A -CB1

  13. The A3 adenosine receptor: history and perspectives.

    PubMed

    Borea, Pier Andrea; Varani, Katia; Vincenzi, Fabrizio; Baraldi, Pier Giovanni; Tabrizi, Mojgan Aghazadeh; Merighi, Stefania; Gessi, Stefania

    2015-01-01

    By general consensus, the omnipresent purine nucleoside adenosine is considered a major regulator of local tissue function, especially when energy supply fails to meet cellular energy demand. Adenosine mediation involves activation of a family of four G protein-coupled adenosine receptors (ARs): A(1), A(2)A, A(2)B, and A(3). The A(3) adenosine receptor (A(3)AR) is the only adenosine subtype to be overexpressed in inflammatory and cancer cells, thus making it a potential target for therapy. Originally isolated as an orphan receptor, A(3)AR presented a twofold nature under different pathophysiologic conditions: it appeared to be protective/harmful under ischemic conditions, pro/anti-inflammatory, and pro/antitumoral depending on the systems investigated. Until recently, the greatest and most intriguing challenge has been to understand whether, and in which cases, selective A(3) agonists or antagonists would be the best choice. Today, the choice has been made and A(3)AR agonists are now under clinical development for some disorders including rheumatoid arthritis, psoriasis, glaucoma, and hepatocellular carcinoma. More specifically, the interest and relevance of these new agents derives from clinical data demonstrating that A(3)AR agonists are both effective and safe. Thus, it will become apparent in the present review that purine scientists do seem to be getting closer to their goal: the incorporation of adenosine ligands into drugs with the ability to save lives and improve human health. PMID:25387804

  14. Blockade of adenosine receptors unmasks a stimulatory effect of ATP on cardiac contractility.

    PubMed Central

    Mantelli, L.; Amerini, S.; Filippi, S.; Ledda, F.

    1993-01-01

    1. The effects of ATP, alpha,beta-methylene ATP and beta,gamma-methylene ATP on the contractile tension of guinea-pig isolated left atria were evaluated. 2. ATP (1-100 microM) produced a concentration-dependent negative inotropic effect; this response was converted to a positive inotropic effect in the presence of the antagonist of adenosine A1 receptors, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 0.1 microM), and in the presence of 8-phenyltheophylline (10 microM), an antagonist of A1 and A2 receptors. 3. The positive inotropic effect of ATP was antagonized by the P2 receptor antagonist, suramin (500 microM). Reactive blue 2 (30-500 microM), a putative P2y receptor antagonist, concentration-dependently reduced and finally abolished the effect of ATP. 4. In the presence of 8-phenyltheophylline, the stable analogues of ATP, alpha,beta-methylene ATP and beta,gamma-methylene ATP (1-30 microM), produced a concentration-dependent increase in atrial contractility of a lesser degree than that induced by ATP. 5. The results suggest that when inhibitory adenosine receptors are blocked, ATP produces a positive inotropic effect, probably mediated by P2y receptor stimulation. PMID:8401938

  15. Prostaglandins and prostaglandin receptor antagonism in migraine.

    PubMed

    Antonova, Maria

    2013-05-01

    Human models of headache may contribute to understanding of prostaglandins' role in migraine pathogenesis. The current thesis investigated the migraine triggering effect of prostaglandin E2 (PGE2) in migraine patients without aura, the efficacy of a novel EP4 receptor antagonist, BGC20-1531, in prevention of PGE2-induced headache and the ability of prostaglandin F2α (PGF2α) to trigger headache without any vasodilatation in healthy volunteers. All studies were designed as double-blind, placebo-controlled, cross-over experiments, where PGE2/PGF2α or saline were infused over 20-25 min. In the study with EP4 receptor antagonist healthy volunteers were pre-treated with two different doses of BGC20-1531 or placebo followed by PGE2 infusion over 25 min. The headache data were collected during the whole study day, whereas the possible vascular changes were measured during the in-hospital phase of 1.5 h. The infusion of PGE2 caused the immediate migraine-like attacks and vasodilatation of the middle cerebral artery in migraine patients without aura. The highly specific and potent EP4 receptor antagonist, BGC20-1531, was not able to attenuate PGE2-induced headache and vasodilatation of both intra- and extra-cerebral arteries. The intravenous infusion of PGF2α did not induce headache or statistically significant vasoconstriction of cerebral arteries in healthy volunteers. Novel data on PGE2-provoked immediate migraine-like attacks suggest that PGE2 may be one of the important final products in the pathogenesis of migraine. The lack of efficacy of EP4 receptor antagonist suggests that a single receptor blockade is not sufficient to block PGE2 responses, hence EP2 receptor should be investigated as a potential drug target for the treatment of migraine. The absence of headache during the PGF2α infusion demonstrates that vasodilating properties are necessary for the induction of headache and migraine. PMID:23673269

  16. 2-Substituted adenosine derivatives: affinity and efficacy at four subtypes of human adenosine receptors.

    PubMed

    Gao, Zhan-Guo; Mamedova, Liaman K; Chen, Peiran; Jacobson, Kenneth A

    2004-11-15

    The affinity and efficacy at four subtypes (A(1), A(2A), A(2B) and A(3)) of human adenosine receptors (ARs) of a wide range of 2-substituted adenosine derivatives were evaluated using radioligand binding assays and a cyclic AMP functional assay in intact CHO cells stably expressing these receptors. Similar to previous studies of the N(6)-position, several 2-substituents were found to be critical structural determinants for the A(3)AR activation. The following adenosine 2-ethers were moderately potent partial agonists (K(i), nM): benzyl (117), 3-chlorobenzyl (72), 2-(3-chlorophenyl)ethyl (41), and 2-(2-naphthyl)ethyl (130). The following adenosine 2-ethers were A(3)AR antagonists: 2,2-diphenylethyl, 2-(2-norbornan)ethyl, R- and S-2-phenylbutyl, and 2-(2-chlorophenyl)ethyl. 2-(S-2-Phenylbutyloxy)adenosine as an A(3)AR antagonist right-shifted the concentration-response curve for the inhibition by NECA of cyclic AMP accumulation with a K(B) value of 212 nM, which is similar to its binding affinity (K(i) = 175 nM). These 2-substituted adenosine derivatives were generally less potent at the A(1)AR in comparison to the A(3)AR, but fully efficacious, with binding K(i) values over 100 nM. The 2-phenylethyl moiety resulted in higher A(3)AR affinity (K(i) in nM) when linked to the 2-position of adenosine through an ether group (54), than when linked through an amine (310) or thioether (1960). 2-[2-(l-Naphthyl)ethyloxy]adenosine (K(i) = 3.8 nM) was found to be the most potent and selective (>50-fold) A(2A) agonist in this series. Mixed A(2A)/A(3)AR agonists have been identified. Interestingly, although most of these compounds were extremely weak at the A(2B)AR, 2-[2-(2-naphthyl)ethyloxy]adenosine (EC(50) = 1.4 microM) and 2-[2-(2-thienyl)-ethyloxy]adenosine (EC(50) = 1.8 microM) were found to be relatively potent A(2B) agonists, although less potent than NECA (EC(50) = 140 nM). PMID:15476669

  17. Involvement of Peripheral Adenosine A2 Receptors in Adenosine A1 Receptor–Mediated Recovery of Respiratory Motor Function After Upper Cervical Spinal Cord Hemisection

    PubMed Central

    James, Elysia; Nantwi, Kwaku D

    2006-01-01

    Background/Objective: In an animal model of spinal cord injury, a latent respiratory motor pathway can be pharmacologically activated through central adenosine A1 receptor antagonism to restore respiratory function after cervical (C2) spinal cord hemisection that paralyzes the hemidiaphragm ipsilateral to injury. Although respiration is modulated by central and peripheral mechanisms, putative involvement of peripheral adenosine A2 receptors in functional recovery in our model is untested. The objective of this study was to assess the effects of peripherally located adenosine A2 receptors on recovery of respiratory function after cervical (C2) spinal cord hemisection. Methods: Respiratory activity was electrophysiologically assessed (under standardized recording conditions) in C2-hemisected adult rats with the carotid bodies intact (H-CBI; n =12) or excised (H-CBE; n =12). Animals were administered the adenosine A2 receptor agonist, CGS-21680, followed by the A1 receptor antagonist, 1, 3-dipropyl-8-cyclopentylxanthine (DPCPX), or administered DPCPX alone. Recovered respiratory activity, characterized as drug-induced activity in the previously quiescent left phrenic nerve of C2-hemisected animals in H-CBI and H-CBE rats, was compared. Recovered respiratory activity was calculated by dividing drug-induced activity in the left phrenic nerve by activity in the right phrenic nerve. Results: Administration of CGS-21680 before DPCPX (n = 6) in H-CBI rats induced a significantly greater recovery (58.5 ± 3.6%) than when DPCPX (42.6 ± 4.6%) was administered (n = 6) alone. In H-CBE rats, prior administration of CGS-21680 (n = 6) did not enhance recovery over that induced by DPCPX (n = 6) alone. Recovery in H-CBE rats amounted to 39.7 ± 3.7% and 38.4 + 4.2%, respectively. Conclusions: Our results suggest that adenosine A2 receptors located in the carotid bodies can enhance the magnitude of adenosine A1 receptor–mediated recovery of respiratory function after C2 hemisection

  18. Identification of possible adenosine receptors in vascular smooth muscle

    SciTech Connect

    Doctrow, S.R.

    1985-01-01

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

  19. Current status of A1 adenosine receptor allosteric enhancers.

    PubMed

    Romagnoli, Romeo; Baraldi, Pier Giovanni; Moorman, Allan R; Borea, Pier Andrea; Varani, Katia

    2015-01-01

    Adenosine is an ubiquitous nucleoside involved in various physiological and pathological functions by stimulating A1, A2A, A2B and A3 adenosine receptors (ARs). Allosteric enhancers to A1ARs may represent novel therapeutic agents because they increase the activity of these receptors by mediating a shift to their active form in the A1AR-G protein ternary complex. In this manner, they are able to amplify the action of endogenous adenosine, which is produced in high concentrations under conditions of metabolic stress. A1AR allosteric enhancers could be used as a justifiable alternative to the exogenous agonists that are characterized by receptor desensitization and downregulation. In this review, an analysis of some of the most interesting allosteric modulators of A1ARs has been reported. PMID:26144263

  20. The Quintiles Prize Lecture 2004. The identification of the adenosine A2B receptor as a novel therapeutic target in asthma.

    PubMed

    Holgate, Stephen T

    2005-08-01

    Adenosine is a powerful bronchoconstrictor of asthmatic, but not normal, airways. In vitro studies on isolated human mast cells and basophils revealed that adenosine and selective analogues augmented inflammatory mediator release from mast cells by stimulating A(2) receptors. Pharmacological blockade of mast cell mediator release in vivo also attenuated adenosine-induced bronchoconstriction, as did theophylline, by adenosine A(2) receptor antagonism. Further in vitro studies revealed that the asthmatic response to adenosine is likely to be mediated via the A(2B) subtype which is selectively antagonised by enprofylline. Studies in animal models, especially mice, have shown a close synergistic interaction between adenosine, Th2 and airway remodelling responses. The recent description of A(2B) receptors on human airway smooth muscle cells that mediate cytokine and chemokine release and induce differentiation of fibroblasts into myofibroblasts strengthens the view that adenosine maybe more than an inflammatory mediator in asthma but also participates in airway wall remodelling in this disease. These data have provided a firm basis for developing adenosine A(2B) receptor antagonists as a new therapeutic approach to this disease. PMID:15980878

  1. The Quintiles Prize Lecture 2004: The identification of the adenosine A2B receptor as a novel therapeutic target in asthma

    PubMed Central

    Holgate, Stephen T

    2005-01-01

    Adenosine is a powerful bronchoconstrictor of asthmatic, but not normal, airways. In vitro studies on isolated human mast cells and basophils revealed that adenosine and selective analogues augmented inflammatory mediator release from mast cells by stimulating A2 receptors. Pharmacological blockade of mast cell mediator release in vivo also attenuated adenosine-induced bronchoconstriction, as did theophylline, by adenosine A2 receptor antagonism. Further in vitro studies revealed that the asthmatic response to adenosine is likely to be mediated via the A2B subtype which is selectively antagonised by enprofylline. Studies in animal models, especially mice, have shown a close synergistic interaction between adenosine, Th2 and airway remodelling responses. The recent description of A2B receptors on human airway smooth muscle cells that mediate cytokine and chemokine release and induce differentiation of fibroblasts into myofibroblasts strengthens the view that adenosine maybe more than an inflammatory mediator in asthma but also participates in airway wall remodelling in this disease. These data have provided a firm basis for developing adenosine A2B receptor antagonists as a new therapeutic approach to this disease. PMID:15980878

  2. 2-Substituted adenosine derivatives: affinity and efficacy at four subtypes of human adenosine receptors

    PubMed Central

    Gao, Zhan-Guo; Mamedova, Liaman K.; Chen, Peiran; Jacobson, Kenneth A.

    2012-01-01

    The affinity and efficacy at four subtypes (A1, A2A, A2B and A3) of human adenosine receptors (ARs) of a wide range of 2-substituted adenosine derivatives were evaluated using radioligand binding assays and a cyclic AMP functional assay in intact CHO cells stably expressing these receptors. Similar to previous studies of the N6-position, several 2-substituents were found to be critical structural determinants for the A3AR activation. The following adenosine 2-ethers were moderately potent partial agonists (Ki, nM): benzyl (117), 3-chlorobenzyl (72), 2-(3-chlorophenyl)ethyl (41), and 2-(2-naphthyl)ethyl (130). The following adenosine 2-ethers were A3AR antagonists: 2,2-diphenylethyl, 2-(2-norbornan)ethyl, R- and S-2-phenylbutyl, and 2-(2-chlorophenyl)ethyl. 2-(S-2-Phenylbutyloxy)a-denosine as an A3AR antagonist right-shifted the concentration–response curve for the inhibition by NECA of cyclic AMP accumulation with a KB value of 212 nM, which is similar to its binding affinity (Ki = 175 nM). These 2-substituted adenosine derivatives were generally less potent at the A1AR in comparison to the A3AR, but fully efficacious, with binding Ki values over 100 nM. The 2-phenylethyl moiety resulted in higher A3AR affinity (Ki in nM) when linked to the 2-position of adenosine through an ether group (54), than when linked through an amine (310) or thioether (1960). 2-[2-(l-Naphthyl)ethyloxy]adenosine (Ki = 3.8 nM) was found to be the most potent and selective (>50-fold) A2A agonist in this series. Mixed A2A/A3AR agonists have been identified. Interestingly, although most of these compounds were extremely weak at the A2BAR, 2-[2-(2-naphthyl)ethyloxy]adenosine (EC50 = 1.4 µM) and 2-[2-(2-thienyl)-ethyloxy]adenosine (EC50 = 1.8 (M) were found to be relatively potent A2B agonists, although less potent than NECA (EC50 = 140 nM). PMID:15476669

  3. ATP- and adenosine-mediated signaling in the central nervous system: adenosine stimulates glutamate release from astrocytes via A2a adenosine receptors.

    PubMed

    Nishizaki, Tomoyuki

    2004-02-01

    Adenosine enhanced intracellular Ca(2+) concentrations in astrocytes via A(2a) adenosine receptors involving protein kinase A (PKA) activation. The Ca(2+) rise is inhibited by brefeldin A, an inhibitor of vesicular transport; but not by neomycin and U73122, phospholipase C inhibitors; xestospongin, an IP(3)-receptor inhibitor; ryanodine, a ryanodine-receptor inhibitor; TMB-8, an endoplasmic reticulum calcium-release blocker; octanol, a gap-junction inhibitor; or cadmium, a non-selective, calcium-channel blocker. Adenosine stimulates astrocytic glutamate release via an A(2a) adenosine receptors/PKA pathway, and the release is inhibited by the vesicular transport inhibitors brefeldin A and bafilomycin A1. A(2a) adenosine receptors and the ensuing PKA events, thus, are endowed with vesicular Ca(2+) release from an unknown intracellular calcium store and vesicular glutamate release from astrocytes. PMID:14978344

  4. Combined Angiotensin Receptor Antagonism and Neprilysin Inhibition.

    PubMed

    Hubers, Scott A; Brown, Nancy J

    2016-03-15

    Heart failure affects ≈5.7 million people in the United States alone. Angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, β-blockers, and aldosterone antagonists have improved mortality in patients with heart failure and reduced ejection fraction, but mortality remains high. In July 2015, the US Food and Drug Administration approved the first of a new class of drugs for the treatment of heart failure: Valsartan/sacubitril (formerly known as LCZ696 and currently marketed by Novartis as Entresto) combines the angiotensin receptor blocker valsartan and the neprilysin inhibitor prodrug sacubitril in a 1:1 ratio in a sodium supramolecular complex. Sacubitril is converted by esterases to LBQ657, which inhibits neprilysin, the enzyme responsible for the degradation of the natriuretic peptides and many other vasoactive peptides. Thus, this combined angiotensin receptor antagonist and neprilysin inhibitor addresses 2 of the pathophysiological mechanisms of heart failure: activation of the renin-angiotensin-aldosterone system and decreased sensitivity to natriuretic peptides. In the Prospective Comparison of ARNI With ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure (PARADIGM-HF) trial, valsartan/sacubitril significantly reduced mortality and hospitalization for heart failure, as well as blood pressure, compared with enalapril in patients with heart failure, reduced ejection fraction, and an elevated circulating level of brain natriuretic peptide or N-terminal pro-brain natriuretic peptide. Ongoing clinical trials are evaluating the role of valsartan/sacubitril in the treatment of heart failure with preserved ejection fraction and hypertension. We review here the mechanisms of action of valsartan/sacubitril, the pharmacological properties of the drug, and its efficacy and safety in the treatment of heart failure and hypertension. PMID:26976916

  5. Chaperoning of the A1-adenosine receptor by endogenous adenosine - an extension of the retaliatory metabolite concept.

    PubMed

    Kusek, Justyna; Yang, Qiong; Witek, Martin; Gruber, Christian W; Nanoff, Christian; Freissmuth, Michael

    2015-01-01

    Cell-permeable orthosteric ligands can assist folding of G protein-coupled receptors in the endoplasmic reticulum (ER); this pharmacochaperoning translates into increased cell surface levels of receptors. Here we used a folding-defective mutant of human A1-adenosine receptor as a sensor to explore whether endogenously produced adenosine can exert a chaperoning effect. This A1-receptor-Y(288)A was retained in the ER of stably transfected human embryonic kidney 293 cells but rapidly reached the plasma membrane in cells incubated with an A1 antagonist. This was phenocopied by raising intracellular adenosine levels with a combination of inhibitors of adenosine kinase, adenosine deaminase, and the equilibrative nucleoside transporter: mature receptors with complex glycosylation accumulated at the cell surface and bound to an A1-selective antagonist with an affinity indistinguishable from the wild-type A1 receptor. The effect of the inhibitor combination was specific, because it did not result in enhanced surface levels of two folding-defective human V2-vasopressin receptor mutants, which were susceptible to pharmacochaperoning by their cognate antagonist. Raising cellular adenosine levels by subjecting cells to hypoxia (5% O2) reproduced chaperoning by the inhibitor combination and enhanced surface expression of A1-receptor-Y(288)A within 1 hour. These findings were recapitulated for the wild-type A1 receptor. Taken together, our observations document that endogenously formed adenosine can chaperone its cognate A1 receptor. This results in a positive feedback loop that has implications for the retaliatory metabolite concept of adenosine action: if chaperoning by intracellular adenosine results in elevated cell surface levels of A1 receptors, these cells will be more susceptible to extracellular adenosine and thus more likely to cope with metabolic distress. PMID:25354767

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

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

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

  7. Effects of xanthine derivatives in a light/dark test in mice and the contribution of adenosine receptors.

    PubMed

    Imaizumi, M; Miyazaki, S; Onodera, K

    1994-11-01

    We investigated the effects of adenosine receptor antagonists, caffeine, theophylline, 8-phenyltheophylline, and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), in a light/dark test in mice. All antagonists decreased the time spent in the light zone in this test, which suggested that these compounds have anxiogenic effects. The anxiogenic effects of theophylline were reduced by pretreatment with CGS 21680, an A2-selective agonist, but not by N6-cyclopentyladenosine (CPA), an A1-selective agonist. However, the antagonism of the theophylline-induced anxiogenic effects by CGS21680 was only observed in the time spent in the light zone, and DPCPX-induced anxiogenic effects were neither reversed by CGS 21680 nor by CPA. Finally, it is notable that xanthine-derived adenosine antagonists tested here commonly showed anxiogenic effects in the light/dark test in mice. It is suggested that there is a minor contribution of adenosine receptors to these effects, although theophylline-induced anxiogenic effects were antagonized by an A2 receptor agonist. PMID:7746025

  8. Hypocretin/orexin antagonism enhances sleep-related adenosine and GABA neurotransmission in rat basal forebrain.

    PubMed

    Vazquez-DeRose, Jacqueline; Schwartz, Michael D; Nguyen, Alexander T; Warrier, Deepti R; Gulati, Srishti; Mathew, Thomas K; Neylan, Thomas C; Kilduff, Thomas S

    2016-03-01

    Hypocretin/orexin (HCRT) neurons provide excitatory input to wake-promoting brain regions including the basal forebrain (BF). The dual HCRT receptor antagonist almorexant (ALM) decreases waking and increases sleep. We hypothesized that HCRT antagonists induce sleep, in part, through disfacilitation of BF neurons; consequently, ALM should have reduced efficacy in BF-lesioned (BFx) animals. To test this hypothesis, rats were given bilateral IgG-192-saporin injections, which predominantly targets cholinergic BF neurons. BFx and intact rats were then given oral ALM, the benzodiazepine agonist zolpidem (ZOL) or vehicle (VEH) at lights-out. ALM was less effective than ZOL at inducing sleep in BFx rats compared to controls. BF adenosine (ADO), γ-amino-butyric acid (GABA), and glutamate levels were then determined via microdialysis from intact, freely behaving rats following oral ALM, ZOL or VEH. ALM increased BF ADO and GABA levels during waking and mixed vigilance states, and preserved sleep-associated increases in GABA under low and high sleep pressure conditions. ALM infusion into the BF also enhanced cortical ADO release, demonstrating that HCRT input is critical for ADO signaling in the BF. In contrast, oral ZOL and BF-infused ZOL had no effect on ADO levels in either BF or cortex. ALM increased BF ADO (an endogenous sleep-promoting substance) and GABA (which is increased during normal sleep), and required an intact BF for maximal efficacy, whereas ZOL blocked sleep-associated BF GABA release, and required no functional contribution from the BF to induce sleep. ALM thus induces sleep by facilitating the neural mechanisms underlying the normal transition to sleep. PMID:25431268

  9. Adenosine receptors and asthma in humans.

    PubMed

    Wilson, C N

    2008-10-01

    According to an executive summary of the GINA dissemination committee report, it is now estimated that approximately 300 million people (5% of the global population or 1 in 20 persons) have asthma. Despite the scientific progress made over the past several decades toward improving our understanding of the pathophysiology of asthma, there is still a great need for improved therapies, particularly oral therapies that enhance patient compliance and that target new mechanisms of action. Adenosine is an important signalling molecule in human asthma. By acting on extracellular G-protein-coupled ARs on a number of different cell types important in the pathophysiology of human asthma, adenosine affects bronchial reactivity, inflammation and airway remodelling. Four AR subtypes (A(1), A(2a), A(2b) and A(3)) have been cloned in humans, are expressed in the lung, and are all targets for drug development for human asthma. This review summarizes what is known about these AR subtypes and their function in human asthma as well as the pros and cons of therapeutic approaches to these AR targets. A number of molecules with high affinity and high selectivity for the human AR subtypes have entered clinical trials or are poised to enter clinical trials as anti-asthma treatments. With the availability of these molecules for testing in humans, the function of ARs in human asthma, as well as the safety and efficacy of approaches to the different AR targets, can now be determined. PMID:18852693

  10. Anxiolytic activity of adenosine receptor activation in mice.

    PubMed

    Jain, N; Kemp, N; Adeyemo, O; Buchanan, P; Stone, T W

    1995-10-01

    1. Purine analogues have been examined for anxiolytic- and anxiogenic-like activity in mice, by use of the elevated plus-maze. 2. The selective A1 receptor agonist, N6-cyclopentyladenosine (CPA) had marked anxiolytic-like activity at 10 and 50 microg kg(-1), with no effect on locomotor performance at these doses. 3. The A1 selective adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (CPX) had no significant effect on anxiety-related measures or locomotor behaviour, but blocked the anxiolytic-like activity of CPA. The hydrophilic xanthine, 8-(p-sulphophenyl) theophylline did not prevent anxiolysis by CPA. 4. Caffeine had anxiogenic-like activity at 30 mg kg(-1) which was prevented by CPA at 50 micro kg(-1). 5. The A2 receptor agonist, N6-[2-(3,5-dimethoxyphenyl)-2(2-methylphenyl)-ethyl]adenosine (DPMA) had no effect on anxiety behaviour but depressed locomotor activity at the highest dose tested of 1 mg kg(-1). The A2 receptor antagonist, 1,3-dimethyl-l-propargylxanthine (DMPX) had no effect on anxiety-related measures or locomotion and did not modify the anxiolytic-like activity of CPA. 6. Administration of DPMA in combination with anxiolytic doses of CPA prevented the anxiolytic-like activity of the latter. 7. The results suggest that the selective activation of central A1 adenosine receptors induces anxiolytic-like behaviour, while the activation of A2 sites causes locomotor depression and reduces the effects of A1 receptor activation. The absence of any effect of CPX alone suggests that the receptors involved in modulating behaviour in the elevated plus-maze in mice are not activated tonically by endogenous adenosine. PMID:8640355

  11. Anxiolytic activity of adenosine receptor activation in mice.

    PubMed Central

    Jain, N.; Kemp, N.; Adeyemo, O.; Buchanan, P.; Stone, T. W.

    1995-01-01

    1. Purine analogues have been examined for anxiolytic- and anxiogenic-like activity in mice, by use of the elevated plus-maze. 2. The selective A1 receptor agonist, N6-cyclopentyladenosine (CPA) had marked anxiolytic-like activity at 10 and 50 microg kg(-1), with no effect on locomotor performance at these doses. 3. The A1 selective adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (CPX) had no significant effect on anxiety-related measures or locomotor behaviour, but blocked the anxiolytic-like activity of CPA. The hydrophilic xanthine, 8-(p-sulphophenyl) theophylline did not prevent anxiolysis by CPA. 4. Caffeine had anxiogenic-like activity at 30 mg kg(-1) which was prevented by CPA at 50 micro kg(-1). 5. The A2 receptor agonist, N6-[2-(3,5-dimethoxyphenyl)-2(2-methylphenyl)-ethyl]adenosine (DPMA) had no effect on anxiety behaviour but depressed locomotor activity at the highest dose tested of 1 mg kg(-1). The A2 receptor antagonist, 1,3-dimethyl-l-propargylxanthine (DMPX) had no effect on anxiety-related measures or locomotion and did not modify the anxiolytic-like activity of CPA. 6. Administration of DPMA in combination with anxiolytic doses of CPA prevented the anxiolytic-like activity of the latter. 7. The results suggest that the selective activation of central A1 adenosine receptors induces anxiolytic-like behaviour, while the activation of A2 sites causes locomotor depression and reduces the effects of A1 receptor activation. The absence of any effect of CPX alone suggests that the receptors involved in modulating behaviour in the elevated plus-maze in mice are not activated tonically by endogenous adenosine. PMID:8640355

  12. A Binding Site Model and Structure-Activity Relationships for the Rat A3 Adenosine Receptor

    PubMed Central

    VAN GALEN, PHILIP J. M.; VAN BERGEN, ANDREW H.; GALLO-RODRIGUEZ, CAROLA; MELMAN, NELI; OLAH, MARK E.; IJZERMAN, AD P.; STILES, GARY L.; JACOBSON, KENNETH A.

    2012-01-01

    SUMMARY A novel adenosine receptor, the A3 receptor, has recently been cloned. We have systematically investigated the hitherto largely unexplored structure-activity relationships (SARs) for binding at A3 receptors, using 125I-N6-2-(4-aminophenyl)ethyladenosine as a radioligand and membranes from Chinese hamster ovary cells stably transfected with the rat A3-cDNA. As is the case for A1 and A2a, receptors, substitutions at the N6 and 5′ positions of adenosine, the prototypic agonist ligand, may yield fairly potent compounds. However, the highest affinity and A3 selectivity is found for N6,5′-disubstituted compounds, in contrast to A1 and A2a receptors. Thus, N6-benzyladenosine-5′-N-ethylcarboxamide is highly potent (Ki, 6.8 nM) and moderately selective (13- and 14-fold versus A1 and A2a). The N6 region of the A3 receptor also appears to tolerate hydrophilic substitutions, in sharp contrast to the other subtypes. Potencies of N6,5′-disubstituted compounds in inhibition of adenylate cyclase via A3 receptors parallel their high affinity in the binding assay. None of the typical xanthine or nonxanthine (A1/A2) antagonists tested show any appreciable affinity for rat A3 receptors. 1,3-Dialkylxanthines did not antagonize the A3 agonist-induced inhibition of adenylate cyclase. A His residue in helix 6 that is absent in A3 receptors but present in A1/A2 receptors may be causal in this respect. In a molecular model for the rat A3 receptor, this mutation, together with an increased bulkiness of residues surrounding the ligand, make antagonist binding unfavorable when compared with a previously developed A1 receptor model. Second, this A3 receptor model predicted similarities with A1 and A2 receptors in the binding requirements for the ribose moiety and that xanthine-7-ribosides would bind to rat A3 receptors. This hypothesis was supported experimentally by the moderate affinity (Ki 6 μM) of 7-riboside of 1,3-dibutylxanthine, which appears to be a partial agonist at

  13. Immunosuppression via adenosine receptor activation by adenosine monophosphate released from apoptotic cells

    PubMed Central

    Yamaguchi, Hiroshi; Maruyama, Toshihiko; Urade, Yoshihiro; Nagata, Shigekazu

    2014-01-01

    Apoptosis is coupled with recruitment of macrophages for engulfment of dead cells, and with compensatory proliferation of neighboring cells. Yet, this death process is silent, and it does not cause inflammation. The molecular mechanisms underlying anti-inflammatory nature of the apoptotic process remains poorly understood. In this study, we found that the culture supernatant of apoptotic cells activated the macrophages to express anti-inflammatory genes such as Nr4a and Thbs1. A high level of AMP accumulated in the apoptotic cell supernatant in a Pannexin1-dependent manner. A nucleotidase inhibitor and A2a adenosine receptor antagonist inhibited the apoptotic supernatant-induced gene expression, suggesting AMP was metabolized to adenosine by an ecto-5’-nucleotidase expressed on macrophages, to activate the macrophage A2a adenosine receptor. Intraperitoneal injection of zymosan into Adora2a- or Panx1-deficient mice produced high, sustained levels of inflammatory mediators in the peritoneal lavage. These results indicated that AMP from apoptotic cells suppresses inflammation as a ‘calm down’ signal. DOI: http://dx.doi.org/10.7554/eLife.02172.001 PMID:24668173

  14. Receptor antagonism/agonism can be uncoupled from pharmacoperone activity.

    PubMed

    Janovick, Jo Ann; Spicer, Timothy P; Smith, Emery; Bannister, Thomas D; Kenakin, Terry; Scampavia, Louis; Conn, P Michael

    2016-10-15

    Pharmacoperones rescue misrouted mutants of the vasopressin receptor type 2 (V2R) and enable them to traffic to the correct biological locus where they function. Previously, a library of nearly 645,000 structures was interrogated with a high throughput screen; pharmacoperones were identified for V2R mutants with a view toward correcting the underlying mutational defects in nephrogenic diabetes insipidus. In the present study, an orthologous assay was used to evaluate hits from the earlier study. We found no consistent relation between antagonism or agonism and pharmacoperone activity. Active pharmacoperones were identified which had minimal antagonistic activity. This increases the therapeutic reach of these drugs, since virtually all pharmacoperone drugs reported to date were selected from peptidomimetic antagonists. Such mixed-activity drugs have a complex pharmacology limiting their therapeutic utility and requiring their removal prior to stimulation of the receptor with agonist. PMID:27389877

  15. Aryl hydrocarbon receptor antagonism and its role in rheumatoid arthritis

    PubMed Central

    Nguyen, Nam Trung; Nakahama, Taisuke; Nguyen, Chi Hung; Tran, Trang Thu; Le, Van Son; Chu, Hoang Ha; Kishimoto, Tadamitsu

    2015-01-01

    Although rheumatoid arthritis (RA) is the most common autoimmune disease, affecting approximately 1% of the population worldwide, its pathogenic mechanisms are poorly understood. Tobacco smoke, an environmental risk factor for RA, contains several ligands of aryl hydrocarbon receptor (Ahr), also known as dioxin receptor. Ahr plays critical roles in the immune system. We previously demonstrated that Ahr in helper T-cells contributes to development of collagen-induced arthritis, a mouse model of RA. Other studies have shown that cigarette smoke condensate and pure Ahr ligands exacerbate RA by altering bone metabolism and inducing proinflammatory responses in fibroblast-like synoviocytes. Consistent with these findings, several Ahr antagonists such as α-naphthoflavone, resveratrol, and GNF351 reverse the effect of Ahr ligands in RA pathogenesis. In this review, we summarize the current knowledge of Ahr function in the immune system and the potential clinical benefits of Ahr antagonism in treating RA. PMID:27186143

  16. Structural Basis for Simvastatin Competitive Antagonism of Complement Receptor 3.

    PubMed

    Jensen, Maria Risager; Bajic, Goran; Zhang, Xianwei; Laustsen, Anne Kjær; Koldsø, Heidi; Skeby, Katrine Kirkeby; Schiøtt, Birgit; Andersen, Gregers R; Vorup-Jensen, Thomas

    2016-08-12

    The complement system is an important part of the innate immune response to infection but may also cause severe complications during inflammation. Small molecule antagonists to complement receptor 3 (CR3) have been widely sought, but a structural basis for their mode of action is not available. We report here on the structure of the human CR3 ligand-binding I domain in complex with simvastatin. Simvastatin targets the metal ion-dependent adhesion site of the open, ligand-binding conformation of the CR3 I domain by direct contact with the chelated Mg(2+) ion. Simvastatin antagonizes I domain binding to the complement fragments iC3b and C3d but not to intercellular adhesion molecule-1. By virtue of the I domain's wide distribution in binding kinetics to ligands, it was possible to identify ligand binding kinetics as discriminator for simvastatin antagonism. In static cellular experiments, 15-25 μm simvastatin reduced adhesion by K562 cells expressing recombinant CR3 and by primary human monocytes, with an endogenous expression of this receptor. Application of force to adhering monocytes potentiated the effects of simvastatin where only a 50-100 nm concentration of the drug reduced the adhesion by 20-40% compared with untreated cells. The ability of simvastatin to target CR3 in its ligand binding-activated conformation is a novel mechanism to explain the known anti-inflammatory effects of this compound, in particular because this CR3 conformation is found in pro-inflammatory environments. Our report points to new designs of CR3 antagonists and opens new perspectives and identifies druggable receptors from characterization of the ligand binding kinetics in the presence of antagonists. PMID:27339893

  17. Adenosine receptor antagonists alter the stability of human epileptic GABAA receptors

    PubMed Central

    Roseti, Cristina; Martinello, Katiuscia; Fucile, Sergio; Piccari, Vanessa; Mascia, Addolorata; Di Gennaro, Giancarlo; Quarato, Pier Paolo; Manfredi, Mario; Esposito, Vincenzo; Cantore, Gianpaolo; Arcella, Antonella; Simonato, Michele; Fredholm, Bertil B.; Limatola, Cristina; Miledi, Ricardo; Eusebi, Fabrizio

    2008-01-01

    We examined how the endogenous anticonvulsant adenosine might influence γ-aminobutyric acid type A (GABAA) receptor stability and which adenosine receptors (ARs) were involved. Upon repetitive activation (GABA 500 μM), GABAA receptors, microtransplanted into Xenopus oocytes from neurosurgically resected epileptic human nervous tissues, exhibited an obvious GABAA-current (IGABA) run-down, which was consistently and significantly reduced by treatment with the nonselective adenosine receptor antagonist CGS15943 (100 nM) or with adenosine deaminase (ADA) (1 units/ml), that inactivates adenosine. It was also found that selective antagonists of A2B (MRS1706, 10 nM) or A3 (MRS1334, 30 nM) receptors reduced IGABA run-down, whereas treatment with the specific A1 receptor antagonist DPCPX (10 nM) was ineffective. The selective A2A receptor antagonist SCH58261 (10 nM) reduced or potentiated IGABA run-down in ≈40% and ≈20% of tested oocytes, respectively. The ADA-resistant, AR agonist 2-chloroadenosine (2-CA) (10 μM) potentiated IGABA run-down but only in ≈20% of tested oocytes. CGS15943 administration again decreased IGABA run-down in patch-clamped neurons from either human or rat neocortex slices. IGABA run-down in pyramidal neurons was equivalent in A1 receptor-deficient and wt neurons but much larger in neurons from A2A receptor-deficient mice, indicating that, in mouse cortex, GABAA-receptor stability is tonically influenced by A2A but not by A1 receptors. IGABA run-down from wt mice was not affected by 2-CA, suggesting maximal ARs activity by endogenous adenosine. Our findings strongly suggest that cortical A2–A3 receptors alter the stability of GABAA receptors, which could offer therapeutic opportunities. PMID:18809912

  18. Pharmacology of the Adenosine A3 Receptor in the Vasculature and Essential Hypertension

    PubMed Central

    Ho, Ming-Fen; Low, Leanne M.; Rose’Meyer, Roselyn B.

    2016-01-01

    Background Essential hypertension is considered to be a multifactorial disorder and its aetiology has yet to be clearly identified. As the adenosine receptors have a significant role in mediating vasodilation, alterations in their structures or signalling pathways may be involved in the development of hypertension. This study aimed to measure the expression of adenosine A3 receptors in a range of cardiovascular tissues and determine whether they could be altered with essential hypertension, and to functionally test responses to adenosine A3 receptor agonists in coronary blood vessels using the isolated perfused heart preparation. Methods mRNA samples from cardiovascular tissues and a range of blood vessels were collected from 10 week old male spontaneously hypertensive rats and age-gender matched Wistar rats (n = 8). The Langendorff heart perfusion preparation was used to characterise adenosine A3 receptor mediated coronary vasodilation in the rat heart. Results Adenosine A3 receptor agonists induced coronary vasodilation. The expression of adenosine A3 receptors in cardiovascular tissues was altered in a tissue-specific pattern. Specifically, down-regulation of adenosine A3 receptor expression occurred in hypertensive hearts, which might be associated with attenuated vasodilator responses observed in coronary vessels to adenosine A3 receptor agonists. Conclusions This study demonstrated alterations in the expression of adenosine A3 receptors occurred in a tissue specific mode, and reduced adenosine A3 receptor mediated coronary vasodilation in hearts from spontaneously hypertensive rats. Our findings with regard to changes in the adenosine A3 receptor in hypertensive hearts suggest that adenosine A3 receptor might play a role in the physiopathology of essential hypertension and potentially open the way to pharmacologic manipulation of vasomotor activity by the use of adenosine A3 receptor agonists. PMID:26907173

  19. Role of adenosine A2B receptors in inflammation

    PubMed Central

    Feoktistov, Igor; Biaggioni, Italo

    2013-01-01

    Recent progress in our understanding of the unique role of A2B receptors in the regulation of inflammation, immunity and tissue repair was considerably facilitated with the introduction of new pharmacological and genetic tools. However, it also led to seemingly conflicting conclusions on the role of A2B adenosine receptors in inflammation with some publications indicating pro-inflammatory effects and others suggesting the opposite. This chapter reviews the functions of A2B receptors in various cell types related to inflammation and integrated effects of A2B receptor modulation in several animal models of inflammation. It is argued that translation of current findings into novel therapies would require a better understanding of A2B receptors functions in diverse types of inflammatory responses in various tissues and at different points of their progression. PMID:21586358

  20. Ischaemic skeletal muscle hyperaemia in the anaesthetized cat: no contribution of A2A adenosine receptors.

    PubMed Central

    Poucher, S M

    1997-01-01

    1. The present study investigated the contribution of the A2A adenosine receptor subtype to the functional hyperaemia response evoked by muscle contraction in anaesthetized cats when muscle blood flow was limited. 2. Application of a stenosis reduced the hindlimb blood flow at rest from 9.67 +/- 1.80 to 5.53 +/- 0.91 ml min(-1) (kg body mass)(-1) and during muscle contraction from 36.80 +/- 2.55 to 11.11 +/- 1.19 ml min(-1) (kg body mass)(-1) (P < 0.001). The force produced by the extensor digitorum longus and tibialis anterior (EDL-TA) muscle groups was also reduced, from 9.66 +/- 0.56 to 4.10 +/- 0.4 N (kg muscle mass)(-1) (P < 0.01). 3. The selective A2A adenosine receptor antagonist ZM241385 (3 mg kg(-1), I.V.) had no effect upon the hindlimb vascular conductance or muscle contraction responses in the presence of the flow-limiting stenosis. 4. In contrast, in the absence of the flow restriction the vascular conductance response was reduced by 27.5 +/- 5.0% (P < 0.05), whilst the isometric force produced by the EDL-TA muscle group was unaffected (pre- vs. post-contraction, 5.8 +/- 0.8 vs. 4.6 +/- 1.0 N (kg muscle mass)(-1) contraction). Oxygen consumption by the contracting hindlimb muscles was maintained (1.71 +/- 0.25 vs. 1.69 +/- 0.26 ml min(-1) (kg body mass)(-1)) by an increase in the oxygen extraction (51.9 +/- 4.9 vs. 66.2 +/- 6.1%; P< 0.01). 5. These results confirm previous data showing that adenosine, acting at the A2A receptor subtype, can contribute up to 30% of the functional hyperaemia response in the hindlimb of anaesthetized cats under free flow conditions. However, when blood flow is limited by a stenosis, antagonism of the A2A adenosine receptor does not affect functional hyperaemia. Images Figure 1 PMID:9097944

  1. Adenosine deaminase inhibition enhances the inotropic response mediated by A1 adenosine receptor in hyperthyroid guinea pig atrium.

    PubMed

    Kemeny-Beke, Adam; Jakab, Anita; Zsuga, Judit; Vecsernyes, Miklos; Karsai, Denes; Pasztor, Fanni; Grenczer, Maria; Szentmiklosi, Andras Jozsef; Berta, Andras; Gesztelyi, Rudolf

    2007-08-01

    The aim of the present study was to test the hypothesis that inhibition of adenosine deaminase (ADA) enhances the efficiency of signal-transduction of myocardial A1 adenosine receptors in hyperthyroidism. The inotropic response to N6-cyclopentyladenosine (CPA), a selective A1 adenosine receptor agonist resistant to ADA, was investigated in the absence or presence of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an ADA and cGMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE2) inhibitor, or of pentostatin (2'-deoxycoformycin; DCF), an exclusive ADA inhibitor, in left atria isolated from eu- or hyperthyroid guinea pigs. Both ADA inhibitors enhanced the effect of CPA only in hyperthyroid atria. EHNA significantly increased the Emax (mean+/-S.E.M.) from 83.8+/-1.2% to 93.4+/-1.2%, while DCF significantly decreased the logEC50 from -7.5+/-0.07 to -7.83+/-0.07 in hyperthyroid samples. Conversely, EHNA also diminished the logEC50 (from -7.5+/-0.07 to -7.65+/-0.07) and DCF also raised the Emax (from 83.8+/-1.2% to 85.7+/-2%) in hyperthyroidism, but these changes were not significant. In conclusion, ADA inhibition moderately but significantly enhanced the efficiency of A(1) adenosine receptor signaling pathway in the hyperthyroid guinea pig atrium. This suggests that elevated intracellular adenosine level caused by ADA inhibition may improve the suppressed responsiveness to A1 adenosine receptor agonists associated with the hyperthyroid state. Alternatively or in addition, the role of decreased concentration of adenosine degradation products cannot be excluded. Furthermore, in the case of EHNA, inhibition of PDE2 also appears to contribute to the enhanced A1 adenosine receptor signaling in the hyperthyroid guinea pig atrium. PMID:17574432

  2. Adenosine A2A receptor dynamics studied with the novel fluorescent agonist Alexa488-APEC

    PubMed Central

    Brand, Frank; Klutz, Athena; Jacobson, Kenneth A.; Fredholm, Bertil B.; Schulte, Gunnar

    2009-01-01

    G protein-coupled receptors, such as the adenosine A2A receptor, are dynamic proteins, which undergo agonist-dependent redistribution from the cell surface to intracellular membranous compartments, such as endosomes. In order to study the kinetics of adenosine A2A receptor redistribution in living cells, we synthesized a novel fluorescent agonist, Alexa488-APEC. Alexa488-APEC binds to adenosine A2A (Ki = 149 ± 27 nM) as well as A3 receptors (Ki= 240 ± 160 nM) but not to adenosine A1 receptors. Further, we characterized the dose-dependent increase in Alexa488-APEC-induced cAMP production as well as cAMP response element binding (CREB) protein phosphorylation, verifying the ligand’s functionality at adenosine A2A but not A2B receptors. In live cell imaging studies, Alexa488-APEC induced adenosine A2A receptor internalization, which was blocked by the competitive reversible antagonist ZM 241385 and hyperosmolaric sucrose. Further, internalized adenosine A2A receptors co-localized with clathrin and Rab5, indicating that agonist stimulation promotes adenosine A2A receptor uptake through a clathrin-dependent mechanism to Rab5-positive endosomes. The basic characterization of Alexa488-APEC provided here showed that it provides a usefultool for tracing adenosine A2A receptors in vitro. PMID:18603240

  3. Adenosine transporters and receptors: key elements for retinal function and neuroprotection.

    PubMed

    Dos Santos-Rodrigues, Alexandre; Pereira, Mariana R; Brito, Rafael; de Oliveira, Nádia A; Paes-de-Carvalho, Roberto

    2015-01-01

    Adenosine is an important neuroactive substance in the central nervous system, including in the retina where subclasses of adenosine receptors and transporters are expressed since early stages of development. Here, we review some evidence showing that adenosine plays important functions in the mature as well as in the developing tissue. Adenosine transporters are divided into equilibrative and concentrative, and the major transporter subtype present in the retina is the ENT1. This transporter is responsible for a bidirectional transport of adenosine and the uptake or release of this nucleoside appears to be regulated by different signaling pathways that are also controlled by activation of adenosine receptors. Adenosine receptors are also key players in retina physiology regulating a variety of functions in the mature and developing tissue. Regulation of excitatory neurotransmitter release and neuroprotection are the main functions played be adenosine in the mature tissue, while regulation of cell survival and neurogenesis are some of the functions played by adenosine in developing retina. Since adenosine is neuroprotective against excitotoxic and metabolic dysfunctions observed in neurological and ocular diseases, the search for adenosine-related drugs regulating adenosine transporters and receptors can be important for advancement of therapeutic strategies against these diseases. PMID:25817878

  4. Adenosine augments interleukin-10 production by microglial cells through an A2B adenosine receptor-mediated process

    PubMed Central

    Koscsó, Balázs; Csóka, Balázs; Selmeczy, Zsolt; Himer, Leonóra; Pacher, Pál; Virág, László; Haskó, György

    2011-01-01

    Microglia are activated by pathogen-associated molecular patterns and produce pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-12, and the anti-inflammatory cytokine IL-10. Adenosine is an endogenous purine nucleoside and is a ligand of four G protein-coupled adenosine receptors (ARs), which are the A1AR, A2AAR, A2BAR and A3AR. ARs have been shown to suppress TNF-α production by microglia, but their role in regulating IL-10 production has not been studied. Here, we demonstrate that adenosine augments IL-10 production by activated murine microglia while suppressing the production of pro-inflammatory cytokines. Since the order of potency of selective AR agonists in inducing IL-10 production was 5′-N-ethylcarboxamidoadenosine (NECA) > N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) > 2-chloro-N6-cyclopentyladenosine (CCPA) ≥ 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethyl-carboxamidoadenosine (CGS21680), and the A2BAR antagonist MRS-1754 prevented the effect of NECA, we conclude that the stimulatory effect of adenosine on IL-10 production is mediated by the A2BAR. Mechanistically, adenosine augmented IL-10 mRNA accumulation by a transcriptional process. Using mutant IL-10 promoter constructs we showed that a CREB-binding region in the promoter mediated the augmenting effect of adenosine on IL-10 transcription. Chromatin immunoprecipitation analysis demonstrated that adenosine induced CREB phosphorylation at the IL-10 promoter. Silencing CREB using lentivirally delivered shRNA blocked the enhancing effect of adenosine on IL-10 production confirming a role for CREB in mediating the stimulatory effect of adenosine on IL-10 production. In addition, adenosine augmented IL-10 production by stimulating p38 MAPK. Collectively, our results establish that A2BARs augment IL-10 production by activated murine microglia. PMID:22116830

  5. Adenosine receptor antagonist and augmented vasodilation during hypoxic exercise.

    PubMed

    Casey, Darren P; Madery, Brandon D; Pike, Tasha L; Eisenach, John H; Dietz, Niki M; Joyner, Michael J; Wilkins, Brad W

    2009-10-01

    We tested the hypothesis that adenosine contributes to augmented skeletal muscle vasodilation during hypoxic exercise. In separate protocols, subjects performed incremental rhythmic forearm exercise (10% and 20% of maximum) during normoxia and normocapnic hypoxia (80% arterial O2 saturation). In protocol 1 (n = 8), subjects received an intra-arterial administration of saline (control) and aminophylline (adenosine receptor antagonist). In protocol 2 (n = 10), subjects received intra-arterial phentolamine (alpha-adrenoceptor antagonist) and combined phentolamine and aminophylline administration. Forearm vascular conductance (FVC; in ml x min(-1).100 mmHg(-1)) was calculated from forearm blood flow (in ml/min) and blood pressure (in mmHg). In protocol 1, the change in FVC (DeltaFVC; change from normoxic baseline) during hypoxic exercise with saline was 172 +/- 29 and 314 +/- 34 ml x min(-1) x 100 mmHg(-1) (10% and 20%, respectively). Aminophylline administration did not affect DeltaFVC during hypoxic exercise at 10% (190 +/- 29 ml x min(-1)x100 mmHg(-1), P = 0.4) or 20% (287 +/- 48 ml x min(-1) x 100 mmHg(-1), P = 0.3). In protocol 2, DeltaFVC due to hypoxic exercise with phentolamine infusion was 313 +/- 30 and 453 +/- 41 ml x min(-1) x 100 mmHg(-1) (10% and 20% respectively). DeltaFVC was similar at 10% (352 +/- 39 ml min(-1) x 100 mmHg(-1), P = 0.8) and 20% (528 +/- 45 ml x min(-1) x 100 mmHg(-1), P = 0.2) hypoxic exercise with combined phentolamine and aminophylline. In contrast, DeltaFVC to exogenous adenosine was reduced by aminophylline administration in both protocols (P < 0.05 for both). These observations suggest that adenosine receptor activation is not obligatory for the augmented hyperemia during hypoxic exercise in humans. PMID:19661449

  6. Blockage of A2A and A3 adenosine receptors decreases the desensitization of human GABAA receptors microtransplanted to Xenopus oocytes

    PubMed Central

    Roseti, Cristina; Palma, Eleonora; Martinello, Katiuscia; Fucile, Sergio; Morace, Roberta; Esposito, Vincenzo; Cantore, Gianpaolo; Arcella, Antonietta; Giangaspero, Felice; Aronica, Eleonora; Mascia, Addolorata; Di Gennaro, Giancarlo; Quarato, Pier Paolo; Manfredi, Mario; Cristalli, Gloria; Lambertucci, Catia; Marucci, Gabriella; Volpini, Rosaria; Limatola, Cristina; Eusebi, Fabrizio

    2009-01-01

    We previously found that the endogenous anticonvulsant adenosine, acting through A2A and A3 adenosine receptors (ARs), alters the stability of currents (IGABA) generated by GABAA receptors expressed in the epileptic human mesial temporal lobe (MTLE). Here we examined whether ARs alter the stability (desensitization) of IGABA expressed in focal cortical dysplasia (FCD) and in periglioma epileptic tissues. The experiments were performed with tissues from 23 patients, using voltage-clamp recordings in Xenopus oocytes microinjected with membranes isolated from human MTLE and FCD tissues or using patch-clamp recordings of pyramidal neurons in epileptic tissue slices. On repetitive activation, the epileptic GABAA receptors revealed instability, manifested by a large IGABA rundown, which in most of the oocytes (≈70%) was obviously impaired by the new A2A antagonists ANR82, ANR94, and ANR152. In most MTLE tissue-microtransplanted oocytes, a new A3 receptor antagonist (ANR235) significantly improved IGABA stability. Moreover, patch-clamped pyramidal neurons from human neocortical slices of periglioma epileptic tissues exhibited altered IGABA rundown on ANR94 treatment. Our findings indicate that antagonizing A2A and A3 receptors increases the IGABA stability in different epileptic tissues and suggest that adenosine derivatives may offer therapeutic opportunities in various forms of human epilepsy. PMID:19721003

  7. The adenosine system modulates Toll-like receptor function: basic mechanisms, clinical correlates and translational opportunities

    PubMed Central

    Coombs, Melanie R. Power; Belderbos, Mirjam E.; Gallington, Leighanne C.; Bont, Louis; Levy, Ofer

    2014-01-01

    Adenosine is an endogenous purine metabolite whose concentration in human blood plasma rises from nanomolar to micromolar during stress or hypoxia. Leukocytes express seven-transmembrane adenosine receptors whose engagement modulates Toll-like receptor-mediated cytokine responses, in part via modulation of intracellular cyclic adenosine monophosphate (cAMP). Adenosine congeners are used clinically to treat arrhythmias and apnea of prematurity. Herein we consider the potential of adenosine congeners as innate immune response modifiers to prevent and/or treat infection. PMID:21342073

  8. The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation.

    PubMed

    Weidmann, Chase A; Raynard, Nathan A; Blewett, Nathan H; Van Etten, Jamie; Goldstrohm, Aaron C

    2014-08-01

    PUF proteins are potent repressors that serve important roles in stem cell maintenance, neurological processes, and embryonic development. These functions are driven by PUF protein recognition of specific binding sites within the 3' untranslated regions of target mRNAs. In this study, we investigated mechanisms of repression by the founding PUF, Drosophila Pumilio, and its human orthologs. Here, we evaluated a previously proposed model wherein the Pumilio RNA binding domain (RBD) binds Argonaute, which in turn blocks the translational activity of the eukaryotic elongation factor 1A. Surprisingly, we found that Argonautes are not necessary for repression elicited by Drosophila and human PUFs in vivo. A second model proposed that the RBD of Pumilio represses by recruiting deadenylases to shorten the mRNA's polyadenosine tail. Indeed, the RBD binds to the Pop2 deadenylase and accelerates deadenylation; however, this activity is not crucial for regulation. Rather, we determined that the poly(A) is necessary for repression by the RBD. Our results reveal that poly(A)-dependent repression by the RBD requires the poly(A) binding protein, pAbp. Furthermore, we show that repression by the human PUM2 RBD requires the pAbp ortholog, PABPC1. Pumilio associates with pAbp but does not disrupt binding of pAbp to the mRNA. Taken together, our data support a model wherein the Pumilio RBD antagonizes the ability of pAbp to promote translation. Thus, the conserved function of the PUF RBD is to bind specific mRNAs, antagonize pAbp function, and promote deadenylation. PMID:24942623

  9. Essential role of adenosine, adenosine A1 receptors, and ATP-sensitive K+ channels in cerebral ischemic preconditioning.

    PubMed Central

    Heurteaux, C; Lauritzen, I; Widmann, C; Lazdunski, M

    1995-01-01

    Preconditioning with sublethal ischemia protects against neuronal damage after subsequent lethal ischemic insults in hippocampal neurons. A pharmacological approach using agonists and antagonists at the adenosine A1 receptor as well as openers and blockers of ATP-sensitive K+ channels has been combined with an analysis of neuronal death and gene expression of subunits of glutamate and gamma-aminobutyric acid receptors, HSP70, c-fos, c-jun, and growth factors. It indicates that the mechanism of ischemic tolerance involves a cascade of events including liberation of adenosine, stimulation of adenosine A1 receptors, and, via these receptors, opening of sulfonylurea-sensitive ATP-sensitive K+ channels. Images Fig. 2 Fig. 3 PMID:7753861

  10. Antagonism of sigma-1 receptors blocks compulsive-like eating.

    PubMed

    Cottone, Pietro; Wang, Xiaofan; Park, Jin Won; Valenza, Marta; Blasio, Angelo; Kwak, Jina; Iyer, Malliga R; Steardo, Luca; Rice, Kenner C; Hayashi, Teruo; Sabino, Valentina

    2012-11-01

    Binge eating disorder is an addiction-like disorder characterized by episodes of rapid and excessive food consumption within discrete periods of time which occur compulsively despite negative consequences. This study was aimed at determining whether antagonism of Sigma-1 receptors (Sig-1Rs) blocked compulsive-like binge eating. We trained male wistar rats to obtain a sugary, highly palatable diet (Palatable group) or a regular chow diet (Chow control group), for 1 h a day under fixed ratio 1 operant conditioning. Following intake stabilization, we evaluated the effects of the selective Sig-1R antagonist BD-1063 on food responding. Using a light/dark conflict test, we also tested whether BD-1063 could block the time spent and the food eaten in an aversive, open compartment, where the palatable diet was offered. Furthermore, we measured Sig-1R mRNA and protein expression in several brain areas of the two groups, 24 h after the last binge session. Palatable rats rapidly developed binge-like eating, escalating the 1 h intake by four times, and doubling the eating rate and the regularity of food responding, compared to Chow rats. BD-1063 dose-dependently reduced binge-like eating and the regularity of food responding, and blocked the increased eating rate in Palatable rats. In the light/dark conflict test, BD-1063 antagonized the increased time spent in the aversive compartment and the increased intake of the palatable diet, without affecting motor activity. Finally, Palatable rats showed reduced Sig-1R mRNA expression in prefrontal and anterior cingulate cortices, and a two-fold increase in Sig-1R protein expression in anterior cingulate cortex compared to control Chow rats. These findings suggest that the Sig-1R system may contribute to the neurobiological adaptations driving compulsive-like eating, opening new avenues of investigation towards pharmacologically treating binge eating disorder. PMID:22713906

  11. Photomodulation of G Protein-Coupled Adenosine Receptors by a Novel Light-Switchable Ligand

    PubMed Central

    2015-01-01

    The adenosinergic system operates through G protein-coupled adenosine receptors, which have become promising therapeutic targets for a wide range of pathological conditions. However, the ubiquity of adenosine receptors and the eventual lack of selectivity of adenosine-based drugs have frequently diminished their therapeutic potential. Accordingly, here we aimed to develop a new generation of light-switchable adenosine receptor ligands that change their intrinsic activity upon irradiation, thus allowing the spatiotemporal control of receptor functioning (i.e., receptor activation/inactivation dependent on location and timing). Therefore, we synthesized an orthosteric, photoisomerizable, and nonselective adenosine receptor agonist, nucleoside derivative MRS5543 containing an aryl diazo linkage on the N6 substituent, which in the dark (relaxed isomer) behaved as a full adenosine A3 receptor (A3R) and partial adenosine A2A receptor (A2AR) agonist. Conversely, upon photoisomerization with blue light (460 nm), it remained a full A3R agonist but became an A2AR antagonist. Interestingly, molecular modeling suggested that structural differences encountered within the third extracellular loop of each receptor could modulate the intrinsic, receptor subtype-dependent, activity. Overall, the development of adenosine receptor ligands with photoswitchable activity expands the pharmacological toolbox in support of research and possibly opens new pharmacotherapeutic opportunities. PMID:25248077

  12. The human D2 dopamine receptor synergizes with the A2A adenosine receptor to stimulate adenylyl cyclase in PC12 cells.

    PubMed

    Kudlacek, Oliver; Just, Herwig; Korkhov, Vladimir M; Vartian, Nina; Klinger, Markus; Pankevych, Halyna; Yang, Qiong; Nanoff, Christian; Freissmuth, Michael; Boehm, Stefan

    2003-07-01

    The adenosine A(2A) receptor and the dopamine D(2) receptor are prototypically coupled to G(s) and G(i)/G(o), respectively. In striatal intermediate spiny neurons, these receptors are colocalized in dendritic spines and act as mutual antagonists. This antagonism has been proposed to occur at the level of the receptors or of receptor-G protein coupling. We tested this model in PC12 cells which endogenously express A(2A) receptors. The human D(2) receptor was introduced into PC12 cells by stable transfection. A(2A)-agonist-mediated inhibition of D(2) agonist binding was absent in PC12 cell membranes but present in HEK293 cells transfected as a control. However, in the resulting PC12 cell lines, the action of the D(2) agonist quinpirole depended on the expression level of the D(2) receptor: at low and high receptor levels, the A(2A)-agonist-induced elevation of cAMP was enhanced and inhibited, respectively. Forskolin-stimulated cAMP formation was invariably inhibited by quinpirole. The effects of quinpirole were abolished by pretreatment with pertussis toxin. A(2A)-receptor-mediated cAMP formation was inhibited by other G(i)/G(o)-coupled receptors that were either endogenously present (P(2y12)-like receptor for ADP) or stably expressed after transfection (A(1) adenosine, metabotropic glutamate receptor-7A). Similarly, voltage activated Ca(2+) channels were inhibited by the endogenous P(2Y) receptor and by the heterologously expressed A(1) receptor but not by the D(2) receptor. These data indicate functional segregation of signaling components. Our observations are thus compatible with the proposed model that D(2) and A(2A) receptors are closely associated, but they highlight the fact that this interaction can also support synergism. PMID:12784121

  13. Glucocorticoid receptor antagonism reverts docetaxel resistance in human prostate cancer

    PubMed Central

    Kroon, Jan; Puhr, Martin; Buijs, Jeroen T; van der Horst, Geertje; Hemmer, Daniëlle M; Marijt, Koen A; Hwang, Ming S; Masood, Motasim; Grimm, Stefan; Storm, Gert; Metselaar, Josbert M; Meijer, Onno C; Culig, Zoran; van der Pluijm, Gabri

    2016-01-01

    Resistance to docetaxel is a major clinical problem in advanced prostate cancer (PCa). Although glucocorticoids (GCs) are frequently used in combination with docetaxel, it is unclear to what extent GCs and their receptor, the glucocorticoid receptor (GR), contribute to the chemotherapy resistance. In this study, we aim to elucidate the role of the GR in docetaxel-resistant PCa in order to improve the current PCa therapies. GR expression was analyzed in a tissue microarray of primary PCa specimens from chemonaive and docetaxel-treated patients, and in cultured PCa cell lines with an acquired docetaxel resistance (PC3-DR, DU145-DR, and 22Rv1-DR). We found a robust overexpression of the GR in primary PCa from docetaxel-treated patients and enhanced GR levels in cultured docetaxel-resistant human PCa cells, indicating a key role of the GR in docetaxel resistance. The capability of the GR antagonists (RU-486 and cyproterone acetate) to revert docetaxel resistance was investigated and revealed significant resensitization of docetaxel-resistant PCa cells for docetaxel treatment in a dose- and time-dependent manner, in which a complete restoration of docetaxel sensitivity was achieved in both androgen receptor (AR)-negative and AR-positive cell lines. Mechanistically, we demonstrated down-regulation of Bcl-xL and Bcl-2 upon GR antagonism, thereby defining potential treatment targets. In conclusion, we describe the involvement of the GR in the acquisition of docetaxel resistance in human PCa. Therapeutic targeting of the GR effectively resensitizes docetaxel-resistant PCa cells. These findings warrant further investigation of the clinical utility of the GR antagonists in the management of patients with advanced and docetaxel-resistant PCa. PMID:26483423

  14. Modulation of bladder function by luminal adenosine turnover and A1 receptor activation

    PubMed Central

    Prakasam, H. Sandeep; Herrington, Heather; Roppolo, James R.; Jackson, Edwin K.

    2012-01-01

    The bladder uroepithelium transmits information to the underlying nervous and musculature systems, is under constant cyclical strain, expresses all four adenosine receptors (A1, A2A, A2B, and A3), and is a site of adenosine production. Although adenosine has a well-described protective effect in several organs, there is a lack of information about adenosine turnover in the uroepithelium or whether altering luminal adenosine concentrations impacts bladder function or overactivity. We observed that the concentration of extracellular adenosine at the mucosal surface of the uroepithelium was regulated by ecto-adenosine deaminase and by equilibrative nucleoside transporters, whereas adenosine kinase and equilibrative nucleoside transporters modulated serosal levels. We further observed that enriching endogenous adenosine by blocking its routes of metabolism or direct activation of mucosal A1 receptors with 2-chloro-N6-cyclopentyladenosine (CCPA), a selective agonist, stimulated bladder activity by lowering the threshold pressure for voiding. Finally, CCPA did not quell bladder hyperactivity in animals with acute cyclophosphamide-induced cystitis but instead exacerbated their irritated bladder phenotype. In conclusion, we find that adenosine levels at both surfaces of the uroepithelium are modulated by turnover, that blocking these pathways or stimulating A1 receptors directly at the luminal surface promotes bladder contractions, and that adenosine further stimulates voiding in animals with cyclophosphamide-induced cystitis. PMID:22552934

  15. Distribution of adenosine receptors in human sclera fibroblasts

    PubMed Central

    Cui, Dongmei; Trier, Klaus; Chen, Xiang; Zeng, Junwen; Yang, Xiao; Hu, Jianmin

    2008-01-01

    Purpose Systemic treatment with adenosine receptor antagonists has been reported to affect the biochemistry and ultrastructure of rabbit sclera. This study was conducted to determine whether adenosine receptors (ADORs) are present in human scleral fibroblasts (HSF). Methods Primary HSF were cultured in vitro and identified with anti-vimentin, anti-keratin, anti-desmin, and anti-S-100 antibodies. Confocal fluorescence microscopy was used to study the distribution of ADORs in the HSF cell lines and in the frozen human scleral sections. ADOR protein expression in HSF and human sclera was confirmed by western blot analysis of cell lysates. Results ADORs were expressed in both HSF and human sclera. This was confirmed by western blot. ADORA1 expression was concentrated in the nucleus. ADORA2A was concentrated mainly in one side of the cytoplasm, and ADORA2B was found both in the nucleus and the cytoplasm. ADORA3 was expressed weakly in the cytoplasm. Conclusions All four subtypes of ADOR were found in HSF and may play a role in scleral remodeling. PMID:18385786

  16. Adenosine protected against pulmonary edema through transporter- and receptor A2-mediated endothelial barrier enhancement

    PubMed Central

    Lu, Qing; Harrington, Elizabeth O.; Newton, Julie; Casserly, Brian; Radin, Gregory; Warburton, Rod; Zhou, Yang; Blackburn, Michael R.

    2010-01-01

    We have previously demonstrated that adenosine plus homocysteine enhanced endothelial basal barrier function and protected against agonist-induced barrier dysfunction in vitro through attenuation of RhoA activation by inhibition of isoprenylcysteine-O-carboxyl methyltransferase. In the current study, we tested the effect of elevated adenosine on pulmonary endothelial barrier function in vitro and in vivo. We noted that adenosine alone dose dependently enhanced endothelial barrier function. While adenosine receptor A1 or A3 antagonists were ineffective, an adenosine transporter inhibitor, NBTI, or a combination of DPMX and MRS1754, antagonists for adenosine receptors A2A and A2B, respectively, partially attenuated the barrier-enhancing effect of adenosine. Similarly, inhibition of both A2A and A2B receptors with siRNA also blunted the effect of adenosine on barrier function. Interestingly, inhibition of both transporters and A2A/A2B receptors completely abolished adenosine-induced endothelial barrier enhancement. The adenosine receptor A2A and A2B agonist, NECA, also significantly enhanced endothelial barrier function. These data suggest that both adenosine transporters and A2A and A2B receptors are necessary for exerting maximal effect of adenosine on barrier enhancement. We also found that adenosine enhanced Rac1 GTPase activity and overexpression of dominant negative Rac1 attenuated adenosine-induced increases in focal adhesion complexes. We further demonstrated that elevation of cellular adenosine by inhibition of adenosine deaminase with Pentostatin significantly enhanced endothelial basal barrier function, an effect that was also associated with enhanced Rac1 GTPase activity and with increased focal adhesion complexes and adherens junctions. Finally, using a non-inflammatory acute lung injury (ALI) model induced by α-naphthylthiourea, we found that administration of Pentostatin, which elevated lung adenosine level by 10-fold, not only attenuated the

  17. Increased adenosine contributes to penile fibrosis, a dangerous feature of priapism, via A2B adenosine receptor signaling

    PubMed Central

    Wen, Jiaming; Jiang, Xianzhen; Dai, Yingbo; Zhang, Yujin; Tang, Yuxin; Sun, Hong; Mi, Tiejuan; Phatarpekar, Prasad V.; Kellems, Rodney E.; Blackburn, Michael R.; Xia, Yang

    2010-01-01

    Priapism is a condition of persistent penile erection in the absence of sexual excitation. Of men with sickle cell disease (SCD), 40% display priapism. The disorder is a dangerous and urgent condition, given its association with penile fibrosis and eventual erectile dysfunction. Current strategies to prevent its progression are poor because of a lack of fundamental understanding of the molecular mechanisms for penile fibrosis in priapism. Here we demonstrate that increased adenosine is a novel causative factor contributing to penile fibrosis in two independent animal models of priapism, adenosine deaminase (ADA)-deficient mice and SCD transgenic mice. An important finding is that chronic reduction of adenosine by ADA enzyme therapy successfully attenuated penile fibrosis in both mouse models, indicating an essential role of increased adenosine in penile fibrosis and a novel therapeutic possibility for this serious complication. Subsequently, we identified that both mice models share a similar fibrotic gene expression profile in penile tissue (including procollagen I, TGF-β1, and plasminogen activator inhibitor-1 mRNA), suggesting that they share similar signaling pathways for progression to penile fibrosis. Thus, in an effort to decipher specific cell types and underlying mechanism responsible for adenosine-mediated penile fibrosis, we purified corpus cavernosal fibroblast cells (CCFCs), the major cell type involved in this process, from wild-type mice. Quantitative RT-PCR showed that the major receptor expressed in these cells is the adenosine receptor A2BR. Based on this fact, we further purified CCFCs from A2BR-deficient mice and demonstrated that A2BR is essential for excess adenosine-mediated penile fibrosis. Finally, we revealed that TGF-β functions downstream of the A2BR to increase CCFC collagen secretion and proliferation. Overall, our studies identify an essential role of increased adenosine in the pathogenesis of penile fibrosis via A2BR signaling and

  18. Cloning, expression and pharmacological characterization of rabbit adenosine A1 and A3 receptors.

    PubMed

    Hill, R J; Oleynek, J J; Hoth, C F; Kiron, M A; Weng, W; Wester, R T; Tracey, W R; Knight, D R; Buchholz, R A; Kennedy, S P

    1997-01-01

    The role of adenosine A1 and A3 receptors in mediating cardioprotection has been studied predominantly in rabbits, yet the pharmacological characteristics of rabbit adenosine A1 and A3 receptor subtypes are unknown. Thus, the rabbit adenosine A3 receptor was cloned and expressed, and its pharmacology was compared with that of cloned adenosine A1 receptors. Stable transfection of rabbit A1 or A3 cDNAs in Chinese hamster ovary-K1 cells resulted in high levels of expression of each of the receptors, as demonstrated by high-affinity binding of the A1/A3 adenosine receptor agonist N6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA). For both receptors, binding of 125I-ABA was inhibited by the GTP analog 5'-guanylimidodiphosphate, and forskolin-stimulated cyclic AMP accumulation was inhibited by the adenosine receptor agonist (R)-phenylisopropyladenosine. The rank orders of potency of adenosine receptor agonists for inhibition of 125I-ABA binding were as follows: rabbit A1, N6-cyclopentyladenosine = (R)-phenylisopropyladenosine > N-ethylcarboxamidoadenosine > or = I-ABA > or = N6-2-(4-aminophenyl) ethyladenosine > > N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > N6-(4-amino-3-benzyl)adenosine; rabbit A3, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > or = I-ABA > > N-ethylcarboxamidoadenosine > N6-2-(4-aminophenyl) ethyladenosine = N6-cyclopentyladenosine = (R)-phenylisopropyladenosine > N6-(4-amino-3-benzyl)adenosine. The adenosine receptor antagonist rank orders were as follow: rabbit A1, 8-cyclopentyl-1,3-dipropylxanthine > 1,3- dipropyl-8-(4-acrylate)phenylxanthine > or = xanthine amine congener > > 8-(p-sulfophenyl)theophylline; rabbit A3, xanthine amine congener > 1,3-dipropyl-8-(4-acrylate)phenylxanthine > or = 8-cyclopentyl-1,3-dipropylxanthine > > 8-(p-sulfophenyl)theophylline. These observations confirm the identity of the expressed proteins as A1 and A3 receptors. The results will facilitate further in-depth studies of the roles of A1 and A3 receptors in

  19. Pyrazolo-triazolo-pyrimidines as adenosine receptor antagonists: Effect of the N-5 bond type on the affinity and selectivity at the four adenosine receptor subtypes

    PubMed Central

    Bolcato, Chiara; Cusan, Claudia; Pastorin, Giorgia; Cacciari, Barbara; Klotz, Karl Norbert; Morizzo, Erika

    2007-01-01

    In the last few years, many efforts have been made to search for potent and selective human A3 adenosine antagonists. In particular, one of the most promising human A3 adenosine receptor antagonists is represented by the pyrazolo-triazolo-pyrimidine family. This class of compounds has been strongly investigated from the point of view of structure-activity relationships. In particular, it has been observed that fundamental requisites for having both potency and selectivity at the human A3 adenosine receptors are the presence of a small substituent at the N8 position and an unsubstitued phenyl carbamoyl moiety at the N5 position. In this study, we report the role of the N5-bond type on the affinity and selectivity at the four adenosine receptor subtypes. The observed structure-activity relationships of this class of antagonists are also exhaustively rationalized using the recently published ligand-based homology modeling approach. PMID:18368532

  20. Adenosine receptor signaling: a key to opening the blood-brain door.

    PubMed

    Bynoe, Margaret S; Viret, Christophe; Yan, Angela; Kim, Do-Geun

    2015-01-01

    The aim of this review is to outline evidence that adenosine receptor (AR) activation can modulate blood-brain barrier (BBB) permeability and the implications for disease states and drug delivery. Barriers of the central nervous system (CNS) constitute a protective and regulatory interface between the CNS and the rest of the organism. Such barriers allow for the maintenance of the homeostasis of the CNS milieu. Among them, the BBB is a highly efficient permeability barrier that separates the brain micro-environment from the circulating blood. It is made up of tight junction-connected endothelial cells with specialized transporters to selectively control the passage of nutrients required for neural homeostasis and function, while preventing the entry of neurotoxic factors. The identification of cellular and molecular mechanisms involved in the development and function of CNS barriers is required for a better understanding of CNS homeostasis in both physiological and pathological settings. It has long been recognized that the endogenous purine nucleoside adenosine is a potent modulator of a large number of neurological functions. More recently, experimental studies conducted with human/mouse brain primary endothelial cells as well as with mouse models, indicate that adenosine markedly regulates BBB permeability. Extracellular adenosine, which is efficiently generated through the catabolism of ATP via the CD39/CD73 ecto-nucleotidase axis, promotes BBB permeability by signaling through A1 and A2A ARs expressed on BBB cells. In line with this hypothesis, induction of AR signaling by selective agonists efficiently augments BBB permeability in a transient manner and promotes the entry of macromolecules into the CNS. Conversely, antagonism of AR signaling blocks the entry of inflammatory cells and soluble factors into the brain. Thus, AR modulation of the BBB appears as a system susceptible to tighten as well as to permeabilize the BBB. Collectively, these findings point

  1. Anthraquinone emodin inhibits human cancer cell invasiveness by antagonizing P2X7 receptors.

    PubMed

    Jelassi, Bilel; Anchelin, Monique; Chamouton, Julie; Cayuela, María Luisa; Clarysse, Lucie; Li, Junying; Goré, Jacques; Jiang, Lin-Hua; Roger, Sébastien

    2013-07-01

    The adenosine 5'-triphosphate (ATP)-gated Ca(2+)-permeable channel P2X7 receptor (P2X7R) is strongly upregulated in many tumors and cancer cells, and has an important role in cancer cell invasion associated with metastases. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an anthraquinone derivative originally isolated from Rheum officinale Baill known for decades to possess anticancer properties. In this study, we examined the effects of emodin on P2X7R-dependent Ca(2+) signaling, extracellular matrix degradation, and in vitro and in vivo cancer cell invasiveness using highly aggressive human cancer cells. Inclusion of emodin at doses ≤10 µM in cell culture had no or very mild effect on the cell viability. ATP elicited increases in intracellular Ca(2+) concentration were reduced by 35 and 60% by 1 and 10 µM emodin, respectively. Emodin specifically inhibited P2X7R-mediated currents with an IC50 of 3 µM and did not inhibit the currents mediated by the other human P2X receptors heterologously expressed in human embryonic kidney (HEK293T) cells. ATP-induced increase in gelatinolytic activity, in cancer cell invasiveness in vitro and in cell morphology changes were prevented by 1 µM emodin. Furthermore, such ATP-evoked effects and inhibition by emodin were almost completely ablated in cancer cells transfected with P2X7R-specific small interfering RNA (siRNA) but not with scrambled siRNA. Finally, the in vivo invasiveness of the P2X7R-positive MDA-MB-435s breast cancer cells, assessed using a zebrafish model of micrometastases, was suppressed by 40 and 50% by 1 and 10 µM emodin. Taken together, these results provide consistent evidence to indicate that emodin inhibits human cancer cell invasiveness by specifically antagonizing the P2X7R. PMID:23524196

  2. Getting personal: Endogenous adenosine receptor signaling in lymphoblastoid cell lines.

    PubMed

    Hillger, J M; Diehl, C; van Spronsen, E; Boomsma, D I; Slagboom, P E; Heitman, L H; IJzerman, A P

    2016-09-01

    Genetic differences between individuals that affect drug action form a challenge in drug therapy. Many drugs target G protein-coupled receptors (GPCRs), and a number of receptor variants have been noted to impact drug efficacy. This, however, has never been addressed in a systematic way, and, hence, we studied real-life genetic variation of receptor function in personalized cell lines. As a showcase we studied adenosine A2A receptor (A2AR) signaling in lymphoblastoid cell lines (LCLs) derived from a family of four from the Netherlands Twin Register (NTR), using a non-invasive label-free cellular assay. The potency of a partial agonist differed significantly for one individual. Genotype comparison revealed differences in two intron SNPs including rs2236624, which has been associated with caffeine-induced sleep disorders. While further validation is needed to confirm genotype-specific effects, this set-up clearly demonstrated that LCLs are a suitable model system to study genetic influences on A2AR response in particular and GPCR responses in general. PMID:27297283

  3. Serotonin 2B Receptor Antagonism Prevents Heritable Pulmonary Arterial Hypertension

    PubMed Central

    Schroer, Alison K.; Chen, Peter; Ryzhova, Larisa M.; Gladson, Santhi; Shay, Sheila; Hutcheson, Joshua D.; Merryman, W. David

    2016-01-01

    Serotonergic anorexigens are the primary pharmacologic risk factor associated with pulmonary arterial hypertension (PAH), and the resulting PAH is clinically indistinguishable from the heritable form of disease, associated with BMPR2 mutations. Both BMPR2 mutation and agonists to the serotonin receptor HTR2B have been shown to cause activation of SRC tyrosine kinase; conversely, antagonists to HTR2B inhibit SRC trafficking and downstream function. To test the hypothesis that a HTR2B antagonist can prevent BMRP2 mutation induced PAH by restricting aberrant SRC trafficking and downstream activity, we exposed BMPR2 mutant mice, which spontaneously develop PAH, to a HTR2B antagonist, SB204741, to block the SRC activation caused by BMPR2 mutation. SB204741 prevented the development of PAH in BMPR2 mutant mice, reduced recruitment of inflammatory cells to their lungs, and reduced muscularization of their blood vessels. By atomic force microscopy, we determined that BMPR2 mutant mice normally had a doubling of vessel stiffness, which was substantially normalized by HTR2B inhibition. SB204741 reduced SRC phosphorylation and downstream activity in BMPR2 mutant mice. Gene expression arrays indicate that the primary changes were in cytoskeletal and muscle contractility genes. These results were confirmed by gel contraction assays showing that HTR2B inhibition nearly normalizes the 400% increase in gel contraction normally seen in BMPR2 mutant smooth muscle cells. Heritable PAH results from increased SRC activation, cellular contraction, and vascular resistance, but antagonism of HTR2B prevents SRC phosphorylation, downstream activity, and PAH in BMPR2 mutant mice. PMID:26863209

  4. Adenosine A1 receptor antagonist prolongs survival in the hypoxic rat.

    PubMed

    Gao, E; Kaplan, J L; Shi, Y; Victain, M; Dalsey, W C; de Garavilla, L

    2001-09-01

    The hypothesis that adenosine A1 receptor (A1AdoR) selective antagonism limits cardiac depression and prolongs survival during acute global hypoxia was tested in a postinsult treatment model using KW-3902 ([8-(noradamantan-3-yl)-1,3-dipropylxanthine]), an A1AdoR selective antagonist. Rats were anesthetized, paralyzed, then ventilated with 8% O2 (hypoxia). In protocol I, 5 min after hypoxia, rats were treated with saline, drug vehicle, or KW-3902 (0.1 mg/kg i.v.). In protocol II, KW-3902 treatment occurred 2.5, 5, or 7.5 min after hypoxia. In protocol I, after hypoxia, left ventricular contractility, heart rate, and systemic mean arterial blood pressure decreased rapidly in saline-and vehicle-treated groups. In contrast, KW-3902 significantly attenuated the decline in these variables. Survival time (the time from the commencement of hypoxia until death) was more prolonged with KW-3902 (109.5 +/- 9.1 min) than with saline (37.6 +/- 5.0 min) or vehicle (35.0 +/- 4.2 min) (p < 0.001). In protocol II, survival time increased from 29.2 +/- 5.5 min in the 7.5-min treatment group to 109.5 +/- 9.5 min (5-min group) and 245.9 +/- 26.1 min (2.5-min group; p < 0.001). KW-3902 prolongs survival in this model, presumably by antagonizing A1AdoR-mediated inhibition of cardiac function. Also, treatment efficacy is highly time dependent. PMID:11486243

  5. Kaempferol inhibits cancer cell growth by antagonizing estrogen-related receptor α and γ activities.

    PubMed

    Wang, Haibin; Gao, Minghui; Wang, Junjian

    2013-11-01

    Kaempferol is a dietary flavonoid that can function as a selective estrogen receptor modulator (SERM). Estrogen-related receptors alpha and gamma (ERRα and ERRγ) are orphan nuclear receptors that play important roles in mitochondrial biogenesis and cancer development. We have shown that kaempferol can functionally antagonize the activities of ERRs based on both response element reporter systems and target gene analysis. Kaempferol modulation of mitochondrial function and suppression cancer cell growth has been confirmed. These findings suggest that kaempferol may exert their anti-cancer activities through antagonizing ERRs activities. PMID:23852933

  6. The adenosine metabolite inosine is a functional agonist of the adenosine A2A receptor with a unique signaling bias.

    PubMed

    Welihinda, Ajith A; Kaur, Manmeet; Greene, Kelly; Zhai, Yongjiao; Amento, Edward P

    2016-06-01

    Inosine is an endogenous purine nucleoside that is produced by catabolism of adenosine. Adenosine has a short half-life (approximately 10s) and is rapidly deaminated to inosine, a stable metabolite with a half-life of approximately 15h. Resembling adenosine, inosine acting through adenosine receptors (ARs) exerts a wide range of anti-inflammatory and immunomodulatory effects in vivo. The immunomodulatory effects of inosine in vivo, at least in part, are mediated via the adenosine A2A receptor (A2AR), an observation that cannot be explained fully by in vitro pharmacological characterization of inosine at the A2AR. It is unclear whether the in vivo effects of inosine are due to inosine or a metabolite of inosine engaging the A2AR. Here, utilizing a combination of label-free, cell-based, and membrane-based functional assays in conjunction with an equilibrium agonist-binding assay we provide evidence for inosine engagement at the A2AR and subsequent activation of downstream signaling events. Inosine-mediated A2AR activation leads to cAMP production with an EC50 of 300.7μM and to extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation with an EC50 of 89.38μM. Our data demonstrate that inosine produces ERK1/2-biased signaling whereas adenosine produces cAMP-biased signaling at the A2AR, highlighting pharmacological differences between these two agonists. Given the in vivo stability of inosine, our data suggest an additional, previously unrecognized, mechanism that utilizes inosine to functionally amplify and prolong A2AR activation in vivo. PMID:26903141

  7. Adenosine A2A receptor deficiency alleviates blast-induced cognitive dysfunction

    PubMed Central

    Ning, Ya-Lei; Yang, Nan; Chen, Xing; Xiong, Ren-Ping; Zhang, Xiu-Zhu; Li, Ping; Zhao, Yan; Chen, Xing-Yun; Liu, Ping; Peng, Yan; Wang, Zheng-Guo; Chen, Jiang-Fan; Zhou, Yuan-Guo

    2013-01-01

    Traumatic brain injury (TBI), particularly explosive blast-induced TBI (bTBI), has become the most prevalent injury among military personnel. The disruption of cognitive function is one of the most serious consequences of bTBI because its long-lasting effects prevent survivors fulfilling their active duty and resuming normal civilian life. However, the mechanisms are poorly understood and there is no treatment available. This study investigated the effects of adenosine A2A receptor (A2AR) on bTBI-induced cognitive deficit, and explored the underlying mechanisms. After being subjected to moderate whole-body blast injury, mice lacking the A2AR (A2AR knockout (KO)) showed less severity and shorter duration of impaired spatial reference memory and working memory than wild-type mice did. In addition, bTBI-induced cortical and hippocampal lesions, as well as proinflammatory cytokine expression, glutamate release, edema, cell loss, and gliosis in both early and prolonged phases of the injury, were significantly attenuated in A2AR KO mice. The results suggest that early injury and chronic neuropathological damages are important mechanisms of bTBI-induced cognitive impairment, and that the impairment can be attenuated by preventing A2AR activation. These findings suggest that A2AR antagonism is a potential therapeutic strategy for mild-to-moderate bTBI and consequent cognitive impairment. PMID:23921902

  8. Association of adenosine receptor gene polymorphisms and in vivo adenosine A1 receptor binding in the human brain.

    PubMed

    Hohoff, Christa; Garibotto, Valentina; Elmenhorst, David; Baffa, Anna; Kroll, Tina; Hoffmann, Alana; Schwarte, Kathrin; Zhang, Weiqi; Arolt, Volker; Deckert, Jürgen; Bauer, Andreas

    2014-12-01

    Adenosine A1 receptors (A1ARs) and the interacting adenosine A2A receptors are implicated in neurological and psychiatric disorders. Variants within the corresponding genes ADORA1 and ADORA2A were shown associated with pathophysiologic alterations, particularly increased anxiety. It is unknown so far, if these variants might modulate the A1AR distribution and availability in different brain regions. In this pilot study, the influence of ADORA1 and ADORA2A variants on in vivo A1AR binding was assessed with the A1AR-selective positron emission tomography (PET) radioligand [(18)F]CPFPX in brains of healthy humans. Twenty-eight normal control subjects underwent PET procedures to calculate the binding potential BPND of [(18)F]CPFPX in cerebral regions and to assess ADORA1 and ADORA2A single nucleotide polymorphism (SNP) effects on regional BPND data. Our results revealed SNPs of both genes associated with [(18)F]CPFPX binding to the A1AR. The strongest effects that withstood even Bonferroni correction of multiple SNP testing were found in non-smoking subjects (N=22) for ADORA2A SNPs rs2236624 and rs5751876 (corr. Pall<0.05). SNP alleles previously identified at risk for increased anxiety like the rs5751876 T-allele corresponded to consistently higher A1AR availability in all brain regions. Our data indicate for the first time that variation of A1AR availability was associated with ADORA SNPs. The finding of increased A1AR availability in regions of the fear network, particularly in ADORA2A risk allele carriers, strongly warrants evaluation and replication in further studies including individuals with increased anxiety. PMID:24943643

  9. Adenosine, type 1 receptors: role in proximal tubule Na+ reabsorption.

    PubMed

    Welch, W J

    2015-01-01

    Adenosine type 1 receptor (A1 -AR) antagonists induce diuresis and natriuresis in experimental animals and humans. Much of this effect is due to inhibition of A1 -ARs in the proximal tubule, which is responsible for 60-70% of the reabsorption of filtered Na(+) and fluid. Intratubular application of receptor antagonists indicates that A1 -AR mediates a portion of Na(+) uptake in PT and PT cells, via multiple transport systems, including Na(+) /H(+) exchanger-3 (NHE3), Na(+) /PO4(-) co-transporter and Na(+) -dependent glucose transporter, SGLT. Renal microperfusion and recollection studies have shown that fluid reabsorption is reduced by A1 -AR antagonists and is lower in A1 -AR KO mice, compared to WT mice. Absolute proximal reabsorption (APR) measured by free-flow micropuncture is equivocal, with studies that show either lower APR or similar APR in A1 -AR KO mice, compared to WT mice. Inhibition of A1 -ARs lowers elevated blood pressure in models of salt-sensitive hypertension, partially due to their effects in the proximal tubule. PMID:25345761

  10. ATP-Sensitive K+ Channels Regulate the Concentrative Adenosine Transporter CNT2 following Activation by A1 Adenosine Receptors

    PubMed Central

    Duflot, Sylvie; Riera, Bárbara; Fernández-Veledo, Sonia; Casadó, Vicent; Norman, Robert I.; Casado, F. Javier; Lluís, Carme; Franco, Rafael; Pastor-Anglada, Marçal

    2004-01-01

    This study describes a novel mechanism of regulation of the high-affinity Na+-dependent adenosine transporter (CNT2) via the activation of A1 adenosine receptors (A1R). This regulation is mediated by the activation of ATP-sensitive K+ (KATP) channels. The high-affinity Na+-dependent adenosine transporter CNT2 and A1R are coexpressed in the basolateral domain of the rat hepatocyte plasma membrane and are colocalized in the rat hepatoma cell line FAO. The transient increase in CNT2-mediated transport activity triggered by (−)-N6-(2-phenylisopropyl)adenosine is fully inhibited by KATP channel blockers and mimicked by a KATP channel opener. A1R agonist activation of CNT2 occurs in both hepatocytes and FAO cells, which express Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B mRNA channel subunits. With the available antibodies against Kir6.X, SUR2A, and SUR2B, it is shown that all of these proteins colocalize with CNT2 and A1R in defined plasma membrane domains of FAO cells. The extent of the purinergic modulation of CNT2 is affected by the glucose concentration, a finding which indicates that glycemia and glucose metabolism may affect this cross-regulation among A1R, CNT2, and KATP channels. These results also suggest that the activation of KATP channels under metabolic stress can be mediated by the activation of A1R. Cell protection under these circumstances may be achieved by potentiation of the uptake of adenosine and its further metabolization to ATP. Mediation of purinergic responses and a connection between the intracellular energy status and the need for an exogenous adenosine supply are novel roles for KATP channels. PMID:15024061

  11. Severe hemorrhage attenuates cardiopulmonary chemoreflex control of regional sympathetic outputs via NTS adenosine receptors.

    PubMed

    Minic, Zeljka; Li, Cailian; O'Leary, Donal S; Scislo, Tadeusz J

    2014-09-15

    Selective stimulation of inhibitory A1 and facilitatory A2a adenosine receptor subtypes located in the nucleus of the solitary tract (NTS) powerfully inhibits cardiopulmonary chemoreflex (CCR) control of regional sympathetic outputs via different mechanisms: direct inhibition of glutamate release and facilitation of an inhibitory neurotransmitter release, respectively. However, it remains unknown whether adenosine naturally released into the NTS has similar inhibitory effects on the CCR as the exogenous agonists do. Our previous study showed that adenosine is released into the NTS during severe hemorrhage and contributes to reciprocal changes of renal (decreases) and adrenal (increases) sympathetic nerve activity observed in this setting. Both A1 and A2a adenosine receptors are involved. Therefore, we tested the hypothesis that, during severe hemorrhage, CCR control of the two sympathetic outputs is attenuated by adenosine naturally released into the NTS. We compared renal and adrenal sympathoinhibitory responses evoked by right atrial injections of 5HT3 receptor agonist phenylbiguanide (2-8 μg/kg) under control conditions, during hemorrhage, and during hemorrhage preceded by blockade of NTS adenosine receptors with bilateral microinjections of 8-(p-sulfophenyl) theophylline (1 nmol/100 nl) in urethane/chloralose anesthetized rats. CCR-mediated inhibition of renal and adrenal sympathetic activity was significantly attenuated during severe hemorrhage despite reciprocal changes in the baseline activity levels, and this attenuation was removed by bilateral blockade of adenosine receptors in the caudal NTS. This confirmed that adenosine endogenously released into the NTS has a similar modulatory effect on integration of cardiovascular reflexes as stimulation of NTS adenosine receptors with exogenous agonists. PMID:25063794

  12. Adenosine 2A receptors modulate reward behaviours for methamphetamine.

    PubMed

    Chesworth, Rose; Brown, Robyn M; Kim, Jee Hyun; Ledent, Catherine; Lawrence, Andrew J

    2016-03-01

    Addiction to methamphetamine (METH) is a global health problem for which there are no approved pharmacotherapies. The adenosine 2A (A2 A ) receptor presents a potential therapeutic target for METH abuse due to its modulatory effects on striatal dopamine and glutamate transmission. Notably, A2 A receptor signalling has been implicated in the rewarding effects of alcohol, cocaine and opiates; yet, the role of this receptor in METH consumption and seeking is essentially unknown. Therefore, the current study used A2 A knockout (KO) mice to assess the role of A2 A in behaviours relevant to METH addiction. METH conditioned place preference was absent in A2 A KO mice compared with wild-type (WT) littermates. Repeated METH treatment produced locomotor sensitization in both genotypes; however, sensitization was attenuated in A2 A KO mice in a dose-related manner. METH intravenous self-administration was intact in A2 A KO mice over a range of doses and schedules of reinforcement. However, the motivation to self-administer was reduced in A2 A KO mice. Regression analysis further supported the observation that the motivation to self-administer METH was reduced in A2 A KO mice even when self-administration was similar to WT mice. Sucrose self-administration was also reduced in A2 A KO mice but only at higher schedules of reinforcement. Collectively, these data suggest that A2 A signalling is critically required to integrate rewarding and motivational properties of both METH and natural rewards. PMID:25612195

  13. Expression of adenosine A2b receptor in rat type II and III taste cells.

    PubMed

    Nishida, Kentaro; Dohi, Yukari; Yamanaka, Yuri; Miyata, Ai; Tsukamoto, Katsunobu; Yabu, Miharu; Ohishi, Akihiro; Nagasawa, Kazuki

    2014-05-01

    We previously demonstrated that equilibrative nucleoside transporter 1 was expressed in taste cells, suggesting the existence of an adenosine signaling system, but whether or not the expression of an adenosine receptor occurs in rat taste buds remains unknown. Therefore, we examined the expression profiles of adenosine receptors and evaluated their functionality in rat circumvallate papillae. Among adenosine receptors, the mRNA for an adenosine A2b receptor (A2bR) was expressed by the rat circumvallate papillae, and its expression level was significantly greater in the circumvallate papillae than in the non-taste lingual epithelium. A2bR-immunoreactivity was detected primarily in type II taste cells, and partial, but significant expression was also observed in type III ones, but there was no immunoreactivity in type I ones. The cAMP generation in isolated epithelium containing taste buds treated with 500 μM adenosine or 10 μM BAY60-6583 was significantly increased compared to in the controls. These findings suggest that adenosine plays a role in signaling transmission via A2bR between taste cells in rats. PMID:24327108

  14. Analysis of the atypical characteristics of adenosine receptors mediating negative inotropic and chronotropic responses of guinea-pig isolated atria and papillary muscles

    PubMed Central

    Gardner, Neil M; Broadley, Kenneth J

    1999-01-01

    Adenosine receptor(s) mediating negative inotropy of paced left atria, isoprenaline-stimulated paced left atria and papillary muscles, and negative chronotropy of spontaneously beating right atria were characterized.Isometric tension of guinea-pig isolated paced left atria and left ventricular papillary muscles and rate of contraction of spontaneously beating right atria were recorded. Papillary muscles were pre-stimulated with isoprenaline (1×10−8 M). Concentration-response curves (CRCs) for tension or rate reduction by N6-cyclopentyladenosine (CPA), the stereoisomers of N6-(2-phenylisopropyl)adenosine ((+)-PIA and (−)-PIA), 5′-(N-carboxamido)adenosine (NECA), N6-2-(4-aminophenyl)ethyladenosine (APNEA) and N6-(3-iodobenzyl)adenosine-5′-N-methyuromide (IB-MECA) revealed a potency order of CPA=(−)-PIA>NECA in right atria and papillary muscles, which is consistent with involvement of A1-receptors. The potency order in left atria was CPA=NECA>(−)-PIA>(+)-PIA>APNEA, which is not typical of A1 adenosine receptors. Weak activity of APNEA and IB-MECA discounts involvement of A3 receptors.pA2 values for the antagonism of CPA by 8(p-sulfophenyl)theophylline (8-SPT) were calculated from Schild plots (log concentration-ratio against log 8-SPT concentration), the unity slopes of which indicated competitive antagonism. The pA2 value in the papillary muscles was significantly higher than for atrial preparations, indicating a possible difference in receptor characteristics between atrial and papillary muscle responses.In left and right atria there was a limit to the displacement of the CPA CRCs at higher concentrations of 8-SPT. The 8-SPT-resistant component of the response is suggested to arise from duality of coupling of a common A1 receptor through either different G proteins or G protein subunits to independent transduction pathways. The results with papillary muscles can be explained by a typical A1 receptor coupled to a single transduction pathway. PMID

  15. Actions of adenosine A1 and A2 receptor antagonists on CFTR antibody-inhibited β-adrenergic mucin secretion response

    PubMed Central

    Pereira, M M C; Lloyd Mills, C; Dormer, R L; McPherson, M A

    1998-01-01

    The cystic fibrosis gene protein, the cystic fibrosis transmembrane conductance regulator (CFTR) acts as a chloride channel and is a key regulator of mucin secretion. The mechanism by which 3-isobutyl-1-methylxanthine (IBMX) corrects the defect in CFTR mediated β-adrenergic stimulation of mucin secretion has not been determined. The present study has investigated the actions of adenosine A1 and A2 receptor antagonists to determine whether ability to stimulate mucin secretion correlates with correction of CFTR antibody inhibited β-adrenergic response and whether excessive cyclic AMP rise is required.CFTR antibodies were introduced into living rat submandibular acini by hypotonic swelling. Following recovery, mucin secretion in response to isoproterenol was measured.The adenosine A1 receptor antagonist, 8 cyclopentyltheophylline (CPT) was a less potent stimulator of mucin secretion than was the A2 receptor antagonist dimethylpropargylxanthine (DMPX). A concentration of CPT close to the Ki for A1 receptor antagonism (10 nM) did not stimulate mucin secretion.DMPX, although a potent stimulator of mucin secretion, did not correct CFTR antibody inhibited mucin secretion.CPT corrected defective CFTR antibody inhibited mucin secretion at a high (1 mM) concentration, suggesting a mechanism other than adenosine receptor antagonism.DMPX potentiated the isoproterenol induced cyclic AMP rise, whereas CPT did not.Correction of the defective CFTR mucin secretion response did not correlate with ability to stimulate mucin secretion and did not require potentiation of β-adrenergic induced increases in cyclic AMP. This affords real promise for the development of a selective drug treatment for cystic fibrosis. PMID:9831904

  16. Identification and function of adenosine A3 receptor in afferent arterioles.

    PubMed

    Lu, Yan; Zhang, Rui; Ge, Ying; Carlstrom, Mattias; Wang, Shaohui; Fu, Yiling; Cheng, Liang; Wei, Jin; Roman, Richard J; Wang, Lei; Gao, Xichun; Liu, Ruisheng

    2015-05-01

    Adenosine plays an important role in regulation of renal microcirculation. All receptors of adenosine, A1, A2A, A2B, and A3, have been found in the kidney. However, little is known about the location and function of the A3 receptor in the kidney. The present study determined the expression and role of A3 receptors in mediating the afferent arteriole (Af-Art) response and studied the interaction of A3 receptors with angiotensin II (ANG II), A1 and A2 receptors on the Af-Art. We found that the A3 receptor expressed in microdissected isolated Af-Art and the mRNA levels of A3 receptor were 59% of A1. In the isolated microperfused Af-Art, A3 receptor agonist IB-MECA did not have a constrictive effect. Activation of A3 receptor dilated the preconstricted Af-Art by norepinephrine and blunted the vasoconstrictive effect of both adenosine A1 receptor activation and ANG II on the Af-Art, respectively. Selective A2 receptor antagonist (both A2A and A2B) had no effect on A3 receptor agonist-induced vasodilation, indicating that the dilatory effect of A3 receptor activation is not mediated by activation of A2 receptor. We conclude that the A3 receptor is expressed in the Af-Art, and activation of the A3 receptor dilates the Af-Art. PMID:25608966

  17. Characterization of agonist radioligand interactions with porcine atrial A1 adenosine receptors

    SciTech Connect

    Leid, M.; Schimerlik, M.I.; Murray, T.F.

    1988-09-01

    The agonist radioligand (-)-N6-(125I)-p-hydroxyphenylisopropyl-adenosine (( 125I)HPIA) was used to characterize adenosine recognition sites in porcine atrial membranes. (125I)HPIA showed saturable binding to an apparently homogeneous population of sites with a maximum binding capacity of 35 +/- 3 fmol/mg of protein and an equilibrium dissociation constant of 2.5 +/- 0.4 nM. Kinetic experiments were performed to address the molecular mechanism of (125I)HPIA binding in porcine atrial membranes. (125I)HPIA apparently interacts with the cardiac adenosine receptor in a simple bimolecular reaction. A kinetically derived (125I) HPIA dissociation constant (2.4 nM) was in good agreement with that parameter measured at equilibrium. Guanyl nucleotides negatively modulated (125I)HPIA binding by increasing its rate of dissociation. This finding is consonant with the formation of a ternary complex in porcine atrial membranes, consisting of ligand, receptor, and guanyl nucleotide-binding protein. Prototypic adenosine receptor agonists and antagonists inhibited specific binding in a manner consistent with the labeling of an A1 adenosine receptor. The results of these experiments suggest that the adenosine receptor present in porcine atrial membranes, as labeled by (125I)HPIA, is of the A1 subtype.

  18. The 2.6 Angstrom Crystal Structure of a Human A[subscript 2A] Adenosine Receptor Bound to an Antagonist

    SciTech Connect

    Jaakola, Veli-Pekka; Griffith, Mark T.; Hanson, Michael A.; Cherezov, Vadim; Chien, Ellen Y.T.; Lane, J. Robert; IJzerman, Adriaan P.; Stevens, Raymond C.

    2009-01-15

    The adenosine class of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A{sub 2A} adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extended conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.

  19. The novel adenosine A2A antagonist Lu AA47070 reverses the motor and motivational effects produced by dopamine D2 receptor blockade.

    PubMed

    Collins, Lyndsey E; Sager, Thomas N; Sams, Anette G; Pennarola, Adam; Port, Russell G; Shahriari, Mona; Salamone, John D

    2012-01-01

    Dopamine D2 and adenosine A(2A) receptors interact to regulate aspects of motor and motivational function, and it has been suggested that adenosine A(2A) antagonists could be useful for the treatment of parkinsonism and depression. The present experiments were performed to characterize the effects of Lu AA47070, which is a phosphonooxymethylene prodrug of a potent and selective adenosine A(2A) receptor antagonist, for its ability to reverse the motor and motivational effects of D2 antagonism. In the first group of studies, Lu AA47070 (3.75-30 mg/kg IP) was assessed for its ability to reverse the effects of the D2 receptor antagonist pimozide (1.0 mg/kg IP) using several measures of motor impairment, including catalepsy, locomotion, and tremulous jaw movements, which is a rodent model of parkinsonian tremor. Lu AA47070 produced a significant reversal of the effects of pimozide on all three measures of parkinsonian motor impairment. In addition, Lu AA47070 was able to reverse the effects of a low dose of the D2 antagonist haloperidol on a concurrent lever pressing/chow feeding task that is used as a measure of effort-related choice behavior. The ability of Lu AA47070 to reverse the effects of D2 receptor blockade suggests that this compound could have potential utility as a treatment for parkinsonism, and for some of the motivational symptoms of depression. PMID:22037410

  20. ENDOTHELIN-A RECEPTOR ANTAGONISM IN EMBRYO CULTURE: WINDOW OF SENSITIVITY AND TIMING OF DEFECT

    EPA Science Inventory

    BRANNEN, K.C., J.M. ROGERS, and E.S. HUNTER, Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina, and Reproductive Toxicology Division, NHEERL, U.S. EPA, Research Triangle Park, North Carolina. Endothelin-A receptor antagonism in embryo culture: w...

  1. Contractile effects and receptor analysis of adenosine-receptors in human detrusor muscle from stable and neuropathic bladders.

    PubMed

    Pakzad, Mahreen; Ikeda, Youko; McCarthy, Carly; Kitney, Darryl G; Jabr, Rita I; Fry, Christopher H

    2016-08-01

    To measure the relative transcription of adenosine receptor subtypes and the contractile effects of adenosine and selective receptor-subtype ligands on detrusor smooth muscle from patients with neuropathic overactive (NDO) and stable bladders and also from guinea-pigs. Contractile function was measured at 37°C in vitro from detrusor smooth muscle strips. Contractions were elicited by superfusate agonists or by electrical field stimulation. Adenosine-receptor (A1, A2A, A2B, A3) transcription was measured by RT-PCR. Adenosine attenuated nerve-mediated responses with equivalent efficacy in human and guinea-pig tissue (pIC50 3.65-3.86); the action was more effective at low (1-8 Hz) compared to high (20-40 Hz) stimulation frequencies in human NDO and guinea-pig tissue. With guinea-pig detrusor the action of adenosine was mirrored by the A1/A2-agonist N-ethylcarboxamidoadenosine (NECA), partly abolished in turn by the A2B-selectve antagonist alloxazine, as well as the A1-selective agonist N6- cyclopentyladenosine (CPA). With detrusor from stable human bladders the effects of NECA and CPA were much smaller than that of adenosine. Adenosine also attenuated carbachol contractures, but mirrored by NECA (in turn blocked by alloxazine) only in guinea-pig tissue. Adenosine receptor subtype transcription was measured in human detrusor and was similar in both groups, except reduced A2A levels in overactive bladder. Suppression of the carbachol contracture in human detrusor is independent of A-receptor activation, in contrast to an A2B-dependent action with guinea-pig tissue. Adenosine also reduced nerve-mediated contractions, by an A1- dependent action suppressing ATP neurotransmitter action. PMID:27185496

  2. A2B Adenosine Receptor Blockade Enhances Macrophage-Mediated Bacterial Phagocytosis and Improves Polymicrobial Sepsis Survival in Mice

    PubMed Central

    Belikoff, Bryan G.; Hatfield, Stephen; Georgiev, Peter; Ohta, Akio; Lukashev, Dmitriy; Buras, Jon A.; Remick, Daniel G.; Sitkovsky, Michail

    2013-01-01

    Antimicrobial treatment strategies must improve to reduce the high mortality rates in septic patients. In noninfectious models of acute inflammation, activation of A2B adenosine receptors (A2BR) in extracellular adenosine-rich microenvironments causes immunosuppression. We examined A2BR in antibacterial responses in the cecal ligation and puncture (CLP) model of sepsis. Antagonism of A2BR significantly increased survival, enhanced bacterial phagocytosis, and decreased IL-6 and MIP-2 (a CXC chemokine) levels after CLP in outbred (ICR/CD-1) mice. During the CLP-induced septic response in A2BR knockout mice, hemodynamic parameters were improved compared with wild-type mice in addition to better survival and decreased plasma IL-6 levels. A2BR deficiency resulted in a dramatic 4-log reduction in peritoneal bacteria. The mechanism of these improvements was due to enhanced macrophage phagocytic activity without augmenting neutrophil phagocytosis of bacteria. Following ex vivo LPS stimulation, septic macrophages from A2BR knockout mice had increased IL-6 and TNF-α secretion compared with wild-type mice. A therapeutic intervention with A2BR blockade was studied by using a plasma biomarker to direct therapy to those mice predicted to die. Pharmacological blockade of A2BR even 32 h after the onset of sepsis increased survival by 65% in those mice predicted to die. Thus, even the late treatment with an A2BR antagonist significantly improved survival of mice (ICR/CD-1) that were otherwise determined to die according to plasma IL-6 levels. Our findings of enhanced bacterial clearance and host survival suggest that antagonism of A2BRs offers a therapeutic target to improve macrophage function in a late treatment protocol that improves sepsis survival. PMID:21242513

  3. Identification of A3 adenosine receptor agonists as novel non-narcotic analgesics.

    PubMed

    Janes, K; Symons-Liguori, A M; Jacobson, K A; Salvemini, D

    2016-04-01

    Chronic pain negatively impacts the quality of life in a variety of patient populations. The current therapeutic repertoire is inadequate in managing patient pain and warrants the development of new therapeutics. Adenosine and its four cognate receptors (A1 , A2A , A2B and A3 ) have important roles in physiological and pathophysiological states, including chronic pain. Preclinical and clinical studies have revealed that while adenosine and agonists of the A1 and A2A receptors have antinociceptive properties, their therapeutic utility is limited by adverse cardiovascular side effects. In contrast, our understanding of the A3 receptor is only in its infancy, but exciting preclinical observations of A3 receptor antinociception, which have been bolstered by clinical trials of A3 receptor agonists in other disease states, suggest pain relief without cardiovascular side effects and with sufficient tolerability. Our goal herein is to briefly discuss adenosine and its receptors in the context of pathological pain and to consider the current data regarding A3 receptor-mediated antinociception. We will highlight recent findings regarding the impact of the A3 receptor on pain pathways and examine the current state of selective A3 receptor agonists used for these studies. The adenosine-to-A3 receptor pathway represents an important endogenous system that can be targeted to provide safe, effective pain relief from chronic pain. PMID:26804983

  4. Role of adenosine A1 and A2A receptors in the alcohol withdrawal syndrome.

    PubMed

    Kaplan, G B; Bharmal, N H; Leite-Morris, K A; Adams, W R

    1999-10-01

    The role of adenosine receptor-mediated signaling was examined in the alcohol withdrawal syndrome. CD-1 mice received a liquid diet containing ethanol (6.7%, v/v) or a control liquid diet that were abruptly discontinued after 14 days of treatment. Mice consuming ethanol showed a progressive increase in signs of intoxication throughout the drinking period. Following abrupt discontinuation of ethanol diet, mice demonstrated reversible signs of handling-induced hyperexcitability that were maximal between 5-8 h. Withdrawing mice received treatment with adenosine receptor agonists at the onset of peak withdrawal (5.5 h) and withdrawal signs were blindly rated (during withdrawal hours 6 and 7). Adenosine A1-receptor agonist R-N6(phenylisopropyl)adenosine (0.15 and 0.3 mg/ kg) reduced withdrawal signs 0.5 and 1.5 h after drug administration in a dose-dependent fashion. Adenosine A2A-selective agonist 2-p-(2-carboxyethyl)phenylethyl-amino-5'-N-ethylcarboxamidoadenosine (0.3 mg/kg) reduced withdrawal signs at both time points. In ethanol-withdrawing mice, there were significant decreases in adenosine transporter sites in striatum without changes in cortex or cerebellum. In ethanol-withdrawing mice, there were no changes in adenosine A1 and A2A receptor concentrations in cortex, striatum, or cerebellum. There appears to be a role for adenosine A1 and A2A receptors in the treatment of the ethanol withdrawal syndrome. Published by Elsevier Science Inc. PMID:10548160

  5. Selective endothelin A receptor antagonism with sitaxentan reduces neointimal lesion size in a mouse model of intraluminal injury

    PubMed Central

    Duthie, Karolina M; Hadoke, Patrick W F; Kirkby, Nicholas S; Miller, Eileen; Ivy, Jessica R; McShane, John F; Lim, Win Gel; Webb, David J

    2015-01-01

    Background and Purpose Endothelin (ET) receptor antagonism reduces neointimal lesion formation in animal models. This investigation addressed the hypothesis that the selective ETA receptor antagonist sitaxentan would be more effective than mixed ETA/B receptor antagonism at inhibiting neointimal proliferation in a mouse model of intraluminal injury. Experimental Approach Antagonism of ETA receptors by sitaxentan (1–100 nM) was assessed in femoral arteries isolated from adult, male C57Bl6 mice using isometric wire myography. Neointimal lesion development was induced by intraluminal injury in mice receiving sitaxentan (ETA antagonist; 15 mg·kg−1·day−1), A192621 (ETB antagonist; 30 mg·kg−1·day−1), the combination of both antagonists or vehicle. Treatment began 1 week before, and continued for 28 days after, surgery. Femoral arteries were then harvested for analysis of lesion size and composition. Key Results Sitaxentan produced a selective, concentration-dependent parallel rightward shift of ET-1-mediated contraction in isolated femoral arteries. Sitaxentan reduced neointimal lesion size, whereas ETB and combined ETA/B receptor antagonism did not. Macrophage and α-smooth muscle actin content were unaltered by ET receptor antagonism but sitaxentan reduced the amount of collagen in lesions. Conclusions and Implications These results suggest that ETA receptor antagonism would be more effective than combined ETA/ETB receptor antagonism at reducing neointimal lesion formation. PMID:25598351

  6. NTS adenosine A2a receptors inhibit the cardiopulmonary chemoreflex control of regional sympathetic outputs via a GABAergic mechanism.

    PubMed

    Minic, Zeljka; O'Leary, Donal S; Scislo, Tadeusz J

    2015-07-01

    Adenosine is a powerful central neuromodulator acting via opposing A1 (inhibitor) and A2a (activator) receptors. However, in the nucleus of the solitary tract (NTS), both adenosine receptor subtypes attenuate cardiopulmonary chemoreflex (CCR) sympathoinhibition of renal, adrenal, and lumbar sympathetic nerve activity and attenuate reflex decreases in arterial pressure and heart rate. Adenosine A1 receptors inhibit glutamatergic transmission in the CCR pathway, whereas adenosine A2a receptors most likely facilitate release of an unknown inhibitory neurotransmitter, which, in turn, inhibits the CCR. We hypothesized that adenosine A2a receptors inhibit the CCR via facilitation of GABA release in the NTS. In urethane-chloralose-anesthetized rats (n = 51), we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of the 5-HT3 receptor agonist phenylbiguanide (1-8 μg/kg) before and after selective stimulation of NTS adenosine A2a receptors [microinjections into the NTS of CGS-21680 (20 pmol/50 nl)] preceded by blockade of GABAA or GABAB receptors in the NTS [bicuculline (10 pmol/100 nl) or SCH-50911 (1 nmol/100 nl)]. Blockade of GABAA receptors virtually abolished adenosine A2a receptor-mediated inhibition of the CCR. GABAB receptors had much weaker but significant effects. These effects were similar for the different sympathetic outputs. We conclude that stimulation of NTS adenosine A2a receptors inhibits CCR-evoked hemodynamic and regional sympathetic reflex responses via a GABA-ergic mechanism. PMID:25910812

  7. Cerebral adenosine A₁ receptors are upregulated in rodent encephalitis.

    PubMed

    Paul, Soumen; Khanapur, Shivashankar; Boersma, Wytske; Sijbesma, Jurgen W; Ishiwata, Kiichi; Elsinga, Philip H; Meerlo, Peter; Doorduin, Janine; Dierckx, Rudi A; van Waarde, Aren

    2014-05-15

    Adenosine A1 receptors (A1Rs) are implied in the modulation of neuroinflammation. Activation of cerebral A1Rs acts as a brake on the microglial response after traumatic brain injury and has neuroprotective properties in animal models of Parkinson's disease and multiple sclerosis. Neuroinflammatory processes in turn may affect the expression of A1Rs, but the available data is limited and inconsistent. Here, we applied an animal model of encephalitis to assess how neuroinflammation affects the expression of A1Rs. Two groups of animals were studied: Infected rats (n=7) were intranasally inoculated with herpes simplex virus-1 (HSV-1, 1 × 10(7) plaque forming units), sham-infected rats (n=6) received only phosphate-buffered saline. Six or seven days later, microPET scans (60 min with arterial blood sampling) were made using the tracer 8-dicyclopropyl-1-(11)C-methyl-3-propyl-xanthine ((11)C-MPDX). Tracer clearance from plasma and partition coefficient (K₁/k₂ estimated from a 2-tissue compartment model fit) were not significantly altered after virus infection. PET tracer distribution volume calculated from a Logan plot was significantly increased in the hippocampus (+37%) and medulla (+27%) of virus infected rats. Tracer binding potential (k₃/k₄ estimated from the model fit) was significantly increased in the cerebellum (+87%) and the medulla (+148%) which may indicate increased A1R expression. This was confirmed by immunohistochemical analysis showing a strong increase of A1R immunoreactivity in the cerebellum of HSV-1-infected rats. Both the quantitative PET data and immunohistochemical analysis indicate that A1Rs are upregulated in brain areas where active virus is present. PMID:24513151

  8. Genetic removal of the A2A adenosine receptor enhances pulmonary inflammation, mucin production, and angiogenesis in adenosine deaminase-deficient mice.

    PubMed

    Mohsenin, Amir; Mi, Tiejuan; Xia, Yang; Kellems, Rodney E; Chen, Jiang-Fan; Blackburn, Michael R

    2007-09-01

    Adenosine is generated at sites of tissue injury where it serves to regulate inflammation and damage. Adenosine signaling has been implicated in the regulation of pulmonary inflammation and damage in diseases such as asthma and chronic obstructive pulmonary disease; however, the contribution of specific adenosine receptors to key immunoregulatory processes in these diseases is still unclear. Mice deficient in the purine catabolic enzyme adenosine deaminase (ADA) develop pulmonary inflammation and mucous metaplasia in association with adenosine elevations making them a useful model for assessing the contribution of specific adenosine receptors to adenosine-mediated pulmonary disease. Studies suggest that the A(2A) adenosine receptor (A(2A)R) functions to limit inflammation and promote tissue protection; however, the contribution of A(2A)R signaling has not been examined in the ADA-deficient model of adenosine-mediated lung inflammation. The purpose of the current study was to examine the contribution of A(2A)R signaling to the pulmonary phenotype seen in ADA-deficient mice. This was accomplished by generating ADA/A(2A)R double knockout mice. Genetic removal of the A(2A)R from ADA-deficient mice resulted in enhanced inflammation comprised largely of macrophages and neutrophils, mucin production in the bronchial airways, and angiogenesis, relative to that seen in the lungs of ADA-deficient mice with the A(2A)R. In addition, levels of the chemokines monocyte chemoattractant protein-1 and CXCL1 were elevated, whereas levels of cytokines such as TNF-alpha and IL-6 were not. There were no compensatory changes in the other adenosine receptors in the lungs of ADA/A(2A)R double knockout mice. These findings suggest that the A(2A)R plays a protective role in the ADA-deficient model of pulmonary inflammation. PMID:17601796

  9. Adenosine A2B receptor modulates intestinal barrier function under hypoxic and ischemia/reperfusion conditions

    PubMed Central

    Yang, Yang; Qiu, Yuan; Wang, Wensheng; Xiao, Weidong; Liang, Hongyin; Zhang, Chaojun; Yang, Hanwenbo; Teitelbaum, Daniel H; Sun, Li-Hua; Yang, Hua

    2014-01-01

    Background: Intestinal barrier function failure from ischemia/reperfusion (I/R) and acute hypoxia has been implicated as a critical determinant in the predisposition to intestinal inflammation and a number of inflammatory disorders. Here, we identified the role of Adenosine A2B receptor (A2BAR) in the regulation of intestinal barrier function under I/R and acute hypoxic conditions. Methods: C57BL/6J mice were used, and were randomized into three groups: Sham, I/R, IR+PSB1115 (a specific A2BAR antagonist) groups. After surgery, the small bowel was harvested for immunohistochemical staining, RNA and protein content, and intestinal permeability analyses. Using an epithelial cell culture model, we investigated the influence of hypoxia on the epithelial function, and the role of A2BAR in the expressions of tight junction and epithelial permeability. The expressions of Claudin-1, occludin and ZO-1 were detected by RT-PCR and Western-Blot. Epithelial barrier function was assessed with transepithelial resistance (TER). Results and conclusions: The A2BAR antagonist, PSB1115, significantly increased tight junction protein expression after intestinal I/R or acute hypoxia conditions. PSB1115 also attenuated the disrupted distribution of TJ proteins. Furthermore, inhibition of A2BAR attenuated the decrease in TER induced by I/R or acute hypoxic conditions, and maintained intestinal barrier function. Antagonism of A2BAR activity improves intestinal epithelial structure and barrier function in a mouse model of intestinal I/R and a cell model of acute hypoxia. These findings support a potentially destructive role for A2BAR under intestinal I/R and acute hypoxic conditions. PMID:24966910

  10. Nucleus tractus solitarii A(2a) adenosine receptors inhibit cardiopulmonary chemoreflex control of sympathetic outputs.

    PubMed

    Minic, Zeljka; O'Leary, Donal S; Scislo, Tadeusz J

    2014-02-01

    Previously we have shown that stimulation of inhibitory A1 adenosine receptors located in the nucleus tractus solitarii (NTS) attenuates cardiopulmonary chemoreflex (CCR) evoked inhibition of renal, adrenal and lumbar sympathetic nerve activity and reflex decreases in arterial pressure and heart rate. Activation of facilitatory A2a adenosine receptors, which dominate over A1 receptors in the NTS, contrastingly alters baseline activity of regional sympathetic outputs: it decreases renal, increases adrenal and does not change lumbar nerve activity. Considering that NTS A2a receptors may facilitate release of inhibitory transmitters we hypothesized that A2a receptors will act in concert with A1 receptors differentially inhibiting regional sympathetic CCR responses (adrenal>lumbar>renal). In urethane/chloralose anesthetized rats (n=38) we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of serotonin 5HT3 receptor agonist, phenylbiguanide, (1-8μg/kg) before and after selective stimulation, blockade or combined blockade and stimulation of NTS A2a adenosine receptors (microinjections into the NTS of CGS-21680 0.2-20pmol/50nl, ZM-241385 40pmol/100nl or ZM-241385+CGS-21680, respectively). We found that stimulation of A2a adenosine receptors uniformly inhibited the regional sympathetic and hemodynamic reflex responses and this effect was abolished by the selective blockade of NTS A2a receptors. This indicates that A2a receptor triggered inhibition of CCR responses and the contrasting shifts in baseline sympathetic activity are mediated via different mechanisms. These data implicate that stimulation of NTS A2a receptors triggers unknown inhibitory mechanism(s) which in turn inhibit transmission in the CCR pathway when adenosine is released into the NTS during severe hypotension. PMID:24216055

  11. Effects of adenosine and adenosine A2A receptor agonist on motor nerve conduction velocity and nerve blood flow in experimental diabetic neuropathy.

    PubMed

    Kumar, Sokindra; Arun, K H S; Kaul, Chaman L; Sharma, Shyam S

    2005-01-01

    This study examined the effects of chronic administration of adenosine and CGS 21680 hydrochloride (adenosine A(2A) receptor agonist) on motor nerve conduction velocity (MNCV), nerve blood flow (NBF) and histology of sciatic nerve in animal model of diabetic neuropathy. Adenosinergic agents were administered for 2 weeks after 6 weeks of streptozotocin-induced (50 mg/kg i.p.) diabetes in male Sprague-Dawley rats. Significant reduction in sciatic MNCV and NBF were observed after 8 weeks in diabetic animals in comparison with control (non diabetic) rats. Adenosine (10 mg/kg, i.p.) significantly improved sciatic MNCV and NBF in diabetic rats. The protective effect of adenosine on MNCV and NBF was completely reversed by theophylline (50 mg/kg, i.p.), a non-selective adenosine receptor antagonist, suggesting that the adenosine effect was mediated via adenosinergic receptors. CGS 21680 (0.1 mg/kg, i.p.) significantly improved NBF; however, MNCV was not significantly improved in diabetic rats. At a dose of 1 mg/kg, neither MNCV nor NBF was improved by CGS 21680 in diabetic rats. ZM 241385 (adenosine A(2A) receptor antagonist) prevented the effect of CGS 21680 (0.1 mg/kg, i.p.). Histological changes observed in sciatic nerve were partially improved by the adenosinergic agents in diabetic rats. Results of the present study, suggest the potential of adenosinergic agents in the therapy of diabetic neuropathy. PMID:15829161

  12. Autoradiographic localization of adenosine receptors in rat brain using (/sup 3/H)cyclohexyladenosine

    SciTech Connect

    Goodman, R.R.; Synder, S.H.

    1982-09-01

    Adenosine (A1) receptor binding sites have been localized in rat brain by an in vitro light microscopic autoradiographic method. The binding of (/sup 3/H)N6-cyclohexyladenosine to slide-mounted rat brain tissue sections has the characteristics of A1 receptors. It is saturable with high affinity and has appropriate pharmacology and stereospecificity. The highest densities of adenosine receptors occur in the molecular layer of the cerebellum, the molecular and polymorphic layers of the hippocampus and dentate gyrus, the medial geniculate body, certain thalamic nuclei, and the lateral septum. High densities also are observed in certain layers of the cerebral cortex, the piriform cortex, the caudate-putamen, the nucleus accumbens, and the granule cell layer of the cerebellum. Most white matter areas, as well as certain gray matter areas, such as the hypothalamus, have negligible receptor concentrations. These localizations suggest possible central nervous system sites of action of adenosine.

  13. 2-Triazole-Substituted Adenosines: A New Class of Selective A3 Adenosine Receptor Agonists, Partial Agonists, and Antagonists

    PubMed Central

    Cosyn, Liesbet; Palaniappan, Krishnan K.; Kim, Soo-Kyung; Duong, Heng T.; Gao, Zhan-Guo; Jacobson, Kenneth A.; Van Calenbergh, Serge

    2016-01-01

    “Click chemistry” was explored to synthesize two series of 2-(1,2,3-triazolyl)adenosine derivatives (1–14). Binding affinity at the human A1, A2A, and A3ARs (adenosine receptors) and relative efficacy at the A3AR were determined. Some triazol-1-yl analogues showed A3AR affinity in the low nanomolar range, a high ratio of A3/A2A selectivity, and a moderate-to-high A3/A1 ratio. The 1,2,3-triazol-4-yl regiomers typically showed decreased A3AR affinity. Sterically demanding groups at the adenine C2 position tended to reduce relative A3AR efficacy. Thus, several 5′-OH derivatives appeared to be selective A3AR antagonists, i.e., 10, with 260-fold binding selectivity in comparison to the A1AR and displaying a characteristic docking mode in an A3AR model. The corresponding 5′-ethyluronamide analogues generally showed increased A3AR affinity and behaved as full agonists, i.e., 17, with 910-fold A3/A1 selectivity. Thus, N6-substituted 2-(1,2,3-triazolyl)-adenosine analogues constitute a novel class of highly potent and selective nucleoside-based A3AR antagonists, partial agonists, and agonists. PMID:17149867

  14. Synaptic mechanisms of adenosine A2A receptor-mediated hyperexcitability in the hippocampus.

    PubMed

    Rombo, Diogo M; Newton, Kathryn; Nissen, Wiebke; Badurek, Sylvia; Horn, Jacqueline M; Minichiello, Liliana; Jefferys, John G R; Sebastiao, Ana M; Lamsa, Karri P

    2015-05-01

    Adenosine inhibits excitatory neurons widely in the brain through adenosine A1 receptor, but activation of adenosine A2A receptor (A2A R) has an opposite effect promoting discharge in neuronal networks. In the hippocampus A2A R expression level is low, and the receptor's effect on identified neuronal circuits is unknown. Using optogenetic afferent stimulation and whole-cell recording from identified postsynaptic neurons we show that A2A R facilitates excitatory glutamatergic Schaffer collateral synapses to CA1 pyramidal cells, but not to GABAergic inhibitory interneurons. In addition, A2A R enhances GABAergic inhibitory transmission between CA1 area interneurons leading to disinhibition of pyramidal cells. Adenosine A2A R has no direct modulatory effect on GABAergic synapses to pyramidal cells. As a result adenosine A2A R activation alters the synaptic excitation - inhibition balance in the CA1 area resulting in increased pyramidal cell discharge to glutamatergic Schaffer collateral stimulation. In line with this, we show that A2A R promotes synchronous pyramidal cell firing in hyperexcitable conditions where extracellular potassium is elevated or following high-frequency electrical stimulation. Our results revealed selective synapse- and cell type specific adenosine A2A R effects in hippocampal CA1 area. The uncovered mechanisms help our understanding of A2A R's facilitatory effect on cortical network activity. PMID:25402014

  15. Interaction of mechanisms involving epoxyeicosatrienoic acids, adenosine receptors, and metabotropic glutamate receptors in neurovascular coupling in rat whisker barrel cortex

    PubMed Central

    Shi, Yanrong; Liu, Xiaoguang; Gebremedhin, Debebe; Falck, John R; Harder, David R; Koehler, Raymond C

    2008-01-01

    Adenosine, astrocyte metabotropic glutamate receptors (mGluRs), and epoxyeicosatrienoic acids (EETs) have been implicated in neurovascular coupling. Although A2A and A2B receptors mediate cerebral vasodilation to adenosine, the role of each receptor in the cerebral blood flow (CBF) response to neural activation remains to be fully elucidated. In addition, adenosine can amplify astrocyte calcium, which may increase arachidonic acid metabolites such as EETs. The interaction of these pathways was investigated by determining if combined treatment with antagonists exerted an additive inhibitory effect on the CBF response. During whisker stimulation of anesthetized rats, the increase in cortical CBF was reduced by approximately half after individual administration of A2B, mGluR and EET antagonists and EET synthesis inhibitors. Combining treatment of either a mGluR antagonist, an EET antagonist, or an EET synthesis inhibitor with an A2B receptor antagonist did not produce an additional decrement in the CBF response. Likewise, the CBF response also remained reduced by ~50% when an EET antagonist was combined with an mGluR antagonist or an mGluR antagonist plus an A2B receptor antagonist. In contrast, A2A and A3 receptor antagonists had no effect on the CBF response to whisker stimulation. We conclude that (1) adenosine A2B receptors, rather than A2A or A3 receptors, play a significant role in coupling cortical CBF to neuronal activity, and (2) the adenosine A2B receptor, mGluR, and EETs signaling pathways are not functionally additive, consistent with the possibility of astrocytic mGluR and adenosine A2B receptor linkage to the synthesis and release of vasodilatory EETs. PMID:17519974

  16. Cloning and expression of an A1 adenosine receptor from rat brain

    SciTech Connect

    Mahan, L.C.; McVittie, L.D.; Smyk-Randall, E.M.; Nakata, H.; Monsma, F.J. Jr.; Gerfen, C.R.; Sibley, D.R. )

    1991-07-01

    The authors have used the polymerase chain reaction technique to selectively amplify guanine nucleotide-binding regulatory protein (G protein)-coupled receptor cDNA sequences from rat striatal mRNA, using sets of highly degenerate primers derived from transmembrane sequences of previously cloned G protein-coupled receptors. A novel cDNA fragment was identified, which exhibits considerable homology to various members of the G protein-coupled receptor family. This fragment was used to isolate a full-length cDNA from a rat striatal library. A 2.2-kilobase clone was obtained that encodes a protein of 326 amino acids with seven transmembrane domains, as predicted by hydropathy analysis. Stably transfected mouse A9-L cells and Chinese hamster ovary cells that expressed mRNA for this clone were screened with putative receptor ligands. Saturable and specific binding sites for the A1 adenosine antagonist (3H)-1,3-dipropyl-8-cyclopentylxanthine were identified on membranes from transfected cells. The rank order of potency and affinities of various adenosine agonist and antagonist ligands confirmed the identity of this cDNA clone as an A1 adenosine receptor. The high affinity binding of A1 adenosine agonists was shown to be sensitive to the nonhydrolyzable GTP analog guanylyl-5{prime}-imidodiphosphate. In adenylyl cyclase assays, adenosine agonists inhibited forskolin-stimulated cAMP production by greater than 50%, in a pharmacologically specific fashion. Northern blot and in situ hybridization analyses of receptor mRNA in brain tissues revealed two transcripts of 5.6 and 3.1 kilobases, both of which were abundant in cortex, cerebellum, hippocampus, and thalamus, with lower levels in olfactory bulb, striatum, mesencephalon, and retina. These regional distribution data are in good agreement with previous receptor autoradiographic studies involving the A1 adenosine receptor.

  17. BDNF-induced presynaptic facilitation of GABAergic transmission in the hippocampus of young adults is dependent of TrkB and adenosine A2A receptors.

    PubMed

    Colino-Oliveira, Mariana; Rombo, Diogo M; Dias, Raquel B; Ribeiro, Joaquim A; Sebastião, Ana M

    2016-06-01

    Brain-derived neurotrophic factor (BDNF) and adenosine are widely recognized as neuromodulators of glutamatergic transmission in the adult brain. Most BDNF actions upon excitatory plasticity phenomena are under control of adenosine A2A receptors (A2ARs). Concerning gamma-aminobutyric acid (GABA)-mediated transmission, the available information refers to the control of GABA transporters. We now focused on the influence of BDNF and the interplay with adenosine on phasic GABAergic transmission. To assess this, we evaluated evoked and spontaneous synaptic currents recorded from CA1 pyramidal cells in acute hippocampal slices from adult rat brains (6 to 10 weeks old). BDNF (10-100 ng/mL) increased miniature inhibitory postsynaptic current (mIPSC) frequency, but not amplitude, as well as increased the amplitude of inhibitory postsynaptic currents (IPSCs) evoked by afferent stimulation. The facilitatory action of BDNF upon GABAergic transmission was lost in the presence of a Trk inhibitor (K252a, 200 nM), but not upon p75(NTR) blockade (anti-p75(NTR) IgG, 50 μg/mL). Moreover, the facilitatory action of BDNF onto GABAergic transmission was also prevented upon A2AR antagonism (SCH 58261, 50 nM). We conclude that BDNF facilitates GABAergic signaling at the adult hippocampus via a presynaptic mechanism that depends on TrkB and adenosine A2AR activation. PMID:26897393

  18. Metabotropic glutamate receptor subtype 5 antagonism in learning and memory

    PubMed Central

    Simonyi, Agnes; Schachtman, Todd R.; Christoffersen, Gert R. J.

    2010-01-01

    Summary The role of the metabotropic glutamate receptor 5 (mGlu5 receptor) in learning and memory and other behaviors are reviewed by examining the influence of selective antagonists and genetic knockout on performance. This receptor is involved in spatial learning, contextual fear conditioning, inhibitory avoidance, fear potentiated startle, and conditioned taste aversion. However, mGlu5 receptor antagonists have proven to be ineffective in other learning tasks, such as the delayed-match-to-position test and a three-hole spatial learning task. Locomotion is often decreased by mGlu5 receptor antagonists; and other behaviors such as social interaction and consummatory responses can also be affected. In mGlu5 receptor knockout mice, performance in contextual fear conditioning and spatial water maze tasks is impaired. Although the available evidence is suggestive of an important contribution of mGlu5 receptors to cognitive functions, further studies are needed, particularly those with in vivo evaluation of the role of mGlu5 receptors in selective brain regions in different stages of memory formation. PMID:20363219

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

    PubMed Central

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

    2014-01-01

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

  20. 2-(1-Hexyn-1-yl)adenosine-induced intraocular hypertension is mediated via K+ channel opening through adenosine A2A receptor in rabbits.

    PubMed

    Konno, Takashi; Uchibori, Takehiro; Nagai, Akihiko; Kogi, Kentaro; Nakahata, Norimichi

    2005-08-22

    The present study was performed to clarify the mechanism of change in intraocular pressure by 2-(1-hexyn-1-yl)adenosine (2-H-Ado), a selective adenosine A2 receptor agonist, in rabbits. 2-H-Ado (0.1%, 50 microl)-induced ocular hypertension (E(max): 7.7 mm Hg) was inhibited by an adenosine A2A receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine, ATP-sensitive K+ channel blocker glibenclamide or 5-hydroxydecanoic acid, but not by an adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A2B receptor antagonist alloxazine or a cyclooxygenase inhibitor indomethacin. The outflow facility induced by 2-H-Ado seems to be independent of increase in intraocular pressure or ATP-sensitive K+ channel. In contrast, the recovery rate in intraocular pressure decreased by hypertonic saline was accelerated by 2-H-Ado, and this response was dependent on ATP-sensitive K+ channel. These results suggest that 2-H-Ado-induced ocular hypertension is mediated via K+ channel opening through adenosine A2A receptor, and this is probably due to aqueous formation, but independent of change in outflow facility or prostaglandin production. PMID:16023100

  1. Adenosine A3 receptor activation is neuroprotective against retinal neurodegeneration.

    PubMed

    Galvao, Joana; Elvas, Filipe; Martins, Tiago; Cordeiro, M Francesca; Ambrósio, António Francisco; Santiago, Ana Raquel

    2015-11-01

    Death of retinal neural cells, namely retinal ganglion cells (RGCs), is a characteristic of several retinal neurodegenerative diseases. Although the role of adenosine A3 receptor (A3R) in neuroprotection is controversial, A3R activation has been reported to afford protection against several brain insults, with few studies in the retina. In vitro models (retinal neural and organotypic cultures) and animal models [ischemia-reperfusion (I-R) and partial optic nerve transection (pONT)] were used to study the neuroprotective properties of A3R activation against retinal neurodegeneration. The A3R selective agonist (2-Cl-IB-MECA, 1 μM) prevented apoptosis (TUNEL(+)-cells) induced by kainate and cyclothiazide (KA + CTZ) in retinal neural cultures (86.5 ± 7.4 and 37.2 ± 6.1 TUNEL(+)-cells/field, in KA + CTZ and KA + CTZ + 2-Cl-IB-MECA, respectively). In retinal organotypic cultures, 2-Cl-IB-MECA attenuated NMDA-induced cell death, assessed by TUNEL (17.3 ± 2.3 and 8.3 ± 1.2 TUNEL(+)-cells/mm(2) in NMDA and NMDA+2-Cl-IB-MECA, respectively) and PI incorporation (ratio DIV4/DIV2 3.3 ± 0.3 and 1.3 ± 0.1 in NMDA and NMDA+2-Cl-IB-MECA, respectively) assays. Intravitreal 2-Cl-IB-MECA administration afforded protection against I-R injury decreasing the number of TUNEL(+) cells by 72%, and increased RGC survival by 57%. Also, intravitreal administration of 2-Cl-IB-MECA inhibited apoptosis (from 449.4 ± 37.8 to 207.6 ± 48.9 annexin-V(+)-cells) and RGC loss (from 1.2 ± 0.6 to 8.1 ± 1.7 cells/mm) induced by pONT. This study demonstrates that 2-Cl-IB-MECA is neuroprotective to the retina, both in vitro and in vivo. Activation of A3R may have great potential in the management of retinal neurodegenerative diseases characterized by RGC death, as glaucoma and diabetic retinopathy, and ischemic diseases. PMID:26297614

  2. Peripheral endothelin A receptor antagonism attenuates carcinoma-induced pain.

    PubMed

    Schmidt, Brian L; Pickering, Victoria; Liu, Stanley; Quang, Phuong; Dolan, John; Connelly, S Thaddeus; Jordan, Richard C K

    2007-05-01

    In this study we investigated the role of endothelin-1 (ET-1) and its peripheral receptor (ET-A) in carcinoma-induced pain in a mouse cancer pain model. Tumors were induced in the hind paw of female mice by local injection of cells derived from a human oral squamous cell carcinoma (SCC). Significant pain, as indicated by reduction in withdrawal thresholds in response to mechanical stimulation, began at four days after SCC inoculation and lasted to 28 days, the last day of measurement. Intra-tumor expression of both ET-1 mRNA and ET-1 protein were significantly upregulated compared to normal tissue, and local administration of the ET-A receptor selective antagonist, BQ-123 (100 microM) significantly elevated withdrawal thresholds, indicating the induction of an antinociceptive effect. These findings support the suggestion that ET-1 and ET-A receptors contribute to the severity of carcinoma-induced soft tissue cancer pain. PMID:16807013

  3. Adenosine A2A Receptors Modulate Acute Injury and Neuroinflammation in Brain Ischemia

    PubMed Central

    Pedata, Felicita; Pugliese, Anna Maria; Coppi, Elisabetta; Dettori, Ilaria; Maraula, Giovanna; Cellai, Lucrezia; Melani, Alessia

    2014-01-01

    The extracellular concentration of adenosine in the brain increases dramatically during ischemia. Adenosine A2A receptor is expressed in neurons and glial cells and in inflammatory cells (lymphocytes and granulocytes). Recently, adenosine A2A receptor emerged as a potential therapeutic attractive target in ischemia. Ischemia is a multifactorial pathology characterized by different events evolving in the time. After ischemia the early massive increase of extracellular glutamate is followed by activation of resident immune cells, that is, microglia, and production or activation of inflammation mediators. Proinflammatory cytokines, which upregulate cell adhesion molecules, exert an important role in promoting recruitment of leukocytes that in turn promote expansion of the inflammatory response in ischemic tissue. Protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. A2A receptors present on central cells and on blood cells account for important effects depending on the time-related evolution of the pathological condition. Evidence suggests that A2A receptor antagonists provide early protection via centrally mediated control of excessive excitotoxicity, while A2A receptor agonists provide protracted protection by controlling massive blood cell infiltration in the hours and days after ischemia. Focus on inflammatory responses provides for adenosine A2A receptor agonists a wide therapeutic time-window of hours and even days after stroke. PMID:25165414

  4. Molecular expression of adenosine receptors in OVCAR-3, Caov-4 and SKOV-3 human ovarian cancer cell lines

    PubMed Central

    Hajiahmadi, S.; Panjehpour, M.; Aghaei, M.; Mousavi, S.

    2015-01-01

    Adenosine receptors (A1, A2a, A2b and A3) have several physiological and pathological roles in cancer cell lines. The present study was carried out to evaluate the mRNA and protein expression profile and functional role of adenosine receptors in OVCAR-3, Caov-4 and SKOV-3 ovarian cancer cell lines. The levels of mRNA and protein expression of A1, A2a, A2b and A3 adenosine receptors in the ovarian cancer cell lines were measured by Real-time PCR and western blotting. The functional roles of adenosine receptors were investigated through measurement of cAMP levels after agonist treatment. The mRNA and protein of all adenosine receptors subtypes were expressed in the ovarian cancer cell lines. Our findings demonstrated that A2b and A3 had the most mRNA and protein expression. Moreover, cAMP assay confirmed the functional role of A2b and A3 adenosine receptors. This findings demonstrated that A2b and A3 subtypes are most important adenosine receptors in humn ovarian cancer cell lines. This information provide a strong possibility into the relationship of A2b and A3 adenosine receptor and ovarian cancer. PMID:26430456

  5. Molecular expression of adenosine receptors in OVCAR-3, Caov-4 and SKOV-3 human ovarian cancer cell lines.

    PubMed

    Hajiahmadi, S; Panjehpour, M; Aghaei, M; Mousavi, S

    2015-01-01

    Adenosine receptors (A1, A2a, A2b and A3) have several physiological and pathological roles in cancer cell lines. The present study was carried out to evaluate the mRNA and protein expression profile and functional role of adenosine receptors in OVCAR-3, Caov-4 and SKOV-3 ovarian cancer cell lines. The levels of mRNA and protein expression of A1, A2a, A2b and A3 adenosine receptors in the ovarian cancer cell lines were measured by Real-time PCR and western blotting. The functional roles of adenosine receptors were investigated through measurement of cAMP levels after agonist treatment. The mRNA and protein of all adenosine receptors subtypes were expressed in the ovarian cancer cell lines. Our findings demonstrated that A2b and A3 had the most mRNA and protein expression. Moreover, cAMP assay confirmed the functional role of A2b and A3 adenosine receptors. This findings demonstrated that A2b and A3 subtypes are most important adenosine receptors in humn ovarian cancer cell lines. This information provide a strong possibility into the relationship of A2b and A3 adenosine receptor and ovarian cancer. PMID:26430456

  6. Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking

    SciTech Connect

    Barrington, W.W.; Jacobson, K.A.; Hutchison, A.J.; Williams, M.; Stiles, G.L. )

    1989-09-01

    A high-affinity iodinated agonist radioligand for the A2 adenosine receptor has been synthesized to facilitate studies of the A2 adenosine receptor binding subunit. The radioligand 125I-labeled PAPA-APEC (125I-labeled 2-(4-(2-(2-((4- aminophenyl)methylcarbonylamino)ethylaminocarbonyl)- ethyl)phenyl)ethylamino-5'-N-ethylcarboxamidoadenosine) was synthesized and found to bind to the A2 adenosine receptor in bovine striatal membranes with high affinity (Kd = 1.5 nM) and A2 receptor selectivity. Competitive binding studies reveal the appropriate A2 receptor pharmacologic potency order with 5'-N-ethylcarboxamidoadenosine (NECA) greater than (-)-N6-((R)-1-methyl- 2-phenylethyl)adenosine (R-PIA) greater than (+)-N6-((S)-1-methyl-2- phenylethyl)adenosine (S-PIA). Adenylate cyclase assays, in human platelet membranes, demonstrate a dose-dependent stimulation of cAMP production. PAPA-APEC (1 microM) produces a 43% increase in cAMP production, which is essentially the same degree of increase produced by 5'-N- ethylcarboxamidoadenosine (the prototypic A2 receptor agonist). These findings combined with the observed guanine nucleotide-mediated decrease in binding suggest that PAPA-APEC is a full A2 agonist. The A2 receptor binding subunit was identified by photoaffinity-crosslinking studies using 125I-labeled PAPA-APEC and the heterobifunctional crosslinking agent N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH). After covalent incorporation, a single specifically radiolabeled protein with an apparent molecular mass of 45 kDa was observed on NaDodSO4/PAGE/autoradiography. Incorporation of 125I-labeled PAPA-APEC into this polypeptide is blocked by agonists and antagonists with the expected potency for A2 receptors and is decreased in the presence of 10(-4) M guanosine 5'-(beta, gamma-imido)triphosphate.

  7. Role of extracellular cysteine residues in the adenosine A2A receptor.

    PubMed

    De Filippo, Elisabetta; Namasivayam, Vigneshwaran; Zappe, Lukas; El-Tayeb, Ali; Schiedel, Anke C; Müller, Christa E

    2016-06-01

    The G protein-coupled A2A adenosine receptor represents an important drug target. Crystal structures and modeling studies indicated that three disulfide bonds are formed between ECL1 and ECL2 (I, Cys71(2.69)-Cys159(45.43); II, Cys74(3.22)-Cys146(45.30), and III, Cys77(3.25)-Cys166(45.50)). However, the A2BAR subtype appears to require only disulfide bond III for proper function. In this study, each of the three disulfide bonds in the A2AAR was disrupted by mutation of one of the cysteine residues to serine. The mutant receptors were stably expressed in Chinese hamster ovary cells and analyzed in cyclic adenosine monophosphate (cAMP) accumulation and radioligand binding studies using structurally diverse agonists: adenosine, NECA, CGS21680, and PSB-15826. Results were rationalized by molecular modeling. The observed effects were dependent on the investigated agonist. Loss of disulfide bond I led to a widening of the orthosteric binding pocket resulting in a strong reduction in the potency of adenosine, but not of NECA or 2-substituted nucleosides. Disruption of disulfide bond II led to a significant reduction in the agonists' efficacy indicating its importance for receptor activation. Disulfide bond III disruption reduced potency and affinity of the small adenosine agonists and NECA, but not of the larger 2-substituted agonists. While all the three disulfide bonds were essential for high potency or efficacy of adenosine, structural modification of the nucleoside could rescue affinity or efficacy at the mutant receptors. At present, it cannot be excluded that formation of the extracellular disulfide bonds in the A2AAR is dynamic. This might add another level of G protein-coupled receptor (GPCR) modulation, in particular for the cysteine-rich A2A and A2BARs. PMID:26969588

  8. Orexin 2 Receptor Antagonism is Sufficient to Promote NREM and REM Sleep from Mouse to Man

    PubMed Central

    Gotter, Anthony L.; Forman, Mark S.; Harrell, Charles M.; Stevens, Joanne; Svetnik, Vladimir; Yee, Ka Lai; Li, Xiaodong; Roecker, Anthony J.; Fox, Steven V.; Tannenbaum, Pamela L.; Garson, Susan L.; Lepeleire, Inge De; Calder, Nicole; Rosen, Laura; Struyk, Arie; Coleman, Paul J.; Herring, W. Joseph; Renger, John J.; Winrow, Christopher J.

    2016-01-01

    Orexin neuropeptides regulate sleep/wake through orexin receptors (OX1R, OX2R); OX2R is the predominant mediator of arousal promotion. The potential for single OX2R antagonism to effectively promote sleep has yet to be demonstrated in humans. MK-1064 is an OX2R-single antagonist. Preclinically, MK-1064 promotes sleep and increases both rapid eye movement (REM) and non-REM (NREM) sleep in rats at OX2R occupancies higher than the range observed for dual orexin receptor antagonists. Similar to dual antagonists, MK-1064 increases NREM and REM sleep in dogs without inducing cataplexy. Two Phase I studies in healthy human subjects evaluated safety, tolerability, pharmacokinetics and sleep-promoting effects of MK-1064, and demonstrated dose-dependent increases in subjective somnolence (via Karolinska Sleepiness Scale and Visual Analogue Scale measures) and sleep (via polysomnography), including increased REM and NREM sleep. Thus, selective OX2R antagonism is sufficient to promote REM and NREM sleep across species, similarly to that seen with dual orexin receptor antagonism. PMID:27256922

  9. Orexin 2 Receptor Antagonism is Sufficient to Promote NREM and REM Sleep from Mouse to Man.

    PubMed

    Gotter, Anthony L; Forman, Mark S; Harrell, Charles M; Stevens, Joanne; Svetnik, Vladimir; Yee, Ka Lai; Li, Xiaodong; Roecker, Anthony J; Fox, Steven V; Tannenbaum, Pamela L; Garson, Susan L; Lepeleire, Inge De; Calder, Nicole; Rosen, Laura; Struyk, Arie; Coleman, Paul J; Herring, W Joseph; Renger, John J; Winrow, Christopher J

    2016-01-01

    Orexin neuropeptides regulate sleep/wake through orexin receptors (OX1R, OX2R); OX2R is the predominant mediator of arousal promotion. The potential for single OX2R antagonism to effectively promote sleep has yet to be demonstrated in humans. MK-1064 is an OX2R-single antagonist. Preclinically, MK-1064 promotes sleep and increases both rapid eye movement (REM) and non-REM (NREM) sleep in rats at OX2R occupancies higher than the range observed for dual orexin receptor antagonists. Similar to dual antagonists, MK-1064 increases NREM and REM sleep in dogs without inducing cataplexy. Two Phase I studies in healthy human subjects evaluated safety, tolerability, pharmacokinetics and sleep-promoting effects of MK-1064, and demonstrated dose-dependent increases in subjective somnolence (via Karolinska Sleepiness Scale and Visual Analogue Scale measures) and sleep (via polysomnography), including increased REM and NREM sleep. Thus, selective OX2R antagonism is sufficient to promote REM and NREM sleep across species, similarly to that seen with dual orexin receptor antagonism. PMID:27256922

  10. Exploring new scaffolds for angiotensin II receptor antagonism.

    PubMed

    Kritsi, Eftichia; Matsoukas, Minos-Timotheos; Potamitis, Constantinos; Karageorgos, Vlasios; Detsi, Anastasia; Magafa, Vasilliki; Liapakis, George; Mavromoustakos, Thomas; Zoumpoulakis, Panagiotis

    2016-09-15

    Nowadays, AT1 receptor (AT1R) antagonists (ARBs) constitute the one of the most prevalent classes of antihypertensive drugs that modulate the renin-angiotensin system (RAS). Their main uses include also treatment of diabetic nephropathy (kidney damage due to diabetes) and congestive heart failure. Towards this direction, our study has been focused on the discovery of novel agents bearing different scaffolds which may evolve as a new class of AT1 receptor antagonists. To fulfill this aim, a combination of computational approaches and biological assays were implemented. Particularly, a pharmacophore model was established and served as a 3D search query to screen the ChEMBL15 database. The reliability and accuracy of virtual screening results were improved by using molecular docking studies. In total, 4 compounds with completely diverse chemical scaffolds from potential ARBs, were picked and tested for their binding affinity to AT1 receptor. Results revealed high nanomolar to micromolar affinity (IC50) for all the compounds. Especially, compound 4 exhibited a binding affinity of 199nM. Molecular dynamics simulations were utilized in an effort to provide a molecular basis of their binding to AT1R in accordance to their biological activities. PMID:27480029

  11. Circadian rhythm in adenosine A1 receptor of mouse cerebral cortex

    SciTech Connect

    Florio, C.; Rosati, A.M.; Traversa, U.; Vertua, R. )

    1991-01-01

    In order to investigate diurnal variation in adenosine A1 receptors binding parameters, Bmax and Kd values of specifically bound N6-cyclohexyl-({sup 3}H)adenosine were determined in the cerebral cortex of mice that had been housed under controlled light-dark cycles for 4 weeks. Significant differences were found for Bmax values measured at 3-hr intervals across a 24-h period, with low Bmax values during the light period and high Bmax values during the dark period. The amplitude between 03.00 and 18.00 hr was 33%. No substantial rhythm was found in the Kd values. It is suggested that the changes in the density of A1 receptors could reflect a physiologically-relevant mechanism by which adenosine exerts its modulatory role in the central nervous system.

  12. Recent developments in A2B adenosine receptor ligands.

    PubMed

    Kalla, Rao V; Zablocki, Jeff; Tabrizi, Mojgan Aghazadeh; Baraldi, Pier Giovanni

    2009-01-01

    A selective, high-affinity A(2B) adenosine receptor (AR) antagonist will be useful as a pharmacological tool to help determine the role of the A(2B)AR in inflammatory diseases and angiogenic diseases. Based on early A(2B)AR-selective ligands with nonoptimal pharmaceutical properties, such as 15 (MRS 1754: K(i)(hA(2B)) = 2 nM; K(i)(hA(1)) = 403 nM; K(i)(hA(2A)) = 503 NM, and K(i)(hA(3)) = 570 nM), several groups have discovered second-generation A(2B)AR ligands that are suitable for development. Scientists at CV Therapeutics have discovered the selective, high-affinity A(2B)AR antagonist 22, a 8-(4-pyrazolyl)-xanthine derivative, (CVT-6883, K(i)(hA(2B)) = 22 nM; K(i)(hA(1)) = 1,940 nM; K(i)(hA(2A)) = 3,280; and K(i)(hA(3)) = 1,070 nM). Compound 22 has demonstrated favorable pharmacokinetic (PK) properties (T(1/2) = 4 h and F > 35% rat), and it is a functional antagonist at the A(2B)AR(K (B) = 6 nM). In a mouse model of asthma, compound 22 demonstrated a dose-dependent efficacy supporting the role of the A(2B)AR in asthma. In two Phase I clinical trails, 22 (CVT-6883) was found to be safe, well tolerated, and suitable for once-daily dosing. Baraldi et al. have independently discovered a selective, high-affinity A(2B)AR antagonist, 30 (MRE2029F20), 8-(5-pyrazolyl)-xanthine (K(i)(hA(2B)) = 5.5 nM; K(i)(hA(1)) = 200 nM; K(i)(hA(2A), A(3)) > 1,000, that has been selected for development in conjunction with King Pharmaceuticals. Compound 30 has been demonstrated to be a functional antagonist of the A(2B)AR, and it has been radiolabeled for use in pharmacological studies. A third compound, 58 (LAS-38096), is a 2-aminopyrimidine derivative (discovered by the Almirall group) that has high A(2B)AR affinity and selectivity (K(i)(hA(2B)) = 17 nM; K(i)(hA(1)) > 1,000 nM; K(i)(hA(2A)) > 2,500; and K(i)(hA(3)) > 1,000 nM), and 58 has been moved into preclinical safety testing. A fourth selective, high-affinity A(2B)AR antagonist, 54 (OSIP339391 K(i))(hA(2B)) = 0.5 nM; K(i))(hA(1

  13. Differences in adenosine A-1 and A-2 receptor density revealed by autoradiography in methylxanthine-sensitive and insensitive mice

    SciTech Connect

    Jarvis, M.F.; Williams, M.

    1988-07-01

    Two strains of inbred mice, CBA/J and SWR/J, have been identified which are, respectively, sensitive and insensitive to the behavioral and toxic effects of methylxanthines. Autoradiographic analyses of brain adenosine receptors were conducted with (/sup 3/H)CHA to label adenosine A-1 receptors and (/sup 3/H)NECA, in the presence of 50 nM CPA, to label adenosine A-2 receptors. For both mouse strains, adenosine A-1 receptors were most highly concentrated in the hippocampus and cerebellum whereas adenosine A-2 receptors were selectively localized in the striatum. CBA/J mice displayed a 30% greater density of adenosine A-1 receptors in the hippocampal CA-1 and CA-3 regions and in the cerebellum as compared to the SWR/J mice. The number of A-2 receptors (Bmax) was 40% greater in the striatum and olfactory tubercle of CBA/J as compared to SWR/J mice. No significant regional differences in A-1 or A-2 receptor affinities were observed between these inbred strains of mice. These results indicate that the differential sensitivity to methylxanthines between these mouse strains may reflect a genetically mediated difference in regional adenosine receptor densities.

  14. MINERALOCORTICOID RECEPTOR ANTAGONISM CONFERS CARDIOPROTECTION IN HEART FAILURE

    PubMed Central

    Seawell, Michael R.; Darazi, Fahed Al; Farah, Victor; Ramanathan, Kodangudi B.; Newman, Kevin P.; Bhattacharya, Syamal K.; Weber, Karl T.

    2012-01-01

    The symptoms and signs constituting the congestive heart failure (CHF) syndrome have their pathophysiologic origins rooted in a salt-avid renal state mediated by effector hormones of the renin-angiotensin-aldosterone and adrenergic nervous systems. Controlled clinical trials, conducted over the past decade in patients having minimally to markedly severe symptomatic heart failure, have demonstrated the efficacy of a pharmacologic regimen that interferes with these hormones, including aldosterone receptor binding with either spironolactone or eplerenone. Potential pathophysiologic mechanisms which have not hitherto been considered involved for the salutary responses and cardioprotection provided by these mineralocorticoid receptor antagonists are reviewed herein. In particular, we focus on the less well-recognized impact of catecholamines and aldosterone on mono- and divalent cation dyshomeostasis which leads to hypokalemia, hypomagnesemia, ionized hypocalcemia with secondary hyperparathyroidism and hypozincemia. Attendant adverse cardiac consequences include a delay in myocardial repolarization with increased propensity for supra- and ventricular arrhythmias and compromised antioxidant defenses with increased susceptibility to nonischemic cardiomyocyte necrosis. PMID:23114591

  15. An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor

    PubMed Central

    Dagdas, Yasin F; Belhaj, Khaoula; Maqbool, Abbas; Chaparro-Garcia, Angela; Pandey, Pooja; Petre, Benjamin; Tabassum, Nadra; Cruz-Mireles, Neftaly; Hughes, Richard K; Sklenar, Jan; Win, Joe; Menke, Frank; Findlay, Kim; Banfield, Mark J; Kamoun, Sophien; Bozkurt, Tolga O

    2016-01-01

    Plants use autophagy to safeguard against infectious diseases. However, how plant pathogens interfere with autophagy-related processes is unknown. Here, we show that PexRD54, an effector from the Irish potato famine pathogen Phytophthora infestans, binds host autophagy protein ATG8CL to stimulate autophagosome formation. PexRD54 depletes the autophagy cargo receptor Joka2 out of ATG8CL complexes and interferes with Joka2's positive effect on pathogen defense. Thus, a plant pathogen effector has evolved to antagonize a host autophagy cargo receptor to counteract host defenses. DOI: http://dx.doi.org/10.7554/eLife.10856.001 PMID:26765567

  16. The adenosine A2B receptor is involved in anion secretion in human pancreatic duct Capan-1 epithelial cells.

    PubMed

    Hayashi, M; Inagaki, A; Novak, I; Matsuda, H

    2016-07-01

    Adenosine modulates a wide variety of biological processes via adenosine receptors. In the exocrine pancreas, adenosine regulates transepithelial anion secretion in duct cells and is considered to play a role in acini-to-duct signaling. To identify the functional adenosine receptors and Cl(-) channels important for anion secretion, we herein performed experiments on Capan-1, a human pancreatic duct cell line, using open-circuit Ussing chamber and gramicidin-perforated patch-clamp techniques. The luminal addition of adenosine increased the negative transepithelial potential difference (V te) in Capan-1 monolayers with a half-maximal effective concentration value of approximately 10 μM, which corresponded to the value obtained on whole-cell Cl(-) currents in Capan-1 single cells. The effects of adenosine on V te, an equivalent short-circuit current (I sc), and whole-cell Cl(-) currents were inhibited by CFTRinh-172, a cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel inhibitor. The adenosine A2B receptor agonist, BAY 60-6583, increased I sc and whole-cell Cl(-) currents through CFTR Cl(-) channels, whereas the A2A receptor agonist, CGS 21680, had negligible effects. The A2B receptor antagonist, PSB 603, inhibited the response of I sc to adenosine. Immunohistochemical analysis showed that the A2A and A2B receptors colocalized with Ezrin in the luminal membranes of Capan-1 monolayers and in rat pancreatic ducts. Adenosine elicited the whole-cell Cl(-) currents in guinea pig duct cells. These results demonstrate that luminal adenosine regulates anion secretion by activating CFTR Cl(-) channels via adenosine A2B receptors on the luminal membranes of Capan-1 cells. The present study endorses that purinergic signaling is important in the regulation of pancreatic secretion. PMID:26965147

  17. Mechanisms of the adenosine A2A receptor-induced sensitization of esophageal C fibers.

    PubMed

    Brozmanova, M; Mazurova, L; Ru, F; Tatar, M; Hu, Y; Yu, S; Kollarik, M

    2016-02-01

    Clinical studies indicate that adenosine contributes to esophageal mechanical hypersensitivity in some patients with pain originating in the esophagus. We have previously reported that the esophageal vagal nodose C fibers express the adenosine A2A receptor. Here we addressed the hypothesis that stimulation of the adenosine A2A receptor induces mechanical sensitization of esophageal C fibers by a mechanism involving transient receptor potential A1 (TRPA1). Extracellular single fiber recordings of activity originating in C-fiber terminals were made in the ex vivo vagally innervated guinea pig esophagus. The adenosine A2A receptor-selective agonist CGS21680 induced robust, reversible sensitization of the response to esophageal distention (10-60 mmHg) in a concentration-dependent fashion (1-100 nM). At the half-maximally effective concentration (EC50: ≈3 nM), CGS21680 induced an approximately twofold increase in the mechanical response without causing an overt activation. This sensitization was abolished by the selective A2A antagonist SCH58261. The adenylyl cyclase activator forskolin mimicked while the nonselective protein kinase inhibitor H89 inhibited mechanical sensitization by CGS21680. CGS21680 did not enhance the response to the purinergic P2X receptor agonist α,β-methylene-ATP, indicating that CGS21680 does not nonspecifically sensitize to all stimuli. Mechanical sensitization by CGS21680 was abolished by pretreatment with two structurally different TRPA1 antagonists AP18 and HC030031. Single cell RT-PCR and whole cell patch-clamp studies in isolated esophagus-specific nodose neurons revealed the expression of TRPA1 in A2A-positive C-fiber neurons and demonstrated that CGS21682 potentiated TRPA1 currents evoked by allylisothiocyanate. We conclude that stimulation of the adenosine A2A receptor induces mechanical sensitization of nodose C fibers by a mechanism sensitive to TRPA1 antagonists indicating the involvement of TRPA1. PMID:26564719

  18. Spinal adenosine A2a receptor activation elicits long-lasting phrenic motor facilitation.

    PubMed

    Golder, Francis J; Ranganathan, Lavanya; Satriotomo, Irawan; Hoffman, Michael; Lovett-Barr, Mary Rachael; Watters, Jyoti J; Baker-Herman, Tracy L; Mitchell, Gordon S

    2008-02-27

    Acute intermittent hypoxia elicits a form of spinal, brain-derived neurotrophic factor (BDNF)-dependent respiratory plasticity known as phrenic long-term facilitation. Ligands that activate G(s)-protein-coupled receptors, such as the adenosine 2a receptor, mimic the effects of neurotrophins in vitro by transactivating their high-affinity receptor tyrosine kinases, the Trk receptors. Thus, we hypothesized that A2a receptor agonists would elicit phrenic long-term facilitation by mimicking the effects of BDNF on TrkB receptors. Here we demonstrate that spinal A2a receptor agonists transactivate TrkB receptors in the rat cervical spinal cord near phrenic motoneurons, thus inducing long-lasting (hours) phrenic motor facilitation. A2a receptor activation increased phosphorylation and new synthesis of an immature TrkB protein, induced TrkB signaling through Akt, and strengthened synaptic pathways to phrenic motoneurons. RNA interference targeting TrkB mRNA demonstrated that new TrkB protein synthesis is necessary for A2a-induced phrenic motor facilitation. A2a receptor activation also increased breathing in unanesthetized rats, and improved breathing in rats with cervical spinal injuries. Thus, small, highly permeable drugs (such as adenosine receptor agonists) that transactivate TrkB receptors may provide an effective therapeutic strategy in the treatment of patients with ventilatory control disorders, such as obstructive sleep apnea, or respiratory insufficiency after spinal injury or during neurodegenerative diseases. PMID:18305238

  19. in Silico investigation of the structural requirements for the AMPA receptor antagonism by quinoxaline derivatives

    PubMed Central

    Azam, Faizul; Abugrain, Ismaiel Mohamed; Sanalla, Mohamed Hussin; Elnaas, Radwan Fatahalla; Rajab, Ibrahim Abdassalam Ibn

    2013-01-01

    Glutamate receptors have been implicated in various neurological disorders and their antagonism offers a suitable approach for the treatment of such disorders. The field of drug design and discovery aims to find best medicines to prevent, treat and cure diseases quickly and efficiently. In this regard, computational tools have helped medicinal chemists modify and optimize molecules to potent drug candidates with better pharmacokinetic profiles, and guiding biologists and pharmacologists to explore new disease genes as well as novel drug targets. In the present study, to understand the structural requirements for AMPA receptor antagonism, molecular docking study was performed on 41 structurally diverse antagonists based on quinoxaline nucleus. Lamarckian genetic algorithm methodology was employed for docking simulations using AutoDock 4.2 program. The results obtained signify that the molecular docking approach is reliable and produces a good correlation coefficient (r2 = 0.6) between experimental and docking predicted AMPA receptor antagonistic activity. The aromatic moiety of quinoxaline core has been proved to be vital for hydrophobic contacts exhibiting - interactions in docked conformations. However, polar moieties such as carboxylic group and 1,2,4-triazole moieties were noted to be sites for hydrophilic interactions in terms of hydrogen bonding with the receptor. These analyses can be exploited to design and develop novel AMPA receptor antagonists for the treatment of different neurological disorders. PMID:24250113

  20. Involvement of adenosine A2a receptor in intraocular pressure decrease induced by 2-(1-octyn-1-yl)adenosine or 2-(6-cyano-1-hexyn-1-yl)adenosine.

    PubMed

    Konno, Takashi; Murakami, Akira; Uchibori, Takehiro; Nagai, Akihiko; Kogi, Kentaro; Nakahata, Norimichi

    2005-04-01

    The aim of the present study is to clarify the mechanism for the decrease in intraocular pressure by 2-alkynyladenosine derivatives in rabbits. The receptor binding analysis revealed that 2-(1-octyn-1-yl)adenosine (2-O-Ado) and 2-(6-cyano-1-hexyn-1-yl)adenosine (2-CN-Ado) selectively bound to the A(2a) receptor with a high affinity. Ocular hypotensive responses to 2-O-Ado and 2-CN-Ado were inhibited by the adenosine A(2a)-receptor antagonist 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC), but not by the adenosine A(1)-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or the adenosine A(2b)-receptor antagonist alloxazine. In addition, 2-O-Ado and 2-CN-Ado caused an increase in outflow facility, which was inhibited by CSC, but not by DPCPX or alloxazine. Moreover, 2-O-Ado and 2-CN-Ado increased cAMP in the aqueous humor, and the 2-O-Ado-induced an increase in cAMP was inhibited by CSC. These results suggest that 2-O-Ado and 2-CN-Ado reduced intraocular pressure via an increase in outflow facility. The ocular hypotension may be mainly mediated through the activation of adenosine A(2a) receptor, although a possible involvement of adenosine A(1) receptor cannot be completely ruled out. 2-O-Ado and 2-CN-Ado are useful lead compounds for the treatment of glaucoma. PMID:15821340

  1. Adenosine A2B Receptor: From Cell Biology to Human Diseases.

    PubMed

    Sun, Ying; Huang, Pingbo

    2016-01-01

    Extracellular adenosine is a ubiquitous signaling molecule that modulates a wide array of biological processes. Recently, significant advances have been made in our understanding of A2B adenosine receptor (A2BAR). In this review, we first summarize some of the general characteristics of A2BAR, and then we describe the multiple binding partners of the receptor, such as newly identified α-actinin-1 and p105, and discuss how these associated proteins could modulate A2BAR's functions, including certain seemingly paradoxical functions of the receptor. Growing evidence indicates a critical role of A2BAR in cancer, renal disease, and diabetes, in addition to its importance in the regulation of vascular diseases, and lung disease. Here, we also discuss the role of A2BAR in cancer, renal disease, and diabetes and the potential of the receptor as a target for treating these three diseases. PMID:27606311

  2. Adenosine A2B Receptor: From Cell Biology to Human Diseases

    PubMed Central

    Sun, Ying; Huang, Pingbo

    2016-01-01

    Extracellular adenosine is a ubiquitous signaling molecule that modulates a wide array of biological processes. Recently, significant advances have been made in our understanding of A2B adenosine receptor (A2BAR). In this review, we first summarize some of the general characteristics of A2BAR, and then we describe the multiple binding partners of the receptor, such as newly identified α-actinin-1 and p105, and discuss how these associated proteins could modulate A2BAR's functions, including certain seemingly paradoxical functions of the receptor. Growing evidence indicates a critical role of A2BAR in cancer, renal disease, and diabetes, in addition to its importance in the regulation of vascular diseases, and lung disease. Here, we also discuss the role of A2BAR in cancer, renal disease, and diabetes and the potential of the receptor as a target for treating these three diseases. PMID:27606311

  3. Antagonism of human formyl peptide receptor 1 with natural compounds and their synthetic derivatives.

    PubMed

    Schepetkin, Igor A; Khlebnikov, Andrei I; Kirpotina, Liliya N; Quinn, Mark T

    2016-08-01

    Formyl peptide receptor 1 (FPR1) regulates a wide variety of neutrophil functional responses and plays an important role in inflammation and the pathogenesis of various diseases. To date, a variety of natural and synthetic molecules have been identified as FPR1 ligands. Here, we review current knowledge on natural products and natural product-inspired small molecules reported to antagonize and/or inhibit the FPR1-mediated responses. Based on this literature, additional screening of selected commercially available natural compounds for their ability to inhibit fMLF-induced Ca(2+) mobilization in human neutrophils and FPR1 transfected HL-60 cells, and pharmacophore modeling, natural products with potential as FPR1 antagonists are considered and discussed in this review. The identification and characterization of natural products that antagonize FPR1 activity may have potential for the development of novel therapeutics to limit or alter the outcome of inflammatory processes. PMID:26382576

  4. Suppression of adenosine 2a receptor (A2aR)-mediated adenosine signaling improves disease phenotypes in a mouse model of amyotrophic lateral sclerosis.

    PubMed

    Ng, Seng Kah; Higashimori, Haruki; Tolman, Michaela; Yang, Yongjie

    2015-05-01

    Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease in which the majority of upper and lower motor neurons are degenerated. Despite intensive efforts to identify drug targets and develop neuroprotective strategies, effective therapeutics for ALS remains unavailable. The identification and characterization of novel targets and pathways remain crucial in the development of ALS therapeutics. Adenosine is a major neuromodulator that actively regulates synaptic transmission. Interestingly, adenosine levels are significantly elevated in the cerebrospinal fluid (CSF) of progressing human ALS patients. In the current study, we showed that adenosine 2a receptor (A2aR), but not adenosine 1 receptor (A1R), is highly enriched in spinal (motor) neurons. A2aR expression is also selectively increased at the symptomatic onset in the spinal cords of SOD1G93A mice and end-stage human ALS spinal cords. Interestingly, we found that direct adenosine treatment is sufficient to induce embryonic stem cell-derived motor neuron (ESMN) cell death in cultures. Subsequent pharmacological inhibition and partial genetic ablation of A2aR (A2aR(+/-)) significantly protect ESMN from SOD1G93A(+) astrocyte-induced cell death and delay disease progression of SOD1G93A mice. Taken together, our results provide compelling novel evidence that A2aR-mediated adenosine signaling contributes to the selective spinal motor neuron degeneration observed in the SOD1G93A mouse model of ALS. PMID:25779930

  5. Adenosine A1 receptor inhibits postnatal neurogenesis and sustains astrogliogenesis from the subventricular zone.

    PubMed

    Benito-Muñoz, Monica; Matute, Carlos; Cavaliere, Fabio

    2016-09-01

    We previously demonstrated that activation of ATP P2X receptors during oxygen and glucose deprivation inhibits neuroblast migration and in vitro neurogenesis from the subventricular zone (SVZ). Here, we have studied the effects of adenosine, the natural end-product of ATP hydrolysis, in modulating neurogenesis and gliogenesis from the SVZ. We provide immunochemical, molecular and pharmacological evidence that adenosine via A1 receptors reduces neuronal differentiation of neurosphere cultures generated from postnatal SVZ. Furthermore, activation of A1 receptors induces downregulation of genes related to neurogenesis as demonstrated by gene expression analysis. Specifically, we found that A1 receptors trigger a signaling cascade that, through the release of IL10, turns on the Bmp2/SMAD pathway. Furthermore, activating A1 receptors in SVZ-neural progenitor cells inhibits neurogenesis and stimulates astrogliogenesis as assayed in vitro in neurosphere cultures and in vivo in the olfactory bulb. Together, these data indicate that adenosine acting at A1 receptors negatively regulates adult neurogenesis while promoting astrogliogenesis, and that this feature may be relevant to pathological conditions whereby purines are profusely released. GLIA 2016;64:1465-1478. PMID:27301342

  6. 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

  7. Neuroprotective and anti-inflammatory effects of the adenosine A(2A) receptor antagonist ST1535 in a MPTP mouse model of Parkinson's disease.

    PubMed

    Frau, Lucia; Borsini, Franco; Wardas, Jadwiga; Khairnar, Amit S; Schintu, Nicoletta; Morelli, Micaela

    2011-03-01

    Adenosine A(2A) receptor antagonists are one of the most attractive classes of drug for the treatment of Parkinson's disease (PD) as they are effective in counteracting motor dysfunctions and display neuroprotective and anti-inflammatory effects in animal models of PD. In this study, we evaluated the neuroprotective and anti-inflammatory properties of the adenosine A(2A) receptor antagonist ST1535 in a subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. C57BL/6J mice were repeatedly administered with vehicle, MPTP (20 mg/kg), or MPTP + ST1535 (2 mg/kg). Mice were sacrificed three days after the last administration of MPTP. Immunohistochemistry for tyrosine hydroxylase (TH) and cresyl violet staining were employed to evaluate dopaminergic neuron degeneration in the substantia nigra pars compacta (SNc) and caudate-putamen (CPu). CD11b and glial fibrillary acidic protein (GFAP) immunoreactivity were, respectively, evaluated as markers of microglial and astroglial response in the SNc and CPu. Stereological analysis for TH revealed a 32% loss of dopaminergic neurons in the SNc after repeated MPTP administration, which was completely prevented by ST1535 coadministration. Similarly, CPu decrease in TH (25%) was prevented by ST1535. MPTP treatment induced an intense gliosis in both the SNc and CPu. ST1535 totally prevented CD11b immunoreactivity in both analyzed areas, but only partially blocked GFAP increase in the SNc and CPu. A(2A) receptor antagonism is a new opportunity for improving symptomatic PD treatment. With its neuroprotective effect on dopaminergic neuron toxicity induced by MPTP and its antagonism on glial activation, ST1535 represents a new prospect for a disease-modifying drug. PMID:20665698

  8. Interaction of progesterone receptor with immobilized adenosine triphosphate.

    PubMed

    Moudgil, V K; Toft, D O

    1977-02-22

    Affinity chromatography has been used to study the binding of ATP to cyto-plasmic progesterone receptors of hen oviduct. A resin which selectively binds the receptor protein was prepared by linking ATP covalently to Sepharose 4B through a 6-carbon bridge of adipic acid dihydrazide. Receptor bound to the affinity resin was recovered in a single peak upon gradient elution with KCl (0.2-1 M) or ATP (0-0.1 M). While affinity chromatography was normally accomplished using the [3H]progesterone receptor complex, the hormone was not necessary for ATP binding under the conditions employed. The chromatography of crude receptor preparations allowed up to 100-fold purification with greater than 80% recovery of the receptor. The semipurified receptor appeared intact when analysed by sucrose gradient centrifugation, polyacrylamide gel electrophoresis, and DEAE-cellulose chromatography. The latter procedure separated the receptor into two components, A and B, both of which were capable of binding ATP. Although a specific biochemical role of ATP in hormone receptor action has not been demonstrated, the present studies support this possibility and, in addition, offer a convenient and reliable step for the purification of progesterone receptors. PMID:836885

  9. Bench-to-bedside review: Adenosine receptors – promising targets in acute lung injury?

    PubMed Central

    Schepp, Carsten P; Reutershan, Jörg

    2008-01-01

    Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening disorders that have substantial adverse effects on outcomes in critically ill patients. ALI/ARDS develops in response to pulmonary or extrapulmonary injury and is characterized by increased leakage from the pulmonary microvasculature and excessive infiltration of polymorphonuclear cells into the lung. Currently, no therapeutic strategies are available to control these fundamental pathophysiological processes in human ALI/ARDS. In a variety of animal models and experimental settings, the purine nucleoside adenosine has been demonstrated to regulate both endothelial barrier integrity and polymorphonuclear cell trafficking in the lung. Adenosine exerts its effects through four G-protein-coupled receptors (A1, A2A, A2B, and A3) that are expressed on leukocytes and nonhematopoietic cells, including endothelial and epithelial cells. Each type of adenosine receptor (AR) is characterized by a unique pharmacological and physiological profile. The development of selective AR agonists and antagonists, as well as the generation of gene-deficient mice, has contributed to a growing understanding of the cellular and molecular processes that are critically involved in the development of ALI/ARDS. Adenosine-dependent pathways are involved in both protective and proinflammatory effects, highlighting the need for a detailed characterization of the distinct pathways. This review summarizes current experimental observations on the role of adenosine signaling in the development of acute lung injury and illustrates that adenosine and ARs are promising targets that may be exploited in the development of innovative therapeutic strategies. PMID:18828873

  10. Adenosine receptor antagonists inhibit the development of morphine sensitization in the C57BL/6 mouse.

    PubMed

    Weisberg, S P; Kaplan, G B

    1999-04-01

    We examined the effects of adenosine antagonists on the development of morphine sensitization in C57BL/6 mice. Adenosine antagonists or vehicle were repeatedly co-administered intraperitoneally with morphine (10 mg/kg, s.c.) to mice once every other day for 9 days. Two days later, a 10 mg/kg morphine-only challenge was administered to each group. Consistent with sensitization, mice receiving morphine alone developed enhanced ambulatory activity responses to subsequent morphine administrations and, upon morphine-only challenge, had a significantly greater response to morphine than vehicle pretreated animals. The nonselective adenosine antagonist, caffeine, at 10 and 20 mg/kg but not at 5 mg/kg, attenuated the development of sensitization during co-administration with morphine and also following morphine-only challenge. The adenosine A1 selective antagonist 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX), at 0.001 and 0.002 mg/kg but not at 0.2 mg/kg, similarly attenuated the development of morphine sensitization. We propose a mechanism which involves an adenosine receptor role in the mesolimbic dopamine system. PMID:10320021

  11. Mechanism of Androgen Receptor Antagonism by Bicalutamide in the Treatment of Prostate Cancer

    PubMed Central

    Osguthorpe, D.J.; Hagler, A.T.

    2011-01-01

    The androgen receptor (AR) plays a key role in a regulating gene expression in a variety of tissues, including the prostate. In the latter role it is one of the primary targets in the development of new chemotherapeutics for treatment of prostate cancer, as well as being the target of the most widely prescribed current drug, bicalutamide (Bcu), for this disease. In view of it’s importance, and the absence of a crystal structure for any antagonist-AR complex, we have carried out a series of molecular dynamics based simulations of the AR-Bcu complex and quantum mechanical (QM) calculations of Bcu, to elucidate the structural basis for antagonism of this key target. The structures which emerge show that bicalutamide antagonizes AR by accessing an additional binding pocket (B-site) adjacent to the hormone binding site (HBS), induced by displacing helix 12. This distorts the coactivator binding site and results in the inactivation of transcription. An alternative equienergetic conformational state of bicalutamide was found to bind in an expanded hormone pocket without materially perturbing either helix 12 or the coactivator binding site. Thus both the structural basis of antagonism and the mechanism underlying agonist properties displayed by bicalutamide in different environments may be rationalized in terms of these structures. In addition the antagonist structure and especially the induced second site (B-site) provides a structural framework for the design of novel antiandrogens. PMID:21466228

  12. Expression of calcitonin gene-related peptide, adenosine A2a receptor and adenosine A1 receptor in experiment rat migraine models

    PubMed Central

    LU, WENXIAN; LI, BIN; CHEN, JINBO; SU, YIPENG; DONG, XIAOMENG; SU, XINYANG; GAO, LIXIANG

    2016-01-01

    A migraine is a disabling neurovascular disorder characterized by a unilateral throbbing headache that lasts from 4 to 72 h. The headache is often accompanied by nausea, vomiting, phonophobia and photophobia, and may be worsened by physical exercise. The trigeminovascular system (TVS) is speculated to have an important role in migraines, although the pathophysiology of this disorder remains to be elucidated. Trigeminal ganglion (TG) and spinal trigeminal nucleus caudalis (TNC) are important components of the TVS. Several clinical cases have provided evidence for the involvement of the brainstem in migraine initiation. Electrical stimulation of the trigeminal ganglion (ESTG) in rats can activate TVS during a migraine attack. Calcitonin gene-related peptide (CGRP) is an important vasoactive compound produced following TVS activation. Numerous studies have revealed that adenosine and its receptors have an important role in pain transmission and regulation process. However, only a few studies have examined whether adenosine A2a receptor (A2aR) and adenosine A1 receptor (A1R) are involved in migraine and nociceptive pathways. In the present study, CGRP, A2aR and A1R expression levels were detected in the TG and TNC of ESTG models through reverse transcription-quantitative polymerase chain reaction and western blot analysis. Tianshu capsule (TSC), a type of Chinese medicine, was also used in the ESTG rat models to examine its influence on the three proteins. Results demonstrated that CGRP, A2aR and A1R mediated pain transmission and the regulation process during migraine and the expression of the three proteins was regulated by TSC. PMID:26998280

  13. Impairment of ATP hydrolysis decreases adenosine A1 receptor tonus favoring cholinergic nerve hyperactivity in the obstructed human urinary bladder.

    PubMed

    Silva-Ramos, M; Silva, I; Faria, M; Magalhães-Cardoso, M T; Correia, J; Ferreirinha, F; Correia-de-Sá, P

    2015-12-01

    This study was designed to investigate whether reduced adenosine formation linked to deficits in extracellular ATP hydrolysis by NTPDases contributes to detrusor neuromodulatory changes associated with bladder outlet obstruction in men with benign prostatic hyperplasia (BPH). The kinetics of ATP catabolism and adenosine formation as well as the role of P1 receptor agonists on muscle tension and nerve-evoked [(3)H]ACh release were evaluated in mucosal-denuded detrusor strips from BPH patients (n = 31) and control organ donors (n = 23). The neurogenic release of ATP and [(3)H]ACh was higher (P < 0.05) in detrusor strips from BPH patients. The extracellular hydrolysis of ATP and, subsequent, adenosine formation was slower (t (1/2) 73 vs. 36 min, P < 0.05) in BPH detrusor strips. The A(1) receptor-mediated inhibition of evoked [(3)H]ACh release by adenosine (100 μM), NECA (1 μM), and R-PIA (0.3 μM) was enhanced in BPH bladders. Relaxation of detrusor contractions induced by acetylcholine required 30-fold higher concentrations of adenosine. Despite VAChT-positive cholinergic nerves exhibiting higher A(1) immunoreactivity in BPH bladders, the endogenous adenosine tonus revealed by adenosine deaminase is missing. Restoration of A1 inhibition was achieved by favoring (1) ATP hydrolysis with apyrase (2 U mL(-1)) or (2) extracellular adenosine accumulation with dipyridamole or EHNA, as these drugs inhibit adenosine uptake and deamination, respectively. In conclusion, reduced ATP hydrolysis leads to deficient adenosine formation and A(1) receptor-mediated inhibition of cholinergic nerve activity in the obstructed human bladder. Thus, we propose that pharmacological manipulation of endogenous adenosine levels and/or A(1) receptor activation might be useful to control bladder overactivity in BPH patients. PMID:26521170

  14. Reverse Translation of Clinical Electrophysiological Biomarkers in Behaving Rodents under Acute and Chronic NMDA Receptor Antagonism

    PubMed Central

    Sullivan, Elyse M; Timi, Patricia; Hong, L Elliot; O'Donnell, Patricio

    2015-01-01

    Electroencephalogram (EEG) stands out as a highly translational tool for psychiatric research, yet rodent and human EEG are not typically obtained in the same way. In this study we developed a tool to record skull EEG in awake-behaving rats in a similar manner to how human EEG are obtained and then used this technique to test whether acute NMDA receptor antagonism alters rodent EEG signals in a similar manner as in humans. Acute MK-801 treatment elevated gamma power and reduced beta band power, which closely mirrored EEG data from healthy volunteers receiving acute ketamine. To explore the mechanisms behind these oscillatory changes, we examined the effects of GABA-A receptor blockade, finding that picrotoxin (PTX) recapitulated the decrease in sound-evoked beta oscillations observed with acute MK-801, but did not produce changes in gamma band power. Chronic treatment with either PTX or MK-801 did not affect frequency-specific oscillatory activity when tested 24 h after the last drug injection, but decreased total broadband oscillatory power. Overall, this study validated a novel platform for recording rodent EEG and demonstrated similar oscillatory changes after acute NMDA receptor antagonism in both humans and rodents, suggesting that skull EEG may be a powerful tool for further translational studies. PMID:25176166

  15. Ivermectin Antagonizes Ethanol Inhibition in Purinergic P2X4 Receptors

    PubMed Central

    Popova, Maya; Perkins, Daya; Trudell, James R.; Alkana, Ronald L.; Davies, Daryl L.

    2010-01-01

    ATP-gated purinergic P2X4 receptors (P2X4Rs) are expressed in the central nervous system and are sensitive to ethanol at intoxicating concentrations. P2XRs are trimeric; each subunit consists of two transmembrane (TM) α-helical segments, a large extracellular domain, and intracellular amino and carboxyl terminals. Recent work indicates that position 336 (Met336) in the TM2 segment is critical for ethanol modulation of P2X4Rs. The anthelmintic medication ivermectin (IVM) positively modulates P2X4Rs and is believed to act in the same region as ethanol. The present study tested the hypothesis that IVM can antagonize ethanol action. We investigated IVM and ethanol effects in wild-type and mutant P2X4Rs expressed in Xenopus oocytes by using a two-electrode voltage clamp. IVM antagonized ethanol-induced inhibition of P2X4Rs in a concentration-dependent manner. The size and charge of substitutions at position 336 affected P2X4R sensitivity to both ethanol and IVM. The first molecular model of the rat P2X4R, built onto the X-ray crystal structure of zebrafish P2X4R, revealed a pocket formed by Asp331, Met336, Trp46, and Trp50 that may play a role in the actions of ethanol and IVM. These findings provide the first evidence for IVM antagonism of ethanol effects in P2X4Rs and suggest that the antagonism results from the ability of IVM to interfere with ethanol action on the putative pocket at or near position 336. Taken with the building evidence supporting a role for P2X4Rs in ethanol intake, the present findings suggest that the newly identified alcohol pocket is a potential site for development of medication for alcohol use disorders. PMID:20543096

  16. Functionalized Congeners of 1,4-Dihydropyridines as Antagonist Molecular Probes for A3 Adenosine Receptors

    PubMed Central

    Li, An-Hu; Chang, Louis; Ji, Xiao-duo; Melman, Neli; Jacobson, Kenneth A.

    2012-01-01

    4-Phenylethynyl-6-phenyl-1,4-dihydropyridine derivatives are selective antagonists at human A3 adenosine receptors, with Ki values in a radioligand binding assay vs [125I]AB-MECA [N6-(4-amino-3-iodobenzyl)-5′-N-methylcarbamoyl-adenosine] in the submicromolar range. In this study, functionalized congeners of 1,4-dihydropyridines were designed as chemically reactive adenosine A3 antagonists, for the purpose of synthesizing molecular probes for this receptor subtype. Selectivity of the new analogues for cloned human A3 adenosine receptors was determined in radioligand binding in comparison to binding at rat brain A1 and A2A receptors. Benzyl ester groups at the 3- and/or 5-positions and phenyl groups at the 2- and/or 6-positions were introduced as potential sites for chain attachment. Structure–activity analysis at A3 adenosine receptors indicated that 3,5-dibenzyl esters, but not 2,6-diphenyl groups, are tolerated in binding. Ring substitution of the 5-benzyl ester with a 4-fluorosulfonyl group provided enhanced A3 receptor affinity resulting in a Ki value of 2.42 nM; however, a long-chain derivative containing terminal amine functionalization at the 4-position of the 5-benzyl ester showed only moderate affinity. This sulfonyl fluoride derivative appeared to bind irreversibly to the human A3 receptor (1 h incubation at 100 nM resulting in the loss of 56% of the specific radioligand binding sites), while the binding of other potent dihydropyridines and other antagonists was generally reversible. At the 3-position of the dihydropyridine ring, an amine-functionalized chain attached at the 4-position of a benzyl ester provided higher A3 receptor affinity than the corresponding 5-position isomer. This amine congener was also used as an intermediate in the synthesis of a biotin conjugate, which bound to A3 receptors with a Ki value of 0.60 μM. PMID:10411465

  17. Adenosine A1 Receptor Suppresses Tonic GABAA Receptor Currents in Hippocampal Pyramidal Cells and in a Defined Subpopulation of Interneurons.

    PubMed

    Rombo, Diogo M; Dias, Raquel B; Duarte, Sofia T; Ribeiro, Joaquim A; Lamsa, Karri P; Sebastião, Ana M

    2016-03-01

    Adenosine is an endogenous neuromodulator that decreases excitability of hippocampal circuits activating membrane-bound metabotropic A1 receptor (A1R). The presynaptic inhibitory action of adenosine A1R in glutamatergic synapses is well documented, but its influence on inhibitory GABAergic transmission is poorly known. We report that GABAA receptor (GABAAR)-mediated tonic, but not phasic, transmission is suppressed by A1R in hippocampal neurons. Adenosine A1R activation strongly inhibits GABAAR agonist (muscimol)-evoked currents in Cornu Ammonis 1 (CA1) pyramidal neurons and in a specific subpopulation of interneurons expressing axonal cannabinoid receptor type 1. In addition, A1R suppresses tonic GABAAR currents measured in the presence of elevated ambient GABA as well as in naïve slices. The inhibition of GABAergic currents involves both protein kinase A (PKA) and protein kinase C (PKC) signaling pathways and decreases GABAAR δ-subunit expression. On the contrary, no A1R-mediated modulation was detected in phasic inhibitory postsynaptic currents evoked either by afferent electrical stimulation or by spontaneous quantal release. The results show that A1R modulates extrasynaptic rather than synaptic GABAAR-mediated signaling, and that this modulation selectively occurs in hippocampal pyramidal neurons and in a specific subpopulation of inhibitory interneurons. We conclude that modulation of tonic GABAAR signaling by adenosine A1R in specific neuron types may regulate neuronal gain and excitability in the hippocampus. PMID:25452570

  18. A2B adenosine receptors mediate relaxation of the pig intravesical ureter: adenosine modulation of non adrenergic non cholinergic excitatory neurotransmission

    PubMed Central

    Hernández, Medardo; Barahona, María Victoria; Bustamante, Salvador; García-Sacristán, Albino; Orensanz, Luis M

    1999-01-01

    The present study was designed to characterize the adenosine receptors involved in the relaxation of the pig intravesical ureter, and to investigate the action of adenosine on the non adrenergic non cholinergic (NANC) excitatory ureteral neurotransmission. In U46619 (10−7  M)-contracted strips treated with the adenosine uptake inhibitor, nitrobenzylthioinosine (NBTI, 10−6  M), adenosine and related analogues induced relaxations with the following potency order: 5′-N-ethylcarboxamidoadenosine (NECA)=5′-(N-cyclopropyl)-carboxamidoadenosine (CPCA)=2-chloroadenosine (2-CA)>adenosine>cyclopentyladenosine (CPA)=N6-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide (IB-MECA)=2-[p-(carboxyethyl)-phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS21680). Epithelium removal or incubation with indomethacin (3×10−6  M) and L-NG-nitroarginine (L-NOARG, 3×10−5  M), inhibitors of prostanoids and nitric oxide (NO) synthase, respectively, failed to modify the relaxations to adenosine. 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10−8 M) and 4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385, 3×10−8  M and 10−7  M), A1 and A2A receptor selective antagonists, respectively, did not modify the relaxations to adenosine or NECA. 8-phenyltheophylline (8-PT, 10−5  M) and DPCPX (10−6  M), which block A1/A2-receptors, reduced such relaxations. In strips treated with guanethidine (10−5  M), atropine (10−7  M), L-NOARG (3×10−5  M) and indomethacin (3×10−6  M), both electrical field stimulation (EFS, 5 Hz) and exogenous ATP (10−4  M) induced contractions of preparations. 8-PT (10−5  M) increased both contractions. DPCPX (10−8  M), NECA (10−4  M), CPCA, (10−4  M) and 2-CA (10−4  M) did not alter the contractions to EFS. The present results suggest that adenosine relaxes the pig intravesical ureter, independently of prostanoids

  19. Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice lacking the adenosine A1 receptor.

    PubMed

    Johansson, B; Halldner, L; Dunwiddie, T V; Masino, S A; Poelchen, W; Giménez-Llort, L; Escorihuela, R M; Fernández-Teruel, A; Wiesenfeld-Hallin, Z; Xu, X J; Hårdemark, A; Betsholtz, C; Herlenius, E; Fredholm, B B

    2001-07-31

    Caffeine is believed to act by blocking adenosine A(1) and A(2A) receptors (A(1)R, A(2A)R), indicating that some A(1) receptors are tonically activated. We generated mice with a targeted disruption of the second coding exon of the A(1)R (A(1)R(-/-)). These animals bred and gained weight normally and had a normal heart rate, blood pressure, and body temperature. In most behavioral tests they were similar to A(1)R(+/+) mice, but A(1)R(-/-) mice showed signs of increased anxiety. Electrophysiological recordings from hippocampal slices revealed that both adenosine-mediated inhibition and theophylline-mediated augmentation of excitatory glutamatergic neurotransmission were abolished in A(1)R(-/-) mice. In A(1)R(+/-) mice the potency of adenosine was halved, as was the number of A(1)R. In A(1)R(-/-) mice, the analgesic effect of intrathecal adenosine was lost, and thermal hyperalgesia was observed, but the analgesic effect of morphine was intact. The decrease in neuronal activity upon hypoxia was reduced both in hippocampal slices and in brainstem, and functional recovery after hypoxia was attenuated. Thus A(1)Rs do not play an essential role during development, and although they significantly influence synaptic activity, they play a nonessential role in normal physiology. However, under pathophysiological conditions, including noxious stimulation and oxygen deficiency, they are important. PMID:11470917

  20. Thyroid expression of an A2 adenosine receptor transgene induces thyroid hyperplasia and hyperthyroidism.

    PubMed

    Ledent, C; Dumont, J E; Vassart, G; Parmentier, M

    1992-02-01

    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. PMID:1371462

  1. The use of antagonists to characterize the receptors mediating depolarization of the rat isolated vagus nerve by alpha, beta-methylene adenosine 5'-triphosphate.

    PubMed Central

    Trezise, D. J.; Kennedy, I.; Humphrey, P. P.

    1994-01-01

    1. We have previously found that the P2x-purinoceptor agonist, alpha, beta-methylene adenosine 5'-triphosphate (alpha, beta-methylene ATP), depolarizes the rat cervical vagus nerve, measured with a 'grease-gap' extracellular recording technique. This effect was attenuated by the P2 purinoceptor antagonist, suramin. In the present study we have investigated in more detail the antagonism produced by suramin and have also investigated the actions of two other putative P2 purinoceptor antagonists, cibacron blue and pyridoxal-phosphate-6-azophenyl-2', 5'-disulphonic acid (iso-PPADS). Furthermore, we have studied the interactions between suramin and cibacron blue or iso-PPADS in an attempt to determine whether these antagonists act at a common receptor site. 2. Suramin (1 x 10(-5)-1 x 10(-4) M) produced reversible, concentration-related rightward displacements of the concentration-effect curve to alpha, beta-methylene ATP. Schild analysis of this antagonism yielded a pA2 value of 5.90 with a slope value of 0.47. 3. Cibacron blue (3 x 10(-5)-1 x 10(-4) M) also antagonized depolarizations induced by alpha, beta-methylene ATP. The antagonistic effects of cibacron blue were slow to reach equilibrium but could be readily reversed on washout. At low concentrations for antagonism, cibacron blue (1 x 10(-5) M and 3 x 10(-5) M) produced enhancement of the maximal response to alpha, beta-methylene ATP. At the highest concentration tested (1 x 10(-4) M) the concentration-effect curve to alpha, beta-methylene ATP was shifted to the right in a parallel manner, yielding a pKB estimate of 4.96.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8032652

  2. Orexin-1 receptor blockade dysregulates REM sleep in the presence of orexin-2 receptor antagonism.

    PubMed

    Dugovic, Christine; Shelton, Jonathan E; Yun, Sujin; Bonaventure, Pascal; Shireman, Brock T; Lovenberg, Timothy W

    2014-01-01

    In accordance with the prominent role of orexins in the maintenance of wakefulness via activation of orexin-1 (OX1R) and orexin-2 (OX2R) receptors, various dual OX1/2R antagonists have been shown to promote sleep in animals and humans. While selective blockade of OX2R seems to be sufficient to initiate and prolong sleep, the beneficial effect of additional inhibition of OX1R remains controversial. The relative contribution of OX1R and OX2R to the sleep effects induced by a dual OX1/2R antagonist was further investigated in the rat, and specifically on rapid eye movement (REM) sleep since a deficiency of the orexin system is associated with narcolepsy/cataplexy based on clinical and pre-clinical data. As expected, the dual OX1/2R antagonist SB-649868 was effective in promoting non-REM (NREM) and REM sleep following oral dosing (10 and 30 mg/kg) at the onset of the dark phase. However, a disruption of REM sleep was evidenced by a more pronounced reduction in the onset of REM as compared to NREM sleep, a marked enhancement of the REM/total sleep ratio, and the occurrence of a few episodes of direct wake to REM sleep transitions (REM intrusion). When administered subcutaneously, the OX2R antagonist JNJ-10397049 (10 mg/kg) increased NREM duration whereas the OX1R antagonist GSK-1059865 (10 mg/kg) did not alter sleep. REM sleep was not affected either by OX2R or OX1R blockade alone, but administration of the OX1R antagonist in combination with the OX2R antagonist induced a significant reduction in REM sleep latency and an increase in REM sleep duration at the expense of the time spent in NREM sleep. These results indicate that additional blockade of OX1R to OX2R antagonism elicits a dysregulation of REM sleep by shifting the balance in favor of REM sleep at the expense of NREM sleep that may increase the risk of adverse events. Translation of this hypothesis remains to be tested in the clinic. PMID:24592208

  3. Orexin-1 receptor blockade dysregulates REM sleep in the presence of orexin-2 receptor antagonism

    PubMed Central

    Dugovic, Christine; Shelton, Jonathan E.; Yun, Sujin; Bonaventure, Pascal; Shireman, Brock T.; Lovenberg, Timothy W.

    2014-01-01

    In accordance with the prominent role of orexins in the maintenance of wakefulness via activation of orexin-1 (OX1R) and orexin-2 (OX2R) receptors, various dual OX1/2R antagonists have been shown to promote sleep in animals and humans. While selective blockade of OX2R seems to be sufficient to initiate and prolong sleep, the beneficial effect of additional inhibition of OX1R remains controversial. The relative contribution of OX1R and OX2R to the sleep effects induced by a dual OX1/2R antagonist was further investigated in the rat, and specifically on rapid eye movement (REM) sleep since a deficiency of the orexin system is associated with narcolepsy/cataplexy based on clinical and pre-clinical data. As expected, the dual OX1/2R antagonist SB-649868 was effective in promoting non-REM (NREM) and REM sleep following oral dosing (10 and 30 mg/kg) at the onset of the dark phase. However, a disruption of REM sleep was evidenced by a more pronounced reduction in the onset of REM as compared to NREM sleep, a marked enhancement of the REM/total sleep ratio, and the occurrence of a few episodes of direct wake to REM sleep transitions (REM intrusion). When administered subcutaneously, the OX2R antagonist JNJ-10397049 (10 mg/kg) increased NREM duration whereas the OX1R antagonist GSK-1059865 (10 mg/kg) did not alter sleep. REM sleep was not affected either by OX2R or OX1R blockade alone, but administration of the OX1R antagonist in combination with the OX2R antagonist induced a significant reduction in REM sleep latency and an increase in REM sleep duration at the expense of the time spent in NREM sleep. These results indicate that additional blockade of OX1R to OX2R antagonism elicits a dysregulation of REM sleep by shifting the balance in favor of REM sleep at the expense of NREM sleep that may increase the risk of adverse events. Translation of this hypothesis remains to be tested in the clinic. PMID:24592208

  4. Rat fat-cells have three types of adenosine receptors (Ra, Ri and P). Differential effects of pertussis toxin.

    PubMed Central

    García-Sáinz, J A; Torner, M L

    1985-01-01

    Activation of rat adipocyte R1 adenosine receptors by phenylisopropyladenosine (PIA) decreased cyclic AMP and lipolysis; this effect was blocked in cells from pertussis-toxin-treated rats. In contrast, the ability of 2',5'-dideoxyadenosine to decrease cyclic AMP was not affected by pertussis-toxin treatment. Addition of adenosine deaminase to the medium in which adipocytes from control animals were incubated resulted in activation of lipolysis. Interestingly, adipocytes from toxin-treated rats (which had an already increased basal lipolysis) responded in an opposite fashion to the addition of adenosine deaminase, i.e. the enzyme decreased lipolysis, which suggested that adenosine might be increasing lipolysis in these cells. Studies with the selective agonists N-ethylcarboxamidoadenosine (NECA) and PIA indicated that adenosine increases lipolysis and cyclic AMP accumulation in these cells and that these actions are mediated through Ra adenosine receptors. Adenosine-mediated accumulation of cyclic AMP was also observed in cells preincubated with pertussis toxin (2 micrograms/ml) for 3 h. In these studies NECA was also more effective than PIA. Our results indicate that there are three types of adenosine receptors in fat-cells, whose actions are affected differently by pertussis toxin, i.e. Ri-mediated actions are abolished, Ra-mediated actions are revealed and P-mediated actions are not affected. PMID:3004405

  5. Evidence for deactivation of both ectosolic and cytosolic 5'-nucleotidase by adenosine A1 receptor activation in the rat cardiomyocytes.

    PubMed Central

    Kitakaze, M; Hori, M; Minamino, T; Takashima, S; Komamura, K; Node, K; Kurihara, T; Morioka, T; Sato, H; Inoue, M

    1994-01-01

    Adenosine, an important regulator of many cardiac functions, is produced by ectosolic and cytosolic 5'-nucleotidase. The activity of these enzymes is influenced by several ischemia-sensitive metabolic factors, e.g., ATP, ADP, H+, and inorganic phosphate. However, there is no clear evidence that adenosine itself affects 5'-nucleotidase activity. This study tested whether adenosine decreases the activity of ectosolic and cytosolic 5'-nucleotidase. Cardiomyocytes were isolated from adult male Wistar rats and suspended in the modified Hepes-Tyrode buffer solution. After stabilization, isolated cardiomyocytes were incubated with and without adenosine (10(-9) - 10(-4) M). Ectosolic and cytosolic 5'-nucleotidase activity was decreased by exogenous adenosine (ectosolic 5'-nucleotidase activity, 20.6 +/- 2.3 vs. 8.6 +/- 1.6 mumol/min per 10(6) cells [P < 0.05]; cytosolic 5'-nucleotidase activity, 2.47 +/- 0.58 vs. 1.61 +/- 0.54 mumol/min per 10(6) cells [P < 0.05] at 10(-6) M adenosine) after 30 min. The decrease in ectosolic and cytosolic 5'-nucleotidase activity was inhibited by 8-phenyltheophylline and pertussis toxin, and was mimicked by N6-cyclohexyladenosine, an adenosine A1 receptor agonist. Neither CGS21680C, and A2 receptor agonist, nor cycloheximide deactivated ectosolic and cytosolic 5'-nucleotidase. Thus, we conclude that activation of adenosine A1 receptors is coupled to Gi proteins and attenuates ectosolic and cytosolic 5'-nucleotidase activity in rat cardiomyocytes. Images PMID:7989602

  6. Histamine H4 receptor antagonism reduces hapten-induced scratching behaviour but not inflammation.

    PubMed

    Rossbach, Kristine; Wendorff, Stephanie; Sander, Kerstin; Stark, Holger; Gutzmer, Ralf; Werfel, Thomas; Kietzmann, Manfred; Bäumer, Wolfgang

    2009-01-01

    Effects of the histamine H(4) receptor antagonist JNJ 7777120 (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine) were tested in two models of allergic contact dermatitis. Dermatitis was induced by 2,4-dinitrochlorobenzene and toluene-2,4-diisocyanate, which differ in their Th1-Th2 profile in that way that 2,4-dinitrochlorobenzene is a classical contact allergen with a pronounced Th1-mediated inflammation, while the respiratory chemical allergen toluene-2,4-diisocyanate induces a Th2-dominated inflammation. JNJ 7777120 (15 mg/kg) administered 2 h and 30 min before and 1 h after challenge did not reduce the hapten-induced ear swelling determined 24 h after challenge. This was confirmed by histological evaluation of the ear skin. A repeated administration of the haptens to the rostral part of the back of sensitized animals resulted in a frequent scratching behaviour. An administration of JNJ 7777120 (15 mg/kg) 30 min before challenge reduced this hapten-induced scratching significantly. The H(1) receptor antagonist cetirizine also reduced the scratching bouts in sensitized mice. A combination of H(1) and H(4) receptor antagonists resulted in the strongest inhibition of scratching behaviour associated with allergic dermatitis. These results indicate that H(4) receptor antagonism fails to reduce the allergic inflammatory response but strongly inhibits allergen-induced itch. Thus, a combination of H(4) and H(1) receptor antagonism might be a new strategy to treat pruritus related to allergic diseases like atopic dermatitis. PMID:18647342

  7. Adenosine modulates hypoxia-induced responses in rat PC12 cells via the A2A receptor.

    PubMed

    Kobayashi, S; Conforti, L; Pun, R Y; Millhorn, D E

    1998-04-01

    1. The present study was undertaken to determine the role of adenosine in mediating the cellular responses to hypoxia in rat phaeochromocytoma (PC12) cells, an oxygen-sensitive clonal cell line. 2. Reverse transcriptase polymerase chain reaction studies revealed that PC12 cells express adenosine deaminase (the first catalysing enzyme of adenosine degradation) and the A2A and A2B adenosine receptors, but not the A1 or A3 adenosine receptors. 3. Whole-cell current- and voltage-clamp experiments showed that adenosine attenuated the hypoxia-induced membrane depolarization. The hypoxia-induced suppression of the voltage-sensitive potassium current (IK(V)) was markedly reduced by adenosine. Furthermore, extracellularly applied adenosine increased the peak amplitudes of IK(V) in a concentration-dependent manner. This increase was blocked by pretreatment not only with a non-specific adenosine receptor antagonist, 8-phenyltheophylline (8-PT), but also with a selective A2A receptor antagonist, ZM241385. 4. Ca2+ imaging studies using fura-2 acetoxymethyl ester (fura-2 AM) revealed that the increase in intracellular free Ca2+ during hypoxic exposure was attenuated significantly by adenosine. Voltage-clamp studies showed that adenosine inhibited the voltage-dependent Ca2+ currents (ICa) in a concentration-dependent fashion. This inhibition was also abolished by both 8-PT and ZM241385. 5. The modulation of both IK(V) and ICa by adenosine was prevented by intracellular application of an inhibitor of protein kinase A (PKA), PKA inhibitor fragment (6-22) amide. In addition, the effect of adenosine on either IK(V) or ICa was absent in PKA-deficient PC12 cells. 6. These results indicate that the modulatory effects of adenosine on the hypoxia-induced membrane responses of PC12 cells are likely to be mediated via activation of the A2A receptor, and that the PKA pathway is required for these modulatory actions. We propose that this modulation serves to regulate membrane excitability in

  8. Adenosine modulates hypoxia-induced responses in rat PC12 cells via the A2A receptor

    PubMed Central

    Kobayashi, Shuichi; Conforti, Laura; Pun, Raymund Y K; Millhorn, David E

    1998-01-01

    The present study was undertaken to determine the role of adenosine in mediating the cellular responses to hypoxia in rat phaeochromocytoma (PC12) cells, an oxygen-sensitive clonal cell line. Reverse transcriptase polymerase chain reaction studies revealed that PC12 cells express adenosine deaminase (the first catalysing enzyme of adenosine degradation) and the A2A and A2B adenosine receptors, but not the A1 or A3 adenosine receptors. Whole-cell current- and voltage-clamp experiments showed that adenosine attenuated the hypoxia-induced membrane depolarization. The hypoxia-induced suppression of the voltage-sensitive potassium current (IK(V)) was markedly reduced by adenosine. Furthermore, extracellularly applied adenosine increased the peak amplitudes of IK(V) in a concentration-dependent manner. This increase was blocked by pretreatment not only with a non-specific adenosine receptor antagonist, 8-phenyltheophylline (8-PT), but also with a selective A2A receptor antagonist, ZM241385. Ca2+ imaging studies using fura-2 acetoxymethyl ester (fura-2 AM) revealed that the increase in intracellular free Ca2+ during hypoxic exposure was attenuated significantly by adenosine. Voltage-clamp studies showed that adenosine inhibited the voltage-dependent Ca2+ currents (ICa) in a concentration-dependent fashion. This inhibition was also abolished by both 8-PT and ZM241385. The modulation of both IK(V) and ICa by adenosine was prevented by intracellular application of an inhibitor of protein kinase A (PKA), PKA inhibitor fragment (6–22) amide. In addition, the effect of adenosine on either IK(V) or ICa was absent in PKA-deficient PC12 cells. These results indicate that the modulatory effects of adenosine on the hypoxia-induced membrane responses of PC12 cells are likely to be mediated via activation of the A2A receptor, and that the PKA pathway is required for these modulatory actions. We propose that this modulation serves to regulate membrane excitability in PC12 cells and

  9. Design and evaluation of xanthine based adenosine receptor antagonists: Potential hypoxia targeted immunotherapies

    PubMed Central

    Thomas, Rhiannon; Lee, Joslynn; Chevalier, Vincent; Sadler, Sara; Selesniemi, Kaisa; Hatfield, Stephen; Sitkovsky, Michail; Ondrechen, Mary Jo; Jones, Graham B.

    2015-01-01

    Molecular modeling techniques were applied to the design, synthesis and optimization of a new series of xanthine based adenosine A2A receptor antagonists. The optimized lead compound was converted to a PEG derivative and a functional in vitro bioassay used to confirm efficacy. Additionally, the PEGylated version showed enhanced aqueous solubility and was inert to photoisomerization, a known limitation of existing antagonists of this class. PMID:24126093

  10. Thermostabilisation of an agonist-bound conformation of the human adenosine A(2A) receptor.

    PubMed

    Lebon, Guillaume; Bennett, Kirstie; Jazayeri, Ali; Tate, Christopher G

    2011-06-10

    The adenosine A(2A) receptor (A(2A)R) is a G-protein-coupled receptor that plays a key role in transmembrane signalling mediated by the agonist adenosine. The structure of A(2A)R was determined recently in an antagonist-bound conformation, which was facilitated by the T4 lysozyme fusion in cytoplasmic loop 3 and the considerable stabilisation conferred on the receptor by the bound inverse agonist ZM241385. Unfortunately, the natural agonist adenosine does not sufficiently stabilise the receptor for the formation of diffraction-quality crystals. As a first step towards determining the structure of A(2A)R bound to an agonist, the receptor was thermostabilised by systematic mutagenesis in the presence of the bound agonist [(3)H]5'-N-ethylcarboxamidoadenosine (NECA). Four thermostabilising mutations were identified that when combined to give mutant A(2A)R-GL26, conferred a greater than 200-fold decrease in its rate of unfolding compared to the wild-type receptor. Pharmacological analysis suggested that A(2A)R-GL26 is stabilised in an agonist-bound conformation because antagonists bind with up to 320-fold decreased affinity. None of the thermostabilising mutations are in the ZM241385 binding pocket, suggesting that the mutations affect ligand binding by altering the conformation of the receptor rather than through direct interactions with ligands. A(2A)R-GL26 shows considerable stability in short-chain detergents, which has allowed its purification and crystallisation. PMID:21501622

  11. Neuromedin B receptor antagonism inhibits migration, invasion, and epithelial-mesenchymal transition of breast cancer cells.

    PubMed

    Park, Hyun-Joo; Kim, Mi-Kyoung; Choi, Kyu-Sil; Jeong, Joo-Won; Bae, Soo-Kyung; Kim, Hyung Joon; Bae, Moon-Kyoung

    2016-09-01

    Neuromedin B (NMB) acts as an autocrine growth factor and a pro-angiogenic factor. Its receptor, NMB receptor (NMB-R), is overexpressed in solid tumors. In the present study, we showed that an NMB-R antagonist, PD168368, suppresses migration and invasion of the human breast cancer cell line MDA-MB-231. In addition, PD168368 reduced epithelial-mesenchymal transition (EMT) of breast cancer cells by E-cadherin upregulation and vimentin downregulation. Moreover, we found that PD168368 potently inhibits in vivo metastasis of breast cancer. Taken together, these findings suggest that NMB-R antagonism may be an alternative approach to prevent breast cancer metastasis, and targeting NMB-R may provide a novel therapeutic strategy for breast cancer treatment. PMID:27571778

  12. Tetrahydro-iso-alpha Acids Antagonize Estrogen Receptor Alpha Activity in MCF-7 Breast Cancer Cells

    PubMed Central

    Lempereur, Maëlle; Majewska, Claire; Brunquers, Amandine; Wongpramud, Sumalee; Valet, Bénédicte; Janssens, Philippe; Dillemans, Monique; Van Nedervelde, Laurence; Gallo, Dominique

    2016-01-01

    Tetrahydro-iso-alpha acids commonly called THIAA or Tetra are modified hop acids extracted from hop (Humulus lupulus L.) which are frequently used in brewing industry mainly in order to provide beer bitterness and foam stability. Interestingly, molecular structure of tetrahydro-iso-alpha acids is close to a new type of estrogen receptor alpha (ERα) antagonists aimed at disrupting the binding of coactivators containing an LxxLL motif (NR-box). In this work we show that THIAA decreases estradiol-stimulated proliferation of MCF-7 (ERα-positive breast cancer cells). Besides, we show that it inhibits ERα transcriptional activity. Interestingly, this extract fails to compete with estradiol for ERα binding and does not significantly impact the receptor turnover rate in MCF-7 cells, suggesting that it does not act like classical antiestrogens. Hence, we demonstrate that THIAA is able to antagonize ERα estradiol-induced recruitment of the LxxLL binding motif. PMID:27190515

  13. Tetrahydro-iso-alpha Acids Antagonize Estrogen Receptor Alpha Activity in MCF-7 Breast Cancer Cells.

    PubMed

    Lempereur, Maëlle; Majewska, Claire; Brunquers, Amandine; Wongpramud, Sumalee; Valet, Bénédicte; Janssens, Philippe; Dillemans, Monique; Van Nedervelde, Laurence; Gallo, Dominique

    2016-01-01

    Tetrahydro-iso-alpha acids commonly called THIAA or Tetra are modified hop acids extracted from hop (Humulus lupulus L.) which are frequently used in brewing industry mainly in order to provide beer bitterness and foam stability. Interestingly, molecular structure of tetrahydro-iso-alpha acids is close to a new type of estrogen receptor alpha (ERα) antagonists aimed at disrupting the binding of coactivators containing an LxxLL motif (NR-box). In this work we show that THIAA decreases estradiol-stimulated proliferation of MCF-7 (ERα-positive breast cancer cells). Besides, we show that it inhibits ERα transcriptional activity. Interestingly, this extract fails to compete with estradiol for ERα binding and does not significantly impact the receptor turnover rate in MCF-7 cells, suggesting that it does not act like classical antiestrogens. Hence, we demonstrate that THIAA is able to antagonize ERα estradiol-induced recruitment of the LxxLL binding motif. PMID:27190515

  14. Action of adenosine receptor antagonists on the cardiovascular response to defence area stimulation in the rat.

    PubMed Central

    St Lambert, J H; Dawid-Milner, M S; Silva-Carvalho, L; Spyer, K M

    1994-01-01

    1. The action of adenosine in the mediation of the cardiovascular changes associated with the defence reaction has been investigated in the rat using two A1 receptor antagonists. 2. Cumulative doses of 1,3 dipropyl-cyclopentylxanthine (DPCPX) (0.3-3 mg kg-1) and ethanol (0.03-0.25 ml) and bolus doses of DPCPX (3 mg kg-1) and 8-sulphophenyltheophylline (8-SPT) (20 mg kg-1) were given into alpha-chloralose, paralysed and artificially ventilated rats. Recordings were made of arterial blood pressure and heart rate. 3. Ethanol, the vehicle for DPCPX, failed to modify the magnitude of the defence response; however, cumulative doses of DPCPX produced a dose-dependent decrease in the HDA (hypothalamic defence area)-evoked increase in arterial blood pressure, accompanied by a similar fall in the magnitude of the evoked heart rate response. 4. The evoked rise in arterial blood pressure was reduced significantly by intravenous injection of DPCPX (3 mg kg-1) but not 8-SPT (20 mg kg-1), a purely peripherally acting adenosine antagonist. 5. These results suggest that adenosine acting at A1 receptors located in the central nervous system, is involved in the HDA-evoked pressor response. Whilst the site of action of the A1 receptors is not known, possible locations are discussed. PMID:7812606

  15. Novel Fluorescent Antagonist as a Molecular Probe in A3 Adenosine Receptor Binding Assays Using Flow Cytometry

    PubMed Central

    Kozma, Eszter; Kumar, T. Santhosh; Federico, Stephanie; Phan, Khai; Balasubramanian, Ramachandran; Gao, Zhan-Guo; Paoletta, Silvia; Moro, Stefano; Spalluto, Giampiero; Jacobson, Kenneth A.

    2012-01-01

    The physiological role of the A3 adenosine receptor (AR) was explored in cardiac ischaemia, inflammatory diseases and cancer. We report a new fluorophore-conjugated human (h) A3AR antagonist for application to cell-based assays in ligand discovery and for receptor imaging. Fluorescent pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-ylamine (pyrazolo-triazolo-pyrimidine, PTP) and triazolo[1,5-c]quinazolin-5-yl)amine (triazolo-quinazoline, TQ) AR antagonists were compared. A chain-extended and click-conjugated Alexa Fluor-488 TQ derivative (MRS5449) displayed a radioligand binding Ki value of 6.4 ± 2.5 nM in hA3AR-expressing CHO cell membranes. MRS5449 antagonized hA3AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner (KB 4.8 nM). Using flow cytometry (FCM), MRS5449 saturated hA3ARs with very high specific-to-nonspecific binding ratio with an equilibrium binding constant 5.15 nM, comparable to the Kd value of 6.65 nM calculated from kinetic experiments. Ki values of known AR antagonists in inhibition of MRS5449 binding in whole cell FCM were consistent with radioligand binding in membranes, but agonist binding was 5–20 fold weaker than obtained with agonist radioligand [125I]I-AB-MECA. Further binding analysis of MRS5549 suggested multiple agonist binding states of the A3AR. Molecular docking predicted binding modes of these fluorescent antagonists. Thus, MRS5449 is a useful tool for hA3AR characterization. PMID:22402302

  16. Endogenous adenosine A3 receptor activation selectively alleviates persistent pain states

    PubMed Central

    Little, Joshua W.; Ford, Amanda; Symons-Liguori, Ashley M.; Chen, Zhoumou; Janes, Kali; Doyle, Timothy; Xie, Jennifer; Luongo, Livio; Tosh, Dillip K.; Maione, Sabatino; Bannister, Kirsty; Dickenson, Anthony H.; Vanderah, Todd W.; Porreca, Frank; Jacobson, Kenneth A.

    2015-01-01

    Chronic pain is a global burden that promotes disability and unnecessary suffering. To date, efficacious treatment of chronic pain has not been achieved. Thus, new therapeutic targets are needed. Here, we demonstrate that increasing endogenous adenosine levels through selective adenosine kinase inhibition produces powerful analgesic effects in rodent models of experimental neuropathic pain through the A3 adenosine receptor (A3AR, now known as ADORA3) signalling pathway. Similar results were obtained by the administration of a novel and highly selective A3AR agonist. These effects were prevented by blockade of spinal and supraspinal A3AR, lost in A3AR knock-out mice, and independent of opioid and endocannabinoid mechanisms. A3AR activation also relieved non-evoked spontaneous pain behaviours without promoting analgesic tolerance or inherent reward. Further examination revealed that A3AR activation reduced spinal cord pain processing by decreasing the excitability of spinal wide dynamic range neurons and producing supraspinal inhibition of spinal nociception through activation of serotonergic and noradrenergic bulbospinal circuits. Critically, engaging the A3AR mechanism did not alter nociceptive thresholds in non-neuropathy animals and therefore produced selective alleviation of persistent neuropathic pain states. These studies reveal A3AR activation by adenosine as an endogenous anti-nociceptive pathway and support the development of A3AR agonists as novel therapeutics to treat chronic pain. PMID:25414036

  17. Pair Bond Formation is Impaired by VPAC Receptor Antagonism in the Socially Monogamous Zebra Finch

    PubMed Central

    Kingsbury, Marcy A.; Goodson, James L.

    2014-01-01

    A variety of recent data demonstrate that vasoactive intestinal polypeptide (VIP) and VPAC receptors (which bind VIP, and to a lesser extent, pituitary adenylatecyclase activating peptide) are important for numerous social behaviors in songbirds, including grouping and aggression, although VIP relates to these behaviors in a site-specific manner. In order to determine the global effects of central VPAC receptor activation on social behavior, we here infused a VPAC receptor antagonist or vehicle twice daily into the lateral ventricle of colony-housed male and female zebra finches and quantified a wide range of behaviors. Aggressive behaviors were not altered by ventricular infusions, consistent with known opposing, site-specific relationships of VIP innervation to aggression. Courtship and self-maintenance behaviors were likewise not altered. However, VPAC antagonism produced significant deficits in pair bonding. Antagonist subjects took longer to form a pair bond and were paired for significantly fewer observation sessions relative to control subjects (median 1.5 of 6 observation sessions for antagonist subjects versus 4 for control subjects). Antagonist subjects were also significantly less likely to be paired in the final observation session. Based on the known distribution of VPAC receptors in finches and other vertebrates, we propose that VPAC receptors may mediate pair bonding via a variety of brain areas that are known to be important for the establishment of partner preferences in voles, including the lateral septum, ventral tegmental area, nucleus accumbens and ventral pallidum. PMID:25014003

  18. Dopamine D2/3 receptor antagonism reduces activity-based anorexia.

    PubMed

    Klenotich, S J; Ho, E V; McMurray, M S; Server, C H; Dulawa, S C

    2015-01-01

    Anorexia nervosa (AN) is an eating disorder characterized by severe hypophagia and weight loss, and an intense fear of weight gain. Activity-based anorexia (ABA) refers to the weight loss, hypophagia and paradoxical hyperactivity that develops in rodents exposed to running wheels and restricted food access, and provides a model for aspects of AN. The atypical antipsychotic olanzapine was recently shown to reduce both AN symptoms and ABA. We examined which component of the complex pharmacological profile of olanzapine reduces ABA. Mice received 5-HT(2A/2C), 5-HT3, dopamine D1-like, D2, D3 or D2/3 antagonist treatment, and were assessed for food intake, body weight, wheel running and survival in ABA. D2/3 receptor antagonists eticlopride and amisulpride reduced weight loss and hypophagia, and increased survival during ABA. Furthermore, amisulpride produced larger reductions in weight loss and hypophagia than olanzapine. Treatment with either D3 receptor antagonist SB277011A or D2 receptor antagonist L-741,626 also increased survival. All the other treatments either had no effect or worsened ABA. Overall, selective antagonism of D2 and/or D3 receptors robustly reduces ABA. Studies investigating the mechanisms by which D2 and/or D3 receptors regulate ABA, and the efficacy for D2/3 and/or D3 antagonists to treat AN, are warranted. PMID:26241351

  19. Dopamine D2/3 receptor antagonism reduces activity-based anorexia

    PubMed Central

    Klenotich, S J; Ho, E V; McMurray, M S; Server, C H; Dulawa, S C

    2015-01-01

    Anorexia nervosa (AN) is an eating disorder characterized by severe hypophagia and weight loss, and an intense fear of weight gain. Activity-based anorexia (ABA) refers to the weight loss, hypophagia and paradoxical hyperactivity that develops in rodents exposed to running wheels and restricted food access, and provides a model for aspects of AN. The atypical antipsychotic olanzapine was recently shown to reduce both AN symptoms and ABA. We examined which component of the complex pharmacological profile of olanzapine reduces ABA. Mice received 5-HT2A/2C, 5-HT3, dopamine D1-like, D2, D3 or D2/3 antagonist treatment, and were assessed for food intake, body weight, wheel running and survival in ABA. D2/3 receptor antagonists eticlopride and amisulpride reduced weight loss and hypophagia, and increased survival during ABA. Furthermore, amisulpride produced larger reductions in weight loss and hypophagia than olanzapine. Treatment with either D3 receptor antagonist SB277011A or D2 receptor antagonist L-741,626 also increased survival. All the other treatments either had no effect or worsened ABA. Overall, selective antagonism of D2 and/or D3 receptors robustly reduces ABA. Studies investigating the mechanisms by which D2 and/or D3 receptors regulate ABA, and the efficacy for D2/3 and/or D3 antagonists to treat AN, are warranted. PMID:26241351

  20. The in vivo respiratory phenotype of the adenosine A1 receptor knockout mouse.

    PubMed

    Heitzmann, Dirk; Buehler, Philipp; Schweda, Frank; Georgieff, Michael; Warth, Richard; Thomas, Joerg

    2016-02-01

    The nucleoside adenosine has been implicated in the regulation of respiration, especially during hypoxia in the newborn. In this study the role of adenosine A1 receptors for the control of respiration was investigated in vivo. To this end, respiration of unrestrained adult and neonatal adenosine A1 receptor knockout mice (A1R(-/-)) was measured in a plethysmographic device. Under control conditions (21% O2) and mild hypoxia (12-15% O2) no difference of respiratory parameters was observed between adult wildtype (A1R(+/+)) and A1R(-/-) mice. Under more severe hypoxia (6-10% O2) A1R(+/+) mice showed, after a transient increase of respiration, a decrease of respiration frequency (fR) and tidal volume (VT) leading to a decrease of minute volume (MV). This depression of respiration during severe hypoxia was absent in A1R(-/-) mice which displayed a stimulated respiration as indicated by the enhancement of MV by some 50-60%. During hypercapnia-hyperoxia (3-10% CO2/97-90 % O2), no obvious differences in respiration of A1R(-/-) and A1R(+/+) was observed. In neonatal mice, the respiratory response to hypoxia was surprisingly similar in both genotypes. However, neonatal A1R(-/-) mice appeared to have more frequently periods of apnea during hypoxia and in the post-hypoxic control period. In conclusion, these data indicate that the adenosine A1 receptor is an important molecular component mediating hypoxic depression in adult mice and it appears to stabilize respiration of neonatal mice. PMID:26593641

  1. Adenosine A2a receptors and O2 sensing in development

    PubMed Central

    2011-01-01

    Reduced mitochondrial oxidative phosphorylation, via activation of adenylate kinase and the resulting exponential rise in the cellular AMP/ATP ratio, appears to be a critical factor underlying O2 sensing in many chemoreceptive tissues in mammals. The elevated AMP/ATP ratio, in turn, activates key enzymes that are involved in physiologic adjustments that tend to balance ATP supply and demand. An example is the conversion of AMP to adenosine via 5′-nucleotidase and the resulting activation of adenosine A2A receptors, which are involved in acute oxygen sensing by both carotid bodies and the brain. In fetal sheep, A2A receptors associated with carotid bodies trigger hypoxic cardiovascular chemoreflexes, while central A2A receptors mediate hypoxic inhibition of breathing and rapid eye movements. A2A receptors are also involved in hypoxic regulation of fetal endocrine systems, metabolism, and vascular tone. In developing lambs, A2A receptors play virtually no role in O2 sensing by the carotid bodies, but brain A2A receptors remain critically involved in the roll-off ventilatory response to hypoxia. In adult mammals, A2A receptors have been implicated in O2 sensing by carotid glomus cells, while central A2A receptors likely blunt hypoxic hyperventilation. In conclusion, A2A receptors are crucially involved in the transduction mechanisms of O2 sensing in fetal carotid bodies and brains. Postnatally, central A2A receptors remain key mediators of hypoxic respiratory depression, but they are less critical for O2 sensing in carotid chemoreceptors, particularly in developing lambs. PMID:21677265

  2. Kinin B1 receptor antagonism is equally efficient as angiotensin receptor 1 antagonism in reducing renal fibrosis in experimental obstructive nephropathy, but is not additive

    PubMed Central

    Huart, Antoine; Klein, Julie; Gonzalez, Julien; Buffin-Meyer, Bénédicte; Neau, Eric; Delage, Christine; Calise, Denis; Ribes, David; Schanstra, Joost P.; Bascands, Jean-Loup

    2015-01-01

    Background: Renal tubulointerstitial fibrosis is the pathological hallmark of chronic kidney disease (CKD). Currently, inhibitors of the renin–angiotensin system (RAS) remain the sole therapy in human displaying antifibrotic properties. Further antifibrotic molecules are needed. We have recently reported that the delayed blockade of the bradykinin B1 receptor (B1R) reduced the development of fibrosis in two animal models of renal fibrosis. The usefulness of new drugs also resides in outperforming the gold standards and eventually being additive or complementary to existing therapies. Methods: In this study we compared the efficacy of a B1R antagonist (B1Ra) with that of an angiotensin type 1 receptor antagonist (AT1a) in the unilateral ureteral obstruction (UUO) model of renal fibrosis and determined whether bi-therapy presented higher efficacy than any of the drugs alone. Results: B1R antagonism was as efficient as the gold-standard AT1a treatment. However, bitherapy did not improve the antifibrotic effects at the protein level. We sought for the reason of the absence of this additive effect by studying the expression of a panel of genes involved in the fibrotic process. Interestingly, at the molecular level the different drugs targeted different players of fibrosis that, however, in this severe model did not result in improved reduction of fibrosis at the protein level. Conclusions: As the B1R is induced specifically in the diseased organ and thus potentially displays low side effects it might be an interesting alternative in cases of poor tolerability to RAS inhibitors. PMID:25698969

  3. Presynaptic adenosine A2A receptors dampen cannabinoid CB1 receptor-mediated inhibition of corticostriatal glutamatergic transmission

    PubMed Central

    Ferreira, S G; Gonçalves, F Q; Marques, J M; Tomé, Â R; Rodrigues, R J; Nunes-Correia, I; Ledent, C; Harkany, T; Venance, L; Cunha, R A; Köfalvi, A

    2015-01-01

    Background and Purpose Both cannabinoid CB1 and adenosine A2A receptors (CB1 receptors and A2A receptors) control synaptic transmission at corticostriatal synapses, with great therapeutic importance for neurological and psychiatric disorders. A postsynaptic CB1−A2A receptor interaction has already been elucidated, but the presynaptic A2A receptor-mediated control of presynaptic neuromodulation by CB1 receptors remains to be defined. Because the corticostriatal terminals provide the major input to the basal ganglia, understanding the interactive nature of converging neuromodulation on them will provide us with novel powerful tools to understand the physiology of corticostriatal synaptic transmission and interpret changes associated with pathological conditions. Experimental Approach Pharmacological manipulation of CB1 and A2A receptors was carried out in brain nerve terminals isolated from rats and mice, using flow synaptometry, immunoprecipitation, radioligand binding, ATP and glutamate release measurement. Whole-cell patch-clamp recordings were made in horizontal corticostriatal slices. Key Results Flow synaptometry showed that A2A receptors were extensively co-localized with CB1 receptor-immunopositive corticostriatal terminals and A2A receptors co-immunoprecipitated CB1 receptors in these purified terminals. A2A receptor activation decreased CB1 receptor radioligand binding and decreased the CB1 receptor-mediated inhibition of high-K+-evoked glutamate release in corticostriatal terminals. Accordingly, A2A receptor activation prevented CB1 receptor-mediated paired-pulse facilitation and attenuated the CB1 receptor-mediated inhibition of synaptic transmission in glutamatergic synapses of corticostriatal slices. Conclusions and Implications Activation of presynaptic A2A receptors dampened CB1 receptor-mediated inhibition of corticostriatal terminals. This constitutes a thus far unrecognized mechanism to modulate the potent CB1 receptor-mediated presynaptic

  4. Excess adenosine in murine penile erectile tissues contributes to priapism via A2B adenosine receptor signaling

    PubMed Central

    Mi, Tiejuan; Abbasi, Shahrzad; Zhang, Hong; Uray, Karen; Chunn, Janci L.; Xia, Ling Wei; Molina, Jose G.; Weisbrodt, Norman W.; Kellems, Rodney E.; Blackburn, Michael R.; Xia, Yang

    2008-01-01

    Priapism, abnormally prolonged penile erection in the absence of sexual excitation, is associated with ischemia-mediated erectile tissue damage and subsequent erectile dysfunction. It is common among males with sickle cell disease (SCD), and SCD transgenic mice are an accepted model of the disorder. Current strategies to manage priapism suffer from a poor fundamental understanding of the molecular mechanisms underlying the disorder. Here we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected priapic activity. ADA enzyme therapy successfully corrected the priapic activity both in vivo and in vitro, suggesting that it was dependent on elevated adenosine levels. Further genetic and pharmacologic evidence demonstrated that A2B adenosine receptor–mediated (A2BR-mediated) cAMP and cGMP induction was required for elevated adenosine–induced prolonged penile erection. Finally, priapic activity in SCD transgenic mice was also caused by elevated adenosine levels and A2BR activation. Thus, we have shown that excessive adenosine accumulation in the penis contributes to priapism through increased A2BR signaling in both Ada–/– and SCD transgenic mice. These findings provide insight regarding the molecular basis of priapism and suggest that strategies to either reduce adenosine or block A2BR activation may prove beneficial in the treatment of this disorder. PMID:18340377

  5. Adenosine and Ischemic Preconditioning

    PubMed Central

    Liang, Bruce T.; Swierkosz, Tomasz A.; Herrmann, Howard C.; Kimmel, Stephen; Jacobson, Kenneth A.

    2012-01-01

    Adenosine is released in large amounts during myocardial ischemia and is capable of exerting potent cardioprotective effects in the heart. Although these observations on adenosine have been known for a long time, how adenosine acts to achieve its anti-ischemic effect remains incompletely understood. However, recent advances on the chemistry and pharmacology of adenosine receptor ligands have provided important and novel information on the function of adenosine receptor subtypes in the cardiovascular system. The development of model systems for the cardiac actions of adenosine has yielded important insights into its mechanism of action and have begun to elucidate the sequence of signalling events from receptor activation to the actual exertion of its cardioprotective effect. The present review will focus on the adenosine receptors that mediate the potent anti-ischemic effect of adenosine, new ligands at the receptors, potential molecular signalling mechanisms downstream of the receptor, mediators for cardioprotection, and possible clinical applications in cardiovascular disorders. PMID:10607860

  6. Frondoside A inhibits breast cancer metastasis and antagonizes prostaglandin E receptors EP4 and EP2

    PubMed Central

    Ma, Xinrong; Kundu, Namita; Collin, Peter D; Goloubeva, Olga; Fulton, Amy

    2013-01-01

    Frondoside A, derived from the sea cucumber Cucumaria frondosa has demonstrable anticancer activity in several models, however, the ability of Frondoside A to affect tumor metastasis has not been reported. Using a syngeneic murine model of metastatic breast cancer, we now show that Frondoside A has potent antimetastatic activity. Frondoside A given i.p. to mice bearing mammary gland implanted mammary tumors, inhibits spontaneous tumor metastasis to the lungs. The elevated Cyclooxygenase -2 activity in many malignancies promotes tumor growth and metastasis by producing high levels of PGE2 which acts on the prostaglandin E receptors, chiefly EP4 and EP2. We examined the ability of Frondoside A to modulate the functions of these EP receptors. We now show that Frondoside A antagonizes the prostaglandin E receptors EP2 and EP4. 3H-PGE2 binding to recombinant EP2 or EP4-expressing cells was inhibited by Frondoside A at low μM concentrations. Likewise, EP4 or EP2-linked activation of intracellular cAMP as well as EP4-mediated ERK1/2 activation were also inhibited by Frondoside A. Consistent with the antimetastatic activity observed in vivo, migration of tumor cells in vitro in response to EP4 or EP2 agonists was also inhibited by Frondoside A. These studies identify a new function for an agent with known antitumor activity, and show that the antimetastatic activity may be due in part to a novel mechanism of action. These studies add to the growing body of evidence that Frondoside A may be a promising new agent with potential to treat cancer and may also represent a potential new modality to antagonize EP4. PMID:21761157

  7. Frondoside A inhibits breast cancer metastasis and antagonizes prostaglandin E receptors EP4 and EP2.

    PubMed

    Ma, Xinrong; Kundu, Namita; Collin, Peter D; Goloubeva, Olga; Fulton, Amy M

    2012-04-01

    Frondoside A, derived from the sea cucumber Cucumaria frondosa has demonstrable anticancer activity in several models, however, the ability of Frondoside A to affect tumor metastasis has not been reported. Using a syngeneic murine model of metastatic breast cancer, we now show that Frondoside A has potent antimetastatic activity. Frondoside A given i.p. to mice bearing mammary gland-implanted mammary tumors, inhibits spontaneous tumor metastasis to the lungs. The elevated Cyclooxygenase-2 activity in many malignancies promotes tumor growth and metastasis by producing high levels of PGE(2) which acts on the prostaglandin E receptors, chiefly EP4 and EP2. We examined the ability of Frondoside A to modulate the functions of these EP receptors. We now show that Frondoside A antagonizes the prostaglandin E receptors EP2 and EP4. (3)H-PGE(2) binding to recombinant EP2 or EP4-expressing cells was inhibited by Frondoside A at low μM concentrations. Likewise, EP4 or EP2-linked activation of intracellular cAMP as well as EP4-mediated ERK1/2 activation were also inhibited by Frondoside A. Consistent with the antimetastatic activity observed in vivo, migration of tumor cells in vitro in response to EP4 or EP2 agonists was also inhibited by Frondoside A. These studies identify a new function for an agent with known antitumor activity, and show that the antimetastatic activity may be due in part to a novel mechanism of action. These studies add to the growing body of evidence that Frondoside A may be a promising new agent with potential to treat cancer and may also represent a potential new modality to antagonize EP4. PMID:21761157

  8. Adenosine A(3) receptor agonist acts as a homeostatic regulator of bone marrow hematopoiesis.

    PubMed

    Hofer, Michal; Pospísil, Milan; Znojil, Vladimír; Holá, Jirina; Vacek, Antonín; Streitová, Denisa

    2007-07-01

    The present study was performed to define the optimum conditions of the stimulatory action of the adenosine A(3) receptor agonist, N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), on bone marrow hematopoiesis in mice. Effects of 2-day treatment with IB-MECA given at single doses of 200nmol/kg twice daily were investigated in normal mice and in mice whose femoral bone marrow cells were either depleted or regenerating after pretreatment with the cytotoxic drug 5-fluorouracil. Morphological criteria were used to determine the proliferation state of the granulocytic and erythroid cell systems. Significant negative correlation between the control proliferation state and the increase of cell proliferation after IB-MECA treatment irrespective of the cell lineage investigated was found. The results suggest the homeostatic character of the induced stimulatory effects and the need to respect the functional state of the target tissue when investigating effects of adenosine receptor agonists under in vivo conditions. PMID:17383145

  9. Structural insight into antibody-mediated antagonism of the Glucagon-like peptide-1 Receptor

    PubMed Central

    Hennen, Stephanie; Kodra, János T.; Soroka, Vladyslav; Krogh, Berit O.; Wu, Xiaoai; Kaastrup, Peter; Ørskov, Cathrine; Rønn, Sif G.; Schluckebier, Gerd; Barbateskovic, Silvia; Gandhi, Prafull S.; Reedtz-Runge, Steffen

    2016-01-01

    The Glucagon-like peptide-1 receptor (GLP-1R) is a member of the class B G protein-coupled receptor (GPCR) family and a well-established target for the treatment of type 2 diabetes. The N-terminal extracellular domain (ECD) of GLP-1R is important for GLP-1 binding and the crystal structure of the GLP-1/ECD complex was reported previously. The first structure of a class B GPCR transmembrane (TM) domain was solved recently, but the full length receptor structure is still not well understood. Here we describe the molecular details of antibody-mediated antagonism of the GLP-1R using both in vitro pharmacology and x-ray crystallography. We showed that the antibody Fab fragment (Fab 3F52) blocked the GLP-1 binding site of the ECD directly and thereby acts as a competitive antagonist of native GLP-1. Interestingly, Fab 3F52 also blocked a short peptide agonist believed to engage primarily the transmembrane and extracellular loop region of GLP-1R, whereas functionality of an allosteric small-molecule agonist was not inhibited. This study has implications for the structural understanding of the GLP-1R and related class B GPCRs, which is important for the development of new and improved therapeutics targeting these receptors. PMID:27196125

  10. Structural insight into antibody-mediated antagonism of the Glucagon-like peptide-1 Receptor.

    PubMed

    Hennen, Stephanie; Kodra, János T; Soroka, Vladyslav; Krogh, Berit O; Wu, Xiaoai; Kaastrup, Peter; Ørskov, Cathrine; Rønn, Sif G; Schluckebier, Gerd; Barbateskovic, Silvia; Gandhi, Prafull S; Reedtz-Runge, Steffen

    2016-01-01

    The Glucagon-like peptide-1 receptor (GLP-1R) is a member of the class B G protein-coupled receptor (GPCR) family and a well-established target for the treatment of type 2 diabetes. The N-terminal extracellular domain (ECD) of GLP-1R is important for GLP-1 binding and the crystal structure of the GLP-1/ECD complex was reported previously. The first structure of a class B GPCR transmembrane (TM) domain was solved recently, but the full length receptor structure is still not well understood. Here we describe the molecular details of antibody-mediated antagonism of the GLP-1R using both in vitro pharmacology and x-ray crystallography. We showed that the antibody Fab fragment (Fab 3F52) blocked the GLP-1 binding site of the ECD directly and thereby acts as a competitive antagonist of native GLP-1. Interestingly, Fab 3F52 also blocked a short peptide agonist believed to engage primarily the transmembrane and extracellular loop region of GLP-1R, whereas functionality of an allosteric small-molecule agonist was not inhibited. This study has implications for the structural understanding of the GLP-1R and related class B GPCRs, which is important for the development of new and improved therapeutics targeting these receptors. PMID:27196125

  11. (/sup 125/I)Aminobenzyladenosine, a new radioligand with improved specific binding to adenosine receptors in heart

    SciTech Connect

    Linden, J.; Patel, A.; Sadek, S.

    1985-02-01

    The density of adenosine receptors in membranes derived from rat hearts in 25 times lower than the density of receptors in rat brain membranes. Consequently, adenosine radioligands which are useful in brain such as l-(/sup 3/H)phenylisopropyladenosine, (/sup 3/H)cyclohexyladenosine, (/sup 3/H)-2-chloroadenosine and l-(/sup 125/I)hydroxyphenylisopropyladenosine are of limited usefulness in heart, due to a high ratio of nonspecific to specific binding. We have synthesized a new radioligand, (/sup 125/I)-N6-4-aminobenzyladenosine, which binds to rat heart membranes with one-sixth the nonspecific binding of the other radioligands. (/sup 125/I)-N6-4-aminobenzyladenosine bound to rat ventricle membranes with a K/sub D/ equivalent to that of l-(/sup 125/I)hydroxyphenylisopropyladenosine and a B/sub max/ of 15.2 fmol/mg protein. (/sup 125/I)-N6-4-aminobenzyladenosine bound with a higher affinity to brain (K/sub D/ . 1.93 nM) than to heart membranes (K/sub D/ . 11.6 nM). At the radioligand K/sub D/, 60% of the total (/sup 125/I)-N6-4-aminobenzyladenosine bound to heart membranes was specifically bound. Iodination of aminobenzyladenosine increased its affinity for the adenosine receptor by 22-fold, possibly due to a steric or hydrophobic effect of iodine. The new ligand was found to be a full adenosine agonist based on its ability to inhibit cyclic adenosinemonophosphate accumulation in isolated embryonic chick heart cells and rat adipocytes. (/sup 125/I)-N6-4-Aminobenzyladenosine bound to a single affinity site and was displaced from cardiac and brain adenosine receptors by other adenosine analogues with a potency order of l-phenylisopropyladenosine greater than 5'-N-ethylcarboxamide adenosine. These characteristics suggest that the radioligand binds to an Ri adenosine receptor.

  12. Caffeine-induced behavioral stimulation is dose-dependent and associated with A1 adenosine receptor occupancy.

    PubMed

    Kaplan, G B; Greenblatt, D J; Kent, M A; Cotreau, M M; Arcelin, G; Shader, R I

    1992-05-01

    Caffeine's psychomotor stimulant effects may relate to its blockade of central adenosine receptors. We examined acute caffeine effects on motor activity, adenosine receptor occupancy in vivo, and receptor affinity and density ex vivo. Acute doses of caffeine-sodium benzoate (0, 20, 40, and 60 mg/kg, intraperitoneally [0, 0.10, 0.21, 0.31 mu mol/kg]) were given to CD-1 mice and their activity was measured in an animal activity monitor over a 1-hour period. Adenosine receptor occupancy in vivo was quantified in mice 1 hour postdosage, using the high-affinity, A1 receptor selective adenosine antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine. Adenosine receptor binding affinities and densities were determined from analyses of binding studies in cortical, hippocampal, and brainstem membranes from treated mice (0 and 40 mg/kg caffeine). Caffeine doses of 20 and 40 mg/kg, corresponding to mean brain concentrations of 5 and 17 micrograms/g, increased all horizontal and vertical motor activity measures and stereotypy counts, as compared to doses of 0 and 60 mg/kg. Additionally, all acute caffeine doses significantly altered specific A1 binding in vivo (decreasing binding between 55% and 73% versus vehicle), presumably as it occupied A1 receptors. Therefore, at doses of 20 and 40 mg/kg, caffeine stimulated motor activity as it occupied A1 receptors; at a dose of 60 mg/kg (mean brain concentration of 26 micrograms/g) caffeine had no stimulant effect even though it appeared to occupy A1 receptors. Acute caffeine dosage did not alter ex vivo adenosine receptor binding affinity or density in any brain regions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1599605

  13. Molecular Basis of Ligand Dissociation from the Adenosine A2A Receptor.

    PubMed

    Guo, Dong; Pan, Albert C; Dror, Ron O; Mocking, Tamara; Liu, Rongfang; Heitman, Laura H; Shaw, David E; IJzerman, Adriaan P

    2016-05-01

    How drugs dissociate from their targets is largely unknown. We investigated the molecular basis of this process in the adenosine A2Areceptor (A2AR), a prototypical G protein-coupled receptor (GPCR). Through kinetic radioligand binding experiments, we characterized mutant receptors selected based on molecular dynamic simulations of the antagonist ZM241385 dissociating from the A2AR. We discovered mutations that dramatically altered the ligand's dissociation rate despite only marginally influencing its binding affinity, demonstrating that even receptor features with little contribution to affinity may prove critical to the dissociation process. Our results also suggest that ZM241385 follows a multistep dissociation pathway, consecutively interacting with distinct receptor regions, a mechanism that may also be common to many other GPCRs. PMID:26873858

  14. Pyran Template Approach to the Design of Novel A3 Adenosine Receptor Antagonists

    PubMed Central

    Li, An-Hu; Ji, Xiao-duo; Kim, Hak Sung; Melman, Neli; Jacobson, Kenneth A.

    2016-01-01

    Strategy, Management and Health PolicyVenture Capital Enabling TechnologyPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I–III Regulatory, Quality, ManufacturingPostmarketing Phase IV A3 adenosine receptor antagonists have potential as anti-inflammatory, anti-asthmatic, and anti-ischemic agents. We previously reported the preparation of chemical libraries of 1,4-dihydropyridine (DHP) and pyridine derivatives and identification of members having high affinity at A3 adenosine receptors. These derivatives were synthesized through standard three-component condensation/oxidation reactions, which permitted versatile ring substitution at five positions, i.e., the central ring served as a molecular scaffold for structurally diverse substituents. We extended this template approach from the DHP series to chemically stable pyran derivatives, in which the ring NH is replaced by O and which is similarly derived from a stepwise reaction of three components. Since the orientation of substituent groups may be conformationally similar to the 1,4-DHPs, a direct comparison between the structure activity relationships of key derivatives in binding to adenosine receptors was carried out. Affinity at human A3 receptors expressed in CHO cells was determined vs. binding of [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5′-N-methyl-carbamoyladenosine). There was no potency-enhancing effect, as was observed for DHPs, of 4-styryl, 4-phenylethynyl, or 6-phenyl substitutions. The most potent ligands in this group in binding to human A3 receptors were 6-methyl and 6-phenyl analogs, 3a (MRS 1704) and 4a (MRS 1705), respectively, of 3,5-diethyl 2-methyl-4-phenyl-4H-pyran-3,5-dicarboxylate, which had Ki values of 381 and 583 nM, respectively. These two derivatives were selective for human A3 receptors vs. rat brain A1 receptors by 57-fold and 24-fold, respectively. These derivatives were inactive in binding at rat brain A

  15. (/sup 3/H)-8-cyclopentyl-1,3-dipropylxanthine binding to A1 adenosine receptors of intact rat ventricular myocytes

    SciTech Connect

    Martens, D.; Lohse, M.J.; Schwabe, U.

    1988-09-01

    The purpose of the present study was the identification of A1 adenosine receptors in intact rat ventricular myocytes, which are thought to mediate the negative inotropic effects of adenosine. The adenosine receptor antagonist (/sup 3/H)-8-cyclopentyl-1,3-dipropylxanthine was used as radioligand. Binding of the radioligand to intact myocytes was rapid, reversible, and saturable with a binding capacity of 40,000 binding sites per cell. The dissociation constant of the radioligand was 0.48 nM. The adenosine receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine, xanthine amine congener, and theophylline were competitive inhibitors with affinities in agreement with results obtained for A1 receptors in other tissues. Competition experiments using the adenosine receptor agonists R-N(6)-phenylisopropyladenosine, 5'-N-ethylcarboxamidoadenosine, and S-N(6)-phenylisopropyladenosine gave monophasic displacement curves with Ki values of 50 nM, 440 nM, and 4,300 nM, which agreed well with the GTP-inducible low affinity state in cardiac membranes. The low affinity for agonists was not due to agonist-induced desensitization, and correlated well with the corresponding IC50 values for the inhibition of cyclic AMP accumulation by isoprenaline. It is suggested that only a low affinity state of A1 receptors can be detected in intact rat myocytes due to the presence of high concentrations of guanine nucleotides in intact cells.

  16. Clinical/pharmacological aspect of adenosine A2A receptor antagonist for dyskinesia.

    PubMed

    Kanda, Tomoyuki; Uchida, Shin-ichi

    2014-01-01

    Dopamine replacement therapy using the dopamine precursor, l-3,4-dihydroxyphenylalanine (l-DOPA), with a peripheral dopa decarboxylase inhibitor is the most effective treatment currently available for the symptoms of Parkinson's disease (PD). However, the long-term use of dopaminergic therapies for PD is often limited by the development of motor response complications, such as dyskinesia. Adenosine A2A receptors are a promising nondopaminergic target for the treatment of PD. The treatment of motor response complications involves combinations of regular and controlled release L-DOPA, perhaps with the addition of a COMT inhibitor or the use of a longer-acting dopamine agonist. However, when dyskinesia is already established, the increase in dopaminergic load produced by the addition of a dopamine agonist can result in an increase in the severity and duration of dyskinesia. Currently, there are no well-tolerated antidyskinesia agents available. Amantadine, which may exert its effects through the inhibition of N-methyl-D-aspartate (NMDA) receptors, shows some effects on established dyskinesia. Dyskinesia has a negative impact on the quality of life of patients, sometimes being more disabling than PD itself. Although some patients prefer experiencing dyskinesia than being in the OFF state and unable to move, alternative, more effective therapies are still required for severe disabling dyskinesia to afford patients an improved quality of life while in the ON state. The mechanisms causing and maintaining the dyskinesia have not been clarified. The application of a nondopaminergic approach to modify the basal ganglial activity would be helpful to better understand and treat dyskinesia. The use of an adenosine A2A receptor may provide one such approach. In this literature review, we will summarize the current knowledge from both clinical and nonclinical studies on the effects of adenosine A2A receptor blockade on dyskinesia. PMID:25175964

  17. [Adenosine A2A receptor as a drug target for treatment of sepsis].

    PubMed

    Sivak, K V; Vasin, A V; Egorov, V V; Tsevtkov, V B; Kuzmich, N N; Savina, V A; Kiselev, O I

    2016-01-01

    Sepsis is a generalized infection accompanied by response of the body that manifests in a clinical and laboratory syndrome, namely, in the systemic inflammatory response syndrome (SIRS) from the organism to the infection. Although sepsis is a widespread and life-threatening disease, the assortment of drugs for its treatment is mostly limited by antibiotics. Therefore, the search for new cellular targets for drug therapy of sepsis is an urgent task of modern medicine and pharmacology. One of the most promising targets is the adenosine A(2A) receptor (A(2A)AR). The activation of this receptor, which is mediated by extracellular adenosine, manifests in almost all types of immune cells (lymphocytes, monocytes, macrophages, and dendritic cells) and results in reducing the severity of inflammation and reperfusion injury in various tissues. The activation of adenosine A(2A) receptor inhibits the proliferation of T cells and production of proinflammatory cytokines, which contributes to the activation of the synthesis of anti-inflammatory cytokines, thereby suppressing the systemic response. For this reason, various selective A(2A)AR agonists and antagonists may be considered to be drug candidates for sepsis pharmacotherapy. Nevertheless, they remain only efficient ligands and objects of pre-clinical and clinical trials. This review examines the molecular mechanisms of inflammatory response in sepsis and the structure and functions of A(2A)AR and its role in the pathogenesis of sepsis, as well as examples of using agonists and antagonists of this receptor for the treatment of SIRS and sepsis. PMID:27239843

  18. Elevated Ecto-5’-nucleotidase-Mediated Increased Renal Adenosine Signaling Via A2B Adenosine Receptor Contributes to Chronic Hypertension

    PubMed Central

    Zhang, Weiru; Zhang, Yujin; Wang, Wei; Dai, Yingbo; Ning, Chen; Luo, Renna; Sun, Kaiqi; Glover, Louise; Grenz, Almut; Sun, Hong; Tao, Lijian; Zhang, Wenzheng; Colgan, Sean P.; Blackburn, Michael R.; Eltzschig, Holger K.; Kellems, Rodney E.; Xia, Yang

    2013-01-01

    Rationale Hypertension is the most prevalent life-threatening disease worldwide and is frequently associated with chronic kidney disease (CKD). However, the molecular basis underlying hypertensive CKD is not fully understood. Objective We sought to identify specific factors and signaling pathways that contribute to hypertensive CKD and thereby exacerbate disease progression. Methods and Results Using high-throughput quantitative reverse-transcription polymerase chain reaction profiling, we discovered that the expression level of 5′-ectonucleotidase (CD73), a key enzyme that produces extracellular adenosine, was significantly increased in the kidneys of angiotensin II–infused mice, an animal model of hypertensive nephropathy. Genetic and pharmacological studies in mice revealed that elevated CD73-mediated excess renal adenosine preferentially induced A2B adenosine receptor (ADORA2B) production and that enhanced kidney ADORA2B signaling contributes to angiotensin II–induced hypertension. Similarly, in humans, we found that CD73 and ADORA2B levels were significantly elevated in the kidneys of CKD patients compared with normal individuals and were further elevated in hypertensive CKD patients. These findings led us to further discover that elevated renal CD73 contributes to excess adenosine signaling via ADORA2B activation that directly stimulates endothelin-1 production in a hypoxia-inducible factor-α–dependent manner and underlies the pathogenesis of the disease. Finally, we revealed that hypoxia-inducible factor-α is an important factor responsible for angiotensin II–induced CD73 and ADORA2B expression at the transcriptional level. Conclusions Overall, our studies reveal that angiotensin II–induced renal CD73 promotes the production of renal adenosine that is a prominent driver of hypertensive CKD by enhanced ADORA2B signaling–mediated endothelin-1 induction in a hypoxia-inducible factor-α–dependent manner. The inhibition of excess adenosine

  19. Gene expression and function of adenosine A(2A) receptor in the rat carotid body.

    PubMed

    Kobayashi, S; Conforti, L; Millhorn, D E

    2000-08-01

    The present study was undertaken to determine whether rat carotid bodies express adenosine (Ado) A(2A) receptors and whether this receptor is involved in the cellular response to hypoxia. Our results demonstrate that rat carotid bodies express the A(2A) and A(2B) Ado receptor mRNAs but not the A(1) or A(3) receptor mRNAs as determined by reverse transcriptase-polymerase chain reaction. In situ hybridization confirmed the expression of the A(2A) receptor mRNA. Immunohistochemical studies further showed that the A(2A) receptor is expressed in the carotid body and that it is colocalized with tyrosine hydroxylase in type I cells. Whole cell voltage-clamp studies using isolated type I cells showed that Ado inhibited the voltage-dependent Ca(2+) currents and that this inhibition was abolished by the selective A(2A) receptor antagonist ZM-241385. Ca(2+) imaging studies using fura 2 revealed that exposure to severe hypoxia induced elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in type I cells and that extracellularly applied Ado significantly attenuated the hypoxia-induced elevation of [Ca(2+)](i). Taken together, our findings indicate that A(2A) receptors are present in type I cells and that activation of A(2A) receptors modulates Ca(2+) accumulation during hypoxia. This mechanism may play a role in regulating intracellular Ca(2+) homeostasis and cellular excitability during hypoxia. PMID:10926550

  20. Genomic agonism and phenotypic antagonism between estrogen and progesterone receptors in breast cancer.

    PubMed

    Singhal, Hari; Greene, Marianne E; Tarulli, Gerard; Zarnke, Allison L; Bourgo, Ryan J; Laine, Muriel; Chang, Ya-Fang; Ma, Shihong; Dembo, Anna G; Raj, Ganesh V; Hickey, Theresa E; Tilley, Wayne D; Greene, Geoffrey L

    2016-06-01

    The functional role of progesterone receptor (PR) and its impact on estrogen signaling in breast cancer remain controversial. In primary ER(+) (estrogen receptor-positive)/PR(+) human tumors, we report that PR reprograms estrogen signaling as a genomic agonist and a phenotypic antagonist. In isolation, estrogen and progestin act as genomic agonists by regulating the expression of common target genes in similar directions, but at different levels. Similarly, in isolation, progestin is also a weak phenotypic agonist of estrogen action. However, in the presence of both hormones, progestin behaves as a phenotypic estrogen antagonist. PR remodels nucleosomes to noncompetitively redirect ER genomic binding to distal enhancers enriched for BRCA1 binding motifs and sites that link PR and ER/PR complexes. When both hormones are present, progestin modulates estrogen action, such that responsive transcriptomes, cellular processes, and ER/PR recruitment to genomic sites correlate with those observed with PR alone, but not ER alone. Despite this overall correlation, the transcriptome patterns modulated by dual treatment are sufficiently different from individual treatments, such that antagonism of oncogenic processes is both predicted and observed. Combination therapies using the selective PR modulator/antagonist (SPRM) CDB4124 in combination with tamoxifen elicited 70% cytotoxic tumor regression of T47D tumor xenografts, whereas individual therapies inhibited tumor growth without net regression. Our findings demonstrate that PR redirects ER chromatin binding to antagonize estrogen signaling and that SPRMs can potentiate responses to antiestrogens, suggesting that cotargeting of ER and PR in ER(+)/PR(+) breast cancers should be explored. PMID:27386569

  1. Adenosine A1 Receptors in Mouse Pontine Reticular Formation Depress Breathing, Increase Anesthesia Recovery Time, and Decrease Acetylcholine Release

    PubMed Central

    Gettys, George C.; Liu, Fang; Kimlin, Ed; Baghdoyan, Helen A.; Lydic, Ralph

    2012-01-01

    Background Clinical and preclinical data demonstrate the analgesic actions of adenosine. Central administration of adenosine agonists, however, suppresses arousal and breathing by poorly understood mechanisms. This study tested the two-tailed hypothesis that adenosine A1 receptors in the pontine reticular formation (PRF) of C57BL/6J mice modulate breathing, behavioral arousal, and PRF acetylcholine release. Methods Three sets of experiments used 51 mice. First, breathing was measured by plethysmography after PRF microinjection of the adenosine A1 receptor agonist N6-sulfophenyl adenosine (SPA) or saline. Second, mice were anesthetized with isoflurane and time to recovery of righting response (RoRR) was quantified after PRF microinjection of SPA or saline. Third, acetylcholine release in the PRF was measured before and during microdialysis delivery of SPA, the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), or SPA and DPCPX. Results First, SPA significantly decreased respiratory rate (−18%), tidal volume (−12%) and minute ventilation (−16%). Second, SPA concentration accounted for 76% of the variance in RoRR. Third, SPA concentration accounted for a significant amount of the variance in acetylcholine release (52%), RoRR (98%), and breathing rate (86%). DPCPX alone caused a concentration-dependent increase in acetylcholine, decrease in RoRR, and decrease in breathing rate. Coadministration of SPA and DPCPX blocked the SPA-induced decrease in acetylcholine and increase in RoRR. Conclusions Endogenous adenosine acting at adenosine A1 receptors in the PRF modulates breathing, behavioral arousal, and acetylcholine release. The results support the interpretation that an adenosinergic-cholinergic interaction within the PRF comprises one neurochemical mechanism underlying the wakefulness stimulus for breathing. PMID:23263018

  2. Metabolic mapping of A3 adenosine receptor agonist MRS5980.

    PubMed

    Fang, Zhong-Ze; Tosh, Dilip K; Tanaka, Naoki; Wang, Haina; Krausz, Kristopher W; O'Connor, Robert; Jacobson, Kenneth A; Gonzalez, Frank J

    2015-09-15

    (1S,2R,3S,4R,5S)-4-(2-((5-Chlorothiophen-2-yl)ethynyl)-6-(methylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo[3.1.0]hexane-1-carboxamide (MRS5980) is an A3AR selective agonist containing multiple receptor affinity- and selectivity-enhancing modifications and a therapeutic candidate drug for many inflammatory diseases. Metabolism-related poor pharmacokinetic behavior and toxicities are a major reason for drug R&D failure. Metabolomics with UPLC-MS was employed to profile the metabolism of MRS5980 and MRS5980-induced disruption of endogenous compounds. Recombinant drug-metabolizing enzymes screening experiment were used to determine the enzymes involved in MRS5980 metabolism. Analysis of lipid metabolism-related genes was performed to investigate the reason for MRS5980-induced lipid metabolic disorders. Unsupervised principal components analysis separated the control and MRS5980 treatment groups in feces, urine, and liver samples, but not in bile and serum. The major ions mainly contributing to the separation of feces and urine were oxidized MRS5980, glutathione (GSH) conjugates and cysteine conjugate (degradation product of the GSH conjugates) of MRS5980. The major ions contributing to the group separation of liver samples were phosphatidylcholines. In vitro incubation experiments showed the involvement of CYP3A enzymes in the oxidative metabolism of MRS5980 and direct GSH reactivity of MRS5980. The electrophilic attack by MRS5980 is a minor pathway and did not alter GSH levels in liver or liver histology, and thus may be of minor clinical consequence. Gene expression analysis further showed decreased expression of PC biosynthetic genes choline kinase a and b, which further accelerated conversion of lysophosphatidylcholine to phosphatidylcholines through increasing the expression of lysophosphatidylcholine acyltransferase 3. These data will be useful to guide rational design of drugs targeting A3AR, considering efficacy, metabolic elimination, and

  3. Adenosine A1, but not A2, receptor blockade increases anxiety and arousal in Zebrafish.

    PubMed

    Maximino, Caio; Lima, Monica G; Olivera, Karen R M; Picanço-Diniz, Domingos L W; Herculano, Anderson M

    2011-09-01

    Adenosinergic systems have been implicated in anxiety-like states, as caffeine can induce a state of anxiety in human beings. Caffeine is an antagonist at A(1) and A(2) adenosine receptors but it remains unclear whether anxiety is mediated by one or both of these. As the adenosinergic system is rather conserved, we opted to pursue these questions using zebrafish, a widely used model organism in genetics and developmental biology. Zebrafish adenosine 1. 2A.1 and 2A.2 receptors conserve histidine residues in TM6 and TM7 that are responsible for affinity in bovine A1 receptor. We investigated the effects of caffeine, PACPX (an A(1) receptor antagonist) and 1,3-dimethyl-1-propargylxanthine (DMPX) (an A(2) receptor antagonist) on anxiety-like behaviour and locomotor activity of zebrafish in the scototaxis test as well as evaluated the effects of these drugs on pigment aggregation. Caffeine increased anxiety at the dose of 100 mg/kg, while locomotion at the dose of 10 mg/kg was increased. Both doses of 10 and 100 mg/kg induced pigment aggregation. PACPX, on the other hand, increased anxiety at a dose of 6 mg/kg and induced pigment aggregation at the doses of 0.6 and 6 mg/kg, but did not produce a locomotor effect. DMPX, in turn, increased locomotion at the dose of 6 mg/kg but did not produce any effect on pigment aggregation or anxiety-like behaviour. These results indicate that blockade of A(1)-R, but not A(2)-R, induces anxiety and autonomic arousal, while the blockade of A(2)-R induces hyperlocomotion. Thus, as in rodents, caffeine's anxiogenic and arousing effects are probably mediated by A(1) receptors in zebrafish and its locomotor activating effect is probably mediated by A(2) receptors. PMID:21496211

  4. Recruitment of a Cytoplasmic Chaperone Relay by the A2A Adenosine Receptor*

    PubMed Central

    Bergmayr, Christian; Thurner, Patrick; Keuerleber, Simon; Kudlacek, Oliver; Nanoff, Christian; Freissmuth, Michael; Gruber, Christian W.

    2013-01-01

    The adenosine A2A receptor is a prototypical rhodopsin-like G protein-coupled receptor but has several unique structural features, in particular a long C terminus (of >120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A2A receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A2A receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem affinity purification. The receptor-protein complexes were purified in quantities sufficient for analysis by mass spectrometry. We identified molecular chaperones (heat-shock proteins HSP90α and HSP70-1A) that interact with and retain partially folded A2A receptor prior to ER exit. Complex formation between the A2A receptor and HSP90α (but not HSP90β) and HSP70-1A was confirmed by co-affinity precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A2A receptor. Finally, proteins of the HSP relay machinery (e.g. HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A2A receptor. These observations are consistent with the proposed chaperone/coat protein complex II exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A2A receptor. Release of HSP90 is required prior to recruitment of coat protein complex II components. This prevents premature ER export of partially folded receptors. PMID:23965991

  5. Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory

    PubMed Central

    Orr, Anna G.; Hsiao, Edward C.; Wang, Max M.; Ho, Kaitlyn; Kim, Daniel H.; Wang, Xin; Guo, Weikun; Kang, Jing; Yu, Gui-Qiu; Adame, Anthony; Devidze, Nino; Dubal, Dena B.; Masliah, Eliezer; Conklin, Bruce R.; Mucke, Lennart

    2014-01-01

    Astrocytes express a variety of G protein-coupled receptors and might influence cognitive functions, such as learning and memory. However, the roles of astrocytic Gs-coupled receptors in cognitive function are not known. We found that humans with Alzheimer’s disease (AD) had increased levels of the Gs-coupled adenosine receptor A2A in astrocytes. Conditional genetic removal of these receptors enhanced long-term memory in young and aging mice, and increased the levels of Arc/Arg3.1, an immediate-early gene required for long-term memory. Chemogenetic activation of astrocytic Gs-coupled signaling reduced long-term memory in mice without affecting learning. Similar to humans with AD, aging mice expressing human amyloid precursor protein (hAPP) showed increased levels of astrocytic A2A receptors. Conditional genetic removal of these receptors enhanced memory in aging hAPP mice. Together, these findings establish a regulatory role for astrocytic Gs-coupled receptors in memory and suggest that AD-linked increases in astrocytic A2A receptor levels contribute to memory loss. PMID:25622143

  6. 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.

  7. Antagonism of the thromboxane-prostanoid receptor is cardioprotective against right ventricular pressure overload

    PubMed Central

    Voss, Bryan M.; Pavliv, Leo; de Caestecker, Mark; Hemnes, Anna R.; Carrier, Erica J.

    2016-01-01

    Abstract Right ventricular (RV) failure is the primary cause of death in pulmonary arterial hypertension (PAH) and is a significant cause of morbidity and mortality in other forms of pulmonary hypertension. There are no approved therapies directed at preserving RV function. F-series and E-series isoprostanes are increased in heart failure and PAH, correlate to the severity of disease, and can signal through the thromboxane-prostanoid (TP) receptor, with effects from vasoconstriction to fibrosis. The goal of these studies was to determine whether blockade of the TP receptor with the antagonist CPI211 was beneficial therapeutically in PAH-induced RV dysfunction. Mice with RV dysfunction due to pressure overload by pulmonary artery banding (PAB) were given vehicle or CPI211. Two weeks after PAB, CPI211-treated mice were protected from fibrosis with pressure overload. Gene expression arrays and immunoblotting, quantitative histology and morphometry, and flow cytometric analysis were used to determine the mechanism of CPI211 protection. TP receptor inhibition caused a near normalization of fibrotic area, prevented cellular hypertrophy while allowing increased RV mass, increased expression of antifibrotic thrombospondin-4, and blocked induction of the profibrotic transforming growth factor β (TGF-β) pathway. A thromboxane synthase inhibitor or low-dose aspirin failed to replicate these results, which suggests that a ligand other than thromboxane mediates fibrosis through the TP receptor after pressure overload. This study suggests that TP receptor antagonism may improve RV adaptation in situations of pressure overload by decreasing fibrosis and TGF-β signaling. PMID:27252848

  8. Antagonism of the thromboxane-prostanoid receptor is cardioprotective against right ventricular pressure overload.

    PubMed

    West, James D; Voss, Bryan M; Pavliv, Leo; de Caestecker, Mark; Hemnes, Anna R; Carrier, Erica J

    2016-06-01

    Right ventricular (RV) failure is the primary cause of death in pulmonary arterial hypertension (PAH) and is a significant cause of morbidity and mortality in other forms of pulmonary hypertension. There are no approved therapies directed at preserving RV function. F-series and E-series isoprostanes are increased in heart failure and PAH, correlate to the severity of disease, and can signal through the thromboxane-prostanoid (TP) receptor, with effects from vasoconstriction to fibrosis. The goal of these studies was to determine whether blockade of the TP receptor with the antagonist CPI211 was beneficial therapeutically in PAH-induced RV dysfunction. Mice with RV dysfunction due to pressure overload by pulmonary artery banding (PAB) were given vehicle or CPI211. Two weeks after PAB, CPI211-treated mice were protected from fibrosis with pressure overload. Gene expression arrays and immunoblotting, quantitative histology and morphometry, and flow cytometric analysis were used to determine the mechanism of CPI211 protection. TP receptor inhibition caused a near normalization of fibrotic area, prevented cellular hypertrophy while allowing increased RV mass, increased expression of antifibrotic thrombospondin-4, and blocked induction of the profibrotic transforming growth factor β (TGF-β) pathway. A thromboxane synthase inhibitor or low-dose aspirin failed to replicate these results, which suggests that a ligand other than thromboxane mediates fibrosis through the TP receptor after pressure overload. This study suggests that TP receptor antagonism may improve RV adaptation in situations of pressure overload by decreasing fibrosis and TGF-β signaling. PMID:27252848

  9. Partial adenosine A1 receptor agonism: a potential new therapeutic strategy for heart failure.

    PubMed

    Greene, Stephen J; Sabbah, Hani N; Butler, Javed; Voors, Adriaan A; Albrecht-Küpper, Barbara E; Düngen, Hans-Dirk; Dinh, Wilfried; Gheorghiade, Mihai

    2016-01-01

    Heart failure (HF) represents a global public health and economic problem associated with unacceptable rates of death, hospitalization, and healthcare expenditure. Despite available therapy, HF carries a prognosis comparable to many forms of cancer with a 5-year survival rate of ~50%. The current treatment paradigm for HF with reduced ejection fraction (EF) centers on blocking maladaptive neurohormonal activation and decreasing cardiac workload with therapies that concurrently lower blood pressure and heart rate. Continued development of hemodynamically active medications for stepwise addition to existing therapies carries the risk of limited tolerability and safety. Moreover, this treatment paradigm has thus far failed for HF with preserved EF. Accordingly, development of hemodynamically neutral HF therapies targeting primary cardiac pathologies must be considered. In this context, a partial adenosine A1 receptor (A1R) agonist holds promise as a potentially hemodynamically neutral therapy for HF that could simultaneous improve cardiomyocyte energetics, calcium homeostasis, cardiac structure and function, and long-term clinical outcomes when added to background therapies. In this review, we describe the physiology and pathophysiology of HF as it relates to adenosine agonism, examine the existing body of evidence and biologic rationale for modulation of adenosine A1R activity, and review the current state of drug development of a partial A1R agonist for the treatment of HF. PMID:26701329

  10. History and perspectives of A2A adenosine receptor antagonists as potential therapeutic agents.

    PubMed

    Preti, Delia; Baraldi, Pier Giovanni; Moorman, Allan R; Borea, Pier Andrea; Varani, Katia

    2015-07-01

    Growing evidence emphasizes that the purine nucleoside adenosine plays an active role as a local regulator in different pathologies. Adenosine is a ubiquitous nucleoside involved in various physiological and pathological functions by stimulating A1 , A2A , A2B , and A3 adenosine receptors (ARs). At the present time, the role of A2A ARs is well known in physiological conditions and in a variety of pathologies, including inflammatory tissue damage and neurodegenerative disorders. In particular, the use of selective A2A antagonists has been reported to be potentially useful in the treatment of Parkinson's disease (PD). In this review, A2A AR signal transduction pathways, together with an analysis of the structure-activity relationships of A2A antagonists, and their corresponding pharmacological roles and therapeutic potential have been presented. The initial results from an emerging polypharmacological approach are also analyzed. This approach is based on the optimization of the affinity and/or functional activity of the examined compounds toward multiple targets, such as A1 /A2A ARs and monoamine oxidase-B (MAO-B), both closely implicated in the pathogenesis of PD. PMID:25821194

  11. No effect of nutritional adenosine receptor antagonists on exercise performance in the heat.

    PubMed

    Cheuvront, Samuel N; Ely, Brett R; Kenefick, Robert W; Michniak-Kohn, Bozena B; Rood, Jennifer C; Sawka, Michael N

    2009-02-01

    Nutritional adenosine receptor antagonists can enhance endurance exercise performance in temperate environments, but their efficacy during heat stress is not well understood. This double-blinded, placebo-controlled study compared the effects of an acute dose of caffeine or quercetin on endurance exercise performance during compensable heat stress (40 degrees C, 20-30% rh). On each of three occasions, 10 healthy men each performed 30-min of cycle ergometry at 50% Vo2peak followed by a 15-min performance time trial after receiving either placebo (Group P), caffeine (Group C; 9 mg/kg), or quercetin (Group Q; 2,000 mg). Serial blood samples, physiological (heart rate, rectal, and mean skin body temperatures), perceptual (ratings of perceived exertion, pain, thermal comfort, motivation), and exercise performance measures (total work and pacing strategy) were made. Supplementation with caffeine and quercetin increased preexercise blood concentrations of caffeine (55.62 +/- 4.77 microM) and quercetin (4.76 +/- 2.56 microM) above their in vitro inhibition constants for adenosine receptors. No treatment effects were observed for any physiological or perceptual measures, with the exception of elevated rectal body temperatures (0.20-0.30 degrees C; P < 0.05) for Group C vs. Groups Q and P. Supplementation did not affect total work performed (Groups P: 153.5 +/- 28.3, C: 157.3 +/- 28.9, and Q: 151.1 +/- 31.6 kJ; P > 0.05) or the self-selected pacing strategy employed. These findings indicate that the nutritional adenosine receptor antagonists caffeine and quercetin do not enhance endurance exercise performance during compensable heat stress. PMID:19020291

  12. Adenosine A1 Receptor Antagonist Versus Montelukast on Airway Reactivity and Inflammation

    PubMed Central

    Nadeem, Ahmed; Obiefuna, Peter C.M.; Wilson, Constance N.; Mustafa, S. Jamal

    2006-01-01

    Adenosine produces bronchoconstriction in allergic rabbits, primates, and humans by activating adenosine A1 receptors. Previously, it is reported that a high dose of L-97-1, a water-soluble, small molecule adenosine A1 receptor antagonist, blocks early and late allergic responses, and bronchial hyper-responsiveness to histamine in a hyper-responsive rabbit model of allergic asthma. Effects of a lower dose of L-97-1 are compared to montelukast, a cysteinyl leukotriene-1 receptor antagonist on early allergic response, late allergic response, bronchial hyper-responsiveness, and inflammatory cells in bronchoalveolar lavage (BAL) fluid following house dust mite administration. Rabbits received intraperitoneal injections of house dust mite extract within 24 h of birth followed by booster house dust mite injections. Hyper-responsive rabbits received aerosolized house dust mite (2500 allergen units), 1 h after intragastric administration of L-97-1 (1 mg/kg) or montelukast (0.15 mg/kg) and lung dynamic compliance was measured for 6 h. Lung dynamic compliance was significantly higher following L-97-1 at all time points and with montelukast at 60-300 min following house dust mite (P < 0.05). L-97-1 blocks both early and late allergic responses. Montelukast blocks only the late allergic response. Both L-97-1 and montelukast significantly blocked bronchial hyper-responsiveness at 24 h (P < 0.05). Both L-97-1 and montelukast significantly reduced BAL eosinophils at 6 h and neutrophils at 6 and 24 h (P < 0.05). L-97-1 significantly reduced BAL lymphocytes at 6 and 24 h (P < 0.05). Montelukast significantly reduced BAL macrophages at 6 and 24 h (P < 0.05). By blocking both bronchoconstriction and airway inflammation, L-97-1 may be an effective oral anti-asthma treatment. PMID:17027749

  13. The A2B adenosine receptor protects against inflammation and excessive vascular adhesion

    PubMed Central

    Yang, Dan; Zhang, Ying; Nguyen, Hao G.; Koupenova, Milka; Chauhan, Anil K.; Makitalo, Maria; Jones, Matthew R.; Hilaire, Cynthia St.; Seldin, David C.; Toselli, Paul; Lamperti, Edward; Schreiber, Barbara M.; Gavras, Haralambos; Wagner, Denisa D.; Ravid, Katya

    2006-01-01

    Adenosine has been described as playing a role in the control of inflammation, but it has not been certain which of its receptors mediate this effect. Here, we generated an A2B adenosine receptor–knockout/reporter gene–knock-in (A2BAR-knockout/reporter gene–knock-in) mouse model and showed receptor gene expression in the vasculature and macrophages, the ablation of which causes low-grade inflammation compared with age-, sex-, and strain-matched control mice. Augmentation of proinflammatory cytokines, such as TNF-α, and a consequent downregulation of IκB-α are the underlying mechanisms for an observed upregulation of adhesion molecules in the vasculature of these A2BAR-null mice. Intriguingly, leukocyte adhesion to the vasculature is significantly increased in the A2BAR-knockout mice. Exposure to an endotoxin results in augmented proinflammatory cytokine levels in A2BAR-null mice compared with control mice. Bone marrow transplantations indicated that bone marrow (and to a lesser extent vascular) A2BARs regulate these processes. Hence, we identify the A2BAR as a new critical regulator of inflammation and vascular adhesion primarily via signals from hematopoietic cells to the vasculature, focusing attention on the receptor as a therapeutic target. PMID:16823489

  14. Paeoniflorin Promotes Non-rapid Eye Movement Sleep via Adenosine A1 Receptors.

    PubMed

    Chen, Chang-Rui; Sun, Yu; Luo, Yan-Jia; Zhao, Xin; Chen, Jiang-Fan; Yanagawa, Yuchio; Qu, Wei-Min; Huang, Zhi-Li

    2016-01-01

    Paeoniflorin (PF, C23H28O11), one of the principal active ingredients of Paeonia Radix, exerts depressant effects on the central nervous system. We determined whether PF could modulate sleep behaviors and the mechanisms involved. Electroencephalogram and electromyogram recordings in mice showed that intraperitoneal PF administered at a dose of 25 or 50 mg/kg significantly shortened the sleep latency and increased the amount of non-rapid eye movement (NREM). Immunohistochemical study revealed that PF decreased c-fos expression in the histaminergic tuberomammillary nucleus (TMN). The sleep-promoting effects and changes in c-fos induced by PF were reversed by 8-cyclopentyl-1,3-dimethylxanthine (CPT), an adenosine A1 receptor antagonist, and PF-induced sleep was not observed in adenosine A1 receptor knockout mice. Whole-cell patch clamping in mouse brain slices showed that PF significantly decreased the firing frequency of histaminergic neurons in TMN, which could be completely blocked by CPT. These results indicate that PF increased NREM sleep by inhibiting the histaminergic system via A1 receptors. PMID:26491061

  15. The Second Extracellular Loop of the Adenosine A1 Receptor Mediates Activity of Allosteric Enhancers

    PubMed Central

    Kennedy, Dylan P.; McRobb, Fiona M.; Leonhardt, Susan A.; Purdy, Michael; Figler, Heidi; Marshall, Melissa A.; Chordia, Mahendra; Figler, Robert; Linden, Joel

    2014-01-01

    Allosteric enhancers of the adenosine A1 receptor amplify signaling by orthosteric agonists. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied by an agonist, thereby conferring specificity to stressed or injured tissues that produce adenosine. To explore the mechanism of allosteric enhancer activity, we examined their action on several A1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants, and (4) site-specific mutants. These findings were combined with homology modeling of the A1 receptor and in silico screening of an allosteric enhancer library. The binding modes of known docked allosteric enhancers correlated with the known structure-activity relationship, suggesting that these allosteric enhancers bind to a pocket formed by the second extracellular loop, flanked by residues S150 and M162. We propose a model in which this vestibule controls the entry and efflux of agonists from the orthosteric site and agonist binding elicits a conformational change that enables allosteric enhancer binding. This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists. PMID:24217444

  16. Staurosporine-induced apoptosis in astrocytes is prevented by A1 adenosine receptor activation.

    PubMed

    D'Alimonte, Iolanda; Ballerini, Patrizia; Nargi, Eleonora; Buccella, Silvana; Giuliani, Patricia; Di Iorio, Patrizia; Caciagli, Francesco; Ciccarelli, Renata

    2007-05-11

    Astrocyte apoptosis occurs in acute and chronic pathological processes at the central nervous system and the prevention of astrocyte death may represent an efficacious intervention in protecting neurons against degeneration. Our research shows that rat astrocyte exposure to 100 nM staurosporine for 3h caused apoptotic death accompanied by caspase-3, p38 mitogen-ed protein kinase (MAPK) and glycogen synthase kinase-3beta (GSK3beta) activation. N(6)-chlorocyclopentyladenosine (CCPA, 2.5-75 nM), a selective agonist of A(1) adenosine receptors, added to the cultures 1h prior to staurosporine, induced a dose-dependent anti-apoptotic effect, which was inhibited by the A(1) receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. CCPA also caused a dose- and time-dependent phosphorylation/activation of Akt, a downstream effector of cell survival promoting phosphatidylinositol 3-kinase (PI3K) pathway, which in turn led to inhibition of staurosporine-induced GSK3beta and p38 MAPK activity. Accordingly, the anti-apoptotic effect of CCPA was abolished by culture pre-treatment with LY294002, a selective PI3K inhibitor, pointing out the prevailing role played by PI3K pathway in the protective effect exerted by A(1) receptor activation. Since an abnormal p38 and GSK3beta activity is implicated in acute (stroke) and chronic (Alzheimer's disease) neurodegenerative diseases, the results of the present study provide a hint to better understand adenosine relevance in these disorders. PMID:17400382

  17. Physical origins of remarkable thermostabilization by an octuple mutation for the adenosine A2a receptor

    NASA Astrophysics Data System (ADS)

    Kajiwara, Yuta; Ogino, Takahiro; Yasuda, Satoshi; Takamuku, Yuuki; Murata, Takeshi; Kinoshita, Masahiro

    2016-07-01

    It was experimentally showed that the thermal stability of a membrane protein, the adenosine A2a receptor, was remarkably enhanced by an octuple mutation. Here we theoretically prove that the energy decrease arising from the formation of protein intramolecular hydrogen bonds and the solvent-entropy gain upon protein folding are made substantially larger by the mutation, leading to the remarkable enhancement. The solvent is formed by hydrocarbon groups constituting nonpolar chains of the lipid bilayer within a membrane. The mutation modifies geometric characteristics of the structure so that the solvent crowding can be reduced to a larger extent when the protein folds.

  18. Reengineering the Collision Coupling and Diffusion Mode of the A2A-adenosine Receptor

    PubMed Central

    Keuerleber, Simon; Thurner, Patrick; Gruber, Christian W.; Zezula, Jürgen; Freissmuth, Michael

    2012-01-01

    The A2A-adenosine receptor undergoes restricted collision coupling with its cognate G protein Gs and lacks a palmitoylation site at the end of helix 8 in its intracellular C terminus. We explored the hypothesis that there was a causal link between the absence of a palmitoyl moiety and restricted collision coupling by introducing a palmitoylation site. The resulting mutant A2A-R309C receptor underwent palmitoylation as verified by both mass spectrometry and metabolic labeling. In contrast to the wild type A2A receptor, the concentration-response curve for agonist-induced cAMP accumulation was shifted to the left with increasing expression levels of A2A-R309C receptor, an observation consistent with collision coupling. Single particle tracking of quantum dot-labeled receptors confirmed that wild type and mutant A2A receptor differed in diffusivity and diffusion mode; agonist activation resulted in a decline in mean square displacement of both receptors, but the drop was substantially more pronounced for the wild type receptor. In addition, in the agonist-bound state, the wild type receptor was frequently subject to confinement events (estimated radius 110 nm). These were rarely seen with the palmitoylated A2A-R309C receptor, the preferred diffusion mode of which was a random walk in both the basal and the agonist-activated state. Taken together, the observations link restricted collision coupling to diffusion limits imposed by the absence of a palmitoyl moiety in the C terminus of the A2A receptor. The experiments allowed for visualizing local confinement of an agonist-activated G protein-coupled receptor in an area consistent with the dimensions of a lipid raft. PMID:23071116

  19. Recent improvements in the development of A2B adenosine receptor agonists

    PubMed Central

    Tabrizi, Mojgan Aghazadeh; Fruttarolo, Francesca; Romagnoli, Romeo; Preti, Delia

    2009-01-01

    Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A1, A2A, A2B and A3 (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N6-, C2-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA1Ki = 1050 nM, hA2AKi = 1550 nM, hA2B EC50 = 82 nM, hA3Ki > 5 μM) and its 2-chloro analogue 23 (hA1Ki = 3500 nM, hA2AKi = 4950 nM, hA2B EC50 = 210 nM, hA3Ki > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA1, hA2A, hA3 EC50 > 10 μM; hA2B EC50 = 3 nM) is currently under preclinical-phase investigation for treating coronary

  20. Recent improvements in the development of A2B adenosine receptor agonists

    PubMed Central

    Tabrizi, Mojgan Aghazadeh; Fruttarolo, Francesca; Romagnoli, Romeo; Preti, Delia

    2008-01-01

    Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A1, A2A, A2B and A3 (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N6-, C2-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA1Ki = 1050 nM, hA2AKi = 1550 nM, hA2B EC50 = 82 nM, hA3Ki > 5 μM) and its 2-chloro analogue 23 (hA1Ki = 3500 nM, hA2AKi = 4950 nM, hA2B EC50 = 210 nM, hA3Ki > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA1, hA2A, hA3 EC50 > 10 μM; hA2B EC50 = 3 nM) is currently under preclinical-phase investigation for treating coronary

  1. Past, present and future of A2A adenosine receptor antagonists in the therapy of Parkinson’s disease

    PubMed Central

    Armentero, Marie Therese; Pinna, Annalisa; Ferré, Sergi; Lanciego, José Luis; Müller, Christa E.; Franco, Rafael

    2011-01-01

    Several selective antagonists for adenosine A2A receptors (A2AR) are currently under evaluation in clinical trials (phases I to III) to treat Parkinson’s disease, and they will probably soon reach the market. The usefulness of these antagonists has been deduced from studies demonstrating functional interactions between dopamine D2 and adenosine A2A receptors in the basal ganglia. At present it is believed that A2AR antagonists can be used in combination with the dopamine precursor L-DOPA to minimize the motor symptoms of Parkinson’s patients. However, a considerable body of data indicates that in addition to ameliorating motor symptoms, adenosine A2AR antagonists may also prevent neurodegeneration. Despite these promising indications, one further issue must be considered in order to develop fully optimized anti-parkinsonian drug therapy, namely the existence of receptor (hetero)dimers/oligomers of G protein-coupled receptors, a topic currently the focus of intense debate within the scientific community. Dopamine D2 receptors (D2Rs) expressed in the striatum are known to form heteromers with A2A adenosine receptors. Thus, the development of heteromer-specific A2A receptor antagonists represents a promising strategy for the identification of more selective and safer drugs. PMID:21810444

  2. Neurokinin-1 receptor antagonism attenuates neuronal activity triggered by stress-induced reinstatement of alcohol seeking.

    PubMed

    Schank, J R; Nelson, B S; Damadzic, R; Tapocik, J D; Yao, M; King, C E; Rowe, K E; Cheng, K; Rice, K C; Heilig, M

    2015-12-01

    Substance P (SP) and its cognate neurokinin-1 receptor (NK1R) are involved in alcohol-related behaviors. We have previously reported that NK1R antagonism attenuates stress-induced reinstatement of alcohol seeking and suppresses escalated alcohol self-administration, but does not affect primary reinforcement or cue-induced reinstatement. Here, we administered an NK1R antagonist or vehicle prior to footshock-induced reinstatement of alcohol seeking, and mapped the resulting neuronal activation using Fos immunohistochemistry. As expected, vehicle treated animals exposed to footshock showed induction of Fos immunoreactivity in several regions of the brain stress circuitry, including the amygdala (AMG), nucleus accumbens (NAC), dorsal raphe nucleus (DR), prefrontal cortex (PFC), and bed nucleus of the stria terminalis (BNST). NK1R antagonism selectively suppressed the stress-induced increase in Fos in the DR and NAC shell. In the DR, Fos-induction by stress largely overlapped with tryptophan hydroxylase (TrpH), indicating activation of serotonergic neurons. Of NAC shell neurons activated during stress-induced reinstatement of alcohol seeking, about 30% co-expressed dynorphin (DYN), while 70% co-expressed enkephalin (ENK). Few (<1%) activated NAC shell neurons coexpressed choline acetyltransferase (ChAT), which labels the cholinergic interneurons of this region. Infusion of the NK1R antagonist L822429 into the NAC shell blocked stress-induced reinstatement of alcohol seeking. In contrast, L822429 infusion into the DR had no effect, suggesting that the influence of NK1R signaling on neuronal activity in the DR is indirect. Taken together, our results outline a potential pathway through which endogenous NK1R activation mediates stress-induced alcohol seeking. PMID:26188146

  3. Genomic agonism and phenotypic antagonism between estrogen and progesterone receptors in breast cancer

    PubMed Central

    Singhal, Hari; Greene, Marianne E.; Tarulli, Gerard; Zarnke, Allison L.; Bourgo, Ryan J.; Laine, Muriel; Chang, Ya-Fang; Ma, Shihong; Dembo, Anna G.; Raj, Ganesh V.; Hickey, Theresa E.; Tilley, Wayne D.; Greene, Geoffrey L.

    2016-01-01

    The functional role of progesterone receptor (PR) and its impact on estrogen signaling in breast cancer remain controversial. In primary ER+ (estrogen receptor–positive)/PR+ human tumors, we report that PR reprograms estrogen signaling as a genomic agonist and a phenotypic antagonist. In isolation, estrogen and progestin act as genomic agonists by regulating the expression of common target genes in similar directions, but at different levels. Similarly, in isolation, progestin is also a weak phenotypic agonist of estrogen action. However, in the presence of both hormones, progestin behaves as a phenotypic estrogen antagonist. PR remodels nucleosomes to noncompetitively redirect ER genomic binding to distal enhancers enriched for BRCA1 binding motifs and sites that link PR and ER/PR complexes. When both hormones are present, progestin modulates estrogen action, such that responsive transcriptomes, cellular processes, and ER/PR recruitment to genomic sites correlate with those observed with PR alone, but not ER alone. Despite this overall correlation, the transcriptome patterns modulated by dual treatment are sufficiently different from individual treatments, such that antagonism of oncogenic processes is both predicted and observed. Combination therapies using the selective PR modulator/antagonist (SPRM) CDB4124 in combination with tamoxifen elicited 70% cytotoxic tumor regression of T47D tumor xenografts, whereas individual therapies inhibited tumor growth without net regression. Our findings demonstrate that PR redirects ER chromatin binding to antagonize estrogen signaling and that SPRMs can potentiate responses to antiestrogens, suggesting that cotargeting of ER and PR in ER+/PR+ breast cancers should be explored. PMID:27386569

  4. Kidney-specific reconstitution of the A1 adenosine receptor in A1 adenosine receptor knockout mice reduces renal ischemia–reperfusion injury

    PubMed Central

    Kim, Minjae; Chen, Sean W.C.; Park, Sang Won; Kim, Mihwa; D’Agati, Vivette D.; Yang, Jay; Lee, H. Thomas

    2009-01-01

    Genetic deletion of the adenosine A1 receptor (A1AR) increased renal injury following ischemia-reperfusion injury suggesting that receptor activation is protective in vivo. Here we tested this hypothesis by expressing the human-A1AR in A1AR knockout mice. Renal ischemia-reperfusion was induced in knockout mice 2 days after intrarenal injection of saline or a lentivirus encoding enhanced green fluorescent protein (EGFP) or EGFP-human-A1AR. We found that the latter procedure induced a robust expression of the reporter protein in the kidneys of knockout mice. Mice with kidney-specific human-A1AR reconstitution had significantly lower plasma creatinine, tubular necrosis, apoptosis, and tubular inflammation as evidenced by decreased leukocyte infiltration, pro-inflammatory cytokine, and intercellular adhesion molecule-1 expression in the kidney following injury compared to mice injected with saline or the control lentivirus. Additionally, there were marked disruptions of the proximal tubule epithelial filamentous (F)-actin cytoskeleton in both sets of control mice upon renal injury, whereas the reconstituted mice had better preservation of the renal tubule actin cytoskeleton, which co-localized with the human-A1ARs. Consistent with reduced renal injury, there was a significant increase in heat shock protein-27 expression, also co-localizing with the preserved F-actin cytoskeleton. Our findings suggest that selective expression of cytoprotective A1ARs in the kidney can attenuate renal injury. PMID:19190680

  5. Antagonism of sphingosine-1-phosphate receptors by FTY720 inhibits angiogenesis and tumor vascularization.

    PubMed

    LaMontagne, Kenneth; Littlewood-Evans, Amanda; Schnell, Christian; O'Reilly, Terence; Wyder, Lorenza; Sanchez, Teresa; Probst, Beatrice; Butler, Jeannene; Wood, Alexander; Liau, Gene; Billy, Eric; Theuer, Andreas; Hla, Timothy; Wood, Jeanette

    2006-01-01

    FTY720, a potent immunomodulator, becomes phosphorylated in vivo (FTY-P) and interacts with sphingosine-1-phosphate (S1P) receptors. Recent studies showed that FTY-P affects vascular endothelial growth factor (VEGF)-induced vascular permeability, an important aspect of angiogenesis. We show here that FTY720 has antiangiogenic activity, potently abrogating VEGF- and S1P-induced angiogenesis in vivo in growth factor implant and corneal models. FTY720 administration tended to inhibit primary and significantly inhibited metastatic tumor growth in a mouse model of melanoma growth. In combination with a VEGFR tyrosine kinase inhibitor PTK787/ZK222584, FTY720 showed some additional benefit. FTY720 markedly inhibited tumor-associated angiogenesis, and this was accompanied by decreased tumor cell proliferation and increased apoptosis. In transfected HEK293 cells, FTY-P internalized S1P1 receptors, inhibited their recycling to the cell surface, and desensitized S1P receptor function. Both FTY720 and FTY-P apparently failed to impede VEGF-produced increases in mitogen-activated protein kinase activity in human umbilical vascular endothelial cells (HUVEC), and unlike its activity in causing S1PR internalization, FTY-P did not result in a decrease of surface VEGFR2 levels in HUVEC cells. Pretreatment with FTY720 or FTY-P prevented S1P-induced Ca2+ mobilization and migration in vascular endothelial cells. These data show that functional antagonism of vascular S1P receptors by FTY720 potently inhibits angiogenesis; therefore, this may provide a novel therapeutic approach for pathologic conditions with dysregulated angiogenesis. PMID:16397235

  6. Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats.

    PubMed

    Gowen, M; Stroup, G B; Dodds, R A; James, I E; Votta, B J; Smith, B R; Bhatnagar, P K; Lago, A M; Callahan, J F; DelMar, E G; Miller, M A; Nemeth, E F; Fox, J

    2000-06-01

    Parathyroid hormone (PTH) is an effective bone anabolic agent, but it must be administered parenterally. An orally active anabolic agent would provide a valuable alternative for treating osteoporosis. NPS 2143 is a novel, selective antagonist (a "calcilytic") of the parathyroid cell Ca(2+) receptor. Daily oral administration of NPS 2143 to osteopenic ovariectomized (OVX) rats caused a sustained increase in plasma PTH levels, provoking a dramatic increase in bone turnover but no net change in bone mineral density. Concurrent oral administration of NPS 2143 and subcutaneous infusion of 17beta-estradiol also resulted in increased bone turnover. However, the antiresorptive action of estrogen decreased the extent of bone resorption stimulated by the elevated PTH levels, leading to an increase in bone mass compared with OVX controls or to either treatment alone. Despite the sustained stimulation to the parathyroid gland, parathyroid cells did not undergo hyperplasia. These data demonstrate that an increase in endogenous PTH secretion, induced by antagonism of the parathyroid cell Ca(2+) receptor with a small molecule, leads to a dramatic increase in bone turnover, and they suggest a novel approach to the treatment of osteoporosis. PMID:10841518

  7. Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats

    PubMed Central

    Gowen, Maxine; Stroup, George B.; Dodds, Robert A.; James, Ian E.; Votta, Bart J.; Smith, Brian R.; Bhatnagar, Pradip K.; Lago, Amparo M.; Callahan, James F.; DelMar, Eric G.; Miller, Michael A.; Nemeth, Edward F.; Fox, John

    2000-01-01

    Parathyroid hormone (PTH) is an effective bone anabolic agent, but it must be administered parenterally. An orally active anabolic agent would provide a valuable alternative for treating osteoporosis. NPS 2143 is a novel, selective antagonist (a “calcilytic”) of the parathyroid cell Ca2+ receptor. Daily oral administration of NPS 2143 to osteopenic ovariectomized (OVX) rats caused a sustained increase in plasma PTH levels, provoking a dramatic increase in bone turnover but no net change in bone mineral density. Concurrent oral administration of NPS 2143 and subcutaneous infusion of 17β-estradiol also resulted in increased bone turnover. However, the antiresorptive action of estrogen decreased the extent of bone resorption stimulated by the elevated PTH levels, leading to an increase in bone mass compared with OVX controls or to either treatment alone. Despite the sustained stimulation to the parathyroid gland, parathyroid cells did not undergo hyperplasia. These data demonstrate that an increase in endogenous PTH secretion, induced by antagonism of the parathyroid cell Ca2+ receptor with a small molecule, leads to a dramatic increase in bone turnover, and they suggest a novel approach to the treatment of osteoporosis. PMID:10841518

  8. Hypocretin Receptor 2 Antagonism Dose-Dependently Reduces Escalated Heroin Self-Administration in Rats

    PubMed Central

    Schmeichel, Brooke E; Barbier, Estelle; Misra, Kaushik K; Contet, Candice; Schlosburg, Joel E; Grigoriadis, Dimitri; Williams, John P; Karlsson, Camilla; Pitcairn, Caleb; Heilig, Markus; Koob, George F; Vendruscolo, Leandro F

    2015-01-01

    The hypocretin/orexin (HCRT) system has been associated with both positive and negative drug reinforcement, implicating HCRT receptor 1 (HCRT-R1) signaling in drug-related behaviors for all major drug classes, including opioids. However, to date there are limited studies investigating the role of HCRT receptor 2 (HCRT-R2) signaling in compulsive-like drug seeking. Escalation of drug intake with extended access has been suggested to model the transition from controlled drug use to compulsive-like drug seeking/taking. The current study examined the effects of a HCRT-R2 antagonist, NBI-80713, on heroin self-administration in rats allowed short- (1 h; ShA) or long- (12 h; LgA) access to intravenous heroin self-administration. Results indicate that systemically administered NBI-80713 dose-dependently decreased heroin self-administration in LgA, but not in ShA, animals. Quantitative PCR analyses showed an increase in Hcrtr2 mRNA levels in the central amygdala, a stress-related brain region, of LgA rats. These observations suggest a functional role for HCRT-R2 signaling in compulsive-like heroin self-administration associated with extended access and indicate HCRT-R2 antagonism as a potential pharmacological target for the treatment of heroin dependence. PMID:25367502

  9. Felbamate antagonizes isoniazid- and FG 7142-induced reduction of GABAA receptor function in mouse brain.

    PubMed

    Serra, M; Ghiani, C A; Spano, S; Biggio, G

    1994-11-24

    Injection of the antiepileptic drug, felbamate (2-phenyl-1,3-propanediol dicarbamate), into mice reduced in a dose-dependent manner (150-300 mg/kg i.p.) the isoniazid (200 mg/kg s.c.)-induced increase in ex vivo binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) to cerebral cortical and hippocampal membranes. The same doses of felbamate reduced significantly the number of mice exhibiting isoniazid-induced seizures. A dose of felbamate (50 mg/kg) ineffective in isoniazid-treated mice completely antagonized the increase of [35S]TBPS binding elicited by FG 7142 (N-methyl-beta-carboline-3-carboxamide), a benzodiazepine receptor inverse agonist. The above effects of felbamate resembled those of diazepam. Accordingly, the combination of ineffective doses of felbamate (50 mg/kg) and diazepam (0.2 mg/kg) elicited a marked decrease of [35S]TBPS binding. The results indicate that facilitation of gamma-aminobutyric acid type A (GABAA) receptor function may play a role in the anticonvulsant action of felbamate. PMID:7875235

  10. Localization of the adenosine A1 receptor subtype gene (ADORA1) to chromosome 1q32.1

    SciTech Connect

    Townsend-Nicholson, A.; Schofield, P.R.; Baker, E.

    1995-03-20

    Adenosine, acting through its receptors, exerts effects on almost all organ systems, influencing a diversity of physiological responses, including the inhibition of neurotransmitter release, the modulation of cardiac rhythmicity and contractility, and the potentiation of IgE-dependent mediator release. Adenosine receptors belong to the G protein-coupled receptor superfamily, a class of cell-surface receptors that, when activated, couple to a heterotrimeric G protein complex to effect signal transduction. Molecular cloning and subsequent pharmacological and biochemical analyses have led to the identification of four different subtypes of adenosine receptor. The A3 receptor has been localized to chromosome 3 in the mouse by interspecific backcross analysis, suggesting a human chromosomal localization of 1p13 from known mouse-human linkage homologies. We have previously mapped the A2b adenosine receptor subtype to chromosome 17p11.2-p12 using fluorescence in situ hybridization (FISH) and PCR-based screening of somatic cell hybrid DNAs. A previous report has concluded that the Al and A2a receptor subtypes are localized on chromosome 22q11.2-q13.1 and 11q11-q13, respectively, but conflicts with that of MacCollin et al., who have mapped the A2a gene to chromosome 22. In this report, we show that the human A1 adenosine receptor subtype does not map to chromosome 22q11.2-q13.1, but is instead localized on chromosome 1q32. 13 refs., 1 fig.

  11. Peripheral Adenosine A3 Receptor Activation Causes Regulated Hypothermia in Mice That Is Dependent on Central Histamine H1 Receptors.

    PubMed

    Carlin, Jesse Lea; Tosh, Dilip K; Xiao, Cuiying; Piñol, Ramón A; Chen, Zhoumou; Salvemini, Daniela; Gavrilova, Oksana; Jacobson, Kenneth A; Reitman, Marc L

    2016-02-01

    Adenosine can induce hypothermia, as previously demonstrated for adenosine A1 receptor (A1AR) agonists. Here we use the potent, specific A3AR agonists MRS5698, MRS5841, and MRS5980 to show that adenosine also induces hypothermia via the A3AR. The hypothermic effect of A3AR agonists is independent of A1AR activation, as the effect was fully intact in mice lacking A1AR but abolished in mice lacking A3AR. A3AR agonist-induced hypothermia was attenuated by mast cell granule depletion, demonstrating that the A3AR hypothermia is mediated, at least in part, via mast cells. Central agonist dosing had no clear hypothermic effect, whereas peripheral dosing of a non-brain-penetrant agonist caused hypothermia, suggesting that peripheral A3AR-expressing cells drive the hypothermia. Mast cells release histamine, and blocking central histamine H1 (but not H2 or H4) receptors prevented the hypothermia. The hypothermia was preceded by hypometabolism and mice with hypothermia preferred a cooler environmental temperature, demonstrating that the hypothermic state is a coordinated physiologic response with a reduced body temperature set point. Importantly, hypothermia is not required for the analgesic effects of A3AR agonists, which occur with lower agonist doses. These results support a mechanistic model for hypothermia in which A3AR agonists act on peripheral mast cells, causing histamine release, which stimulates central histamine H1 receptors to induce hypothermia. This mechanism suggests that A3AR agonists will probably not be useful for clinical induction of hypothermia. PMID:26606937

  12. Peripheral Adenosine A3 Receptor Activation Causes Regulated Hypothermia in Mice That Is Dependent on Central Histamine H1 Receptors

    PubMed Central

    Carlin, Jesse Lea; Tosh, Dilip K.; Xiao, Cuiying; Piñol, Ramón A.; Chen, Zhoumou; Salvemini, Daniela; Gavrilova, Oksana; Jacobson, Kenneth A.

    2016-01-01

    Adenosine can induce hypothermia, as previously demonstrated for adenosine A1 receptor (A1AR) agonists. Here we use the potent, specific A3AR agonists MRS5698, MRS5841, and MRS5980 to show that adenosine also induces hypothermia via the A3AR. The hypothermic effect of A3AR agonists is independent of A1AR activation, as the effect was fully intact in mice lacking A1AR but abolished in mice lacking A3AR. A3AR agonist–induced hypothermia was attenuated by mast cell granule depletion, demonstrating that the A3AR hypothermia is mediated, at least in part, via mast cells. Central agonist dosing had no clear hypothermic effect, whereas peripheral dosing of a non–brain-penetrant agonist caused hypothermia, suggesting that peripheral A3AR-expressing cells drive the hypothermia. Mast cells release histamine, and blocking central histamine H1 (but not H2 or H4) receptors prevented the hypothermia. The hypothermia was preceded by hypometabolism and mice with hypothermia preferred a cooler environmental temperature, demonstrating that the hypothermic state is a coordinated physiologic response with a reduced body temperature set point. Importantly, hypothermia is not required for the analgesic effects of A3AR agonists, which occur with lower agonist doses. These results support a mechanistic model for hypothermia in which A3AR agonists act on peripheral mast cells, causing histamine release, which stimulates central histamine H1 receptors to induce hypothermia. This mechanism suggests that A3AR agonists will probably not be useful for clinical induction of hypothermia. PMID:26606937

  13. Activation of the A2A adenosine G-protein-coupled receptor by conformational selection.

    PubMed

    Ye, Libin; Van Eps, Ned; Zimmer, Marco; Ernst, Oliver P; Prosser, R Scott

    2016-05-12

    Conformational selection and induced fit are two prevailing mechanisms to explain the molecular basis for ligand-based activation of receptors. G-protein-coupled receptors are the largest class of cell surface receptors and are important drug targets. A molecular understanding of their activation mechanism is critical for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce. Here we use (19)F nuclear magnetic resonance to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G-protein-coupled receptor. We find an ensemble of four states in equilibrium: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as 'ionic lock') between transmembrane helices 3 and 6; and (2) two active states, S3 and S3', as identified by binding of a G-protein-derived peptide. In contrast to a recent study of the β2-adrenergic receptor, the present approach allowed identification of a second active state for A2AR. Addition of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3', in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equilibria of ligand-dependent G-protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G-protein-coupled receptor in health and disease. PMID:27144352

  14. The A2B adenosine receptor modulates pulmonary hypertension associated with interstitial lung disease

    PubMed Central

    Karmouty-Quintana, Harry; Zhong, Hongyan; Acero, Luis; Weng, Tingting; Melicoff, Ernestina; West, James D.; Hemnes, Anna; Grenz, Almut; Eltzschig, Holger K.; Blackwell, Timothy S.; Xia, Yang; Johnston, Richard A.; Zeng, Dewan; Belardinelli, Luiz; Blackburn, Michael R.

    2012-01-01

    Development of pulmonary hypertension is a common and deadly complication of interstitial lung disease. Little is known regarding the cellular and molecular mechanisms that lead to pulmonary hypertension in patients with interstitial lung disease, and effective treatment options are lacking. The purpose of this study was to examine the adenosine 2B receptor (A2BR) as a regulator of vascular remodeling and pulmonary hypertension secondary to pulmonary fibrosis. To accomplish this, cellular and molecular changes in vascular remodeling were monitored in mice exposed to bleomycin in conjunction with genetic removal of the A2BR or treatment with the A2BR antagonist GS-6201. Results demonstrated that GS-6201 treatment or genetic removal of the A2BR attenuated vascular remodeling and hypertension in our model. Furthermore, direct A2BR activation on vascular cells promoted interleukin-6 and endothelin-1 release. These studies identify a novel mechanism of disease progression to pulmonary hypertension and support the development of A2BR antagonists for the treatment of pulmonary hypertension secondary to interstitial lung disease.—Karmouty-Quintana, H., Zhong, H., Acero, L., Weng, T., Melicoff, E., West, J. D., Hemnes, A., Grenz, A., Eltzschig, H. K., Blackwell, T. S., Xia, Y., Johnston, R. A., Zeng, D., Belardinelli, L., Blackburn, M. R. The A2B adenosine receptor modulates pulmonary hypertension associated with interstitial lung disease. PMID:22415303

  15. Allosteric interactions at adenosine A1 and A3 receptors: new insights into the role of small molecules and receptor dimerization

    PubMed Central

    Hill, Stephen J; May, Lauren T; Kellam, Barrie; Woolard, Jeanette

    2014-01-01

    The purine nucleoside adenosine is present in all cells in tightly regulated concentrations. It is released under a variety of physiological and pathophysiological conditions to facilitate protection and regeneration of tissues. Adenosine acts via specific GPCRs to either stimulate cyclic AMP formation, as exemplified by Gs-protein-coupled adenosine receptors (A2A and A2B), or inhibit AC activity, in the case of Gi/o-coupled adenosine receptors (A1 and A3). Recent advances in our understanding of GPCR structure have provided insights into the conformational changes that occur during receptor activation following binding of agonists to orthosteric (i.e. at the same binding site as an endogenous modulator) and allosteric regulators to allosteric sites (i.e. at a site that is topographically distinct from the endogenous modulator). Binding of drugs to allosteric sites may lead to changes in affinity or efficacy, and affords considerable potential for increased selectivity in new drug development. Herein, we provide an overview of the properties of selective allosteric regulators of the adenosine A1 and A3 receptors, focusing on the impact of receptor dimerization, mechanistic approaches to single-cell ligand-binding kinetics and the effects of A1- and A3-receptor allosteric modulators on in vivo pharmacology. Linked ArticlesThis article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-5 PMID:24024783

  16. An emerging role for adenosine and its receptors in bone homeostasis

    PubMed Central

    Ham, Jack; Evans, Bronwen A. J.

    2012-01-01

    Bone is continually being remodeled and defects in the processes involved lead to bone diseases. Many regulatory factors are known to influence remodeling but other mechanisms, hitherto unknown, may also be involved. Importantly, our understanding of these currently unknown mechanisms may lead to important new therapies for bone disease. It is accepted that purinergic signaling is involved in bone, and our knowledge of this area has increased significantly over the last 15 years, although most of the published work has studied the role of ATP and other signaling molecules via the P2 family of purinergic receptors. During the last few years, however, there has been increased interest within the bone field in the role of P1 receptors where adenosine is the primary signaling molecule. This review will bring together the current information available in relation to this expanding area of research. PMID:23024635

  17. Pharmacological and Therapeutic Effects of A3 Adenosine Receptor (A3AR) Agonists

    PubMed Central

    Fishman, Pnina; Bar-Yehuda, Sara; Liang, Bruce T.; Jacobson, Kenneth A.

    2011-01-01

    The Gi-coupled A3 adenosine receptor (A3AR) mediates anti-inflammatory, anticancer and anti-ischemic protective effects. The receptor is overexpressed in inflammatory and cancer cells, while low expression is found in normal cells, rendering the A3AR as a potential therapeutic target. Highly selective A3AR agonists have been synthesized and molecular recognition in the binding site has been characterized. The present review summarizes preclinical and clinical human studies demonstrating that A3AR agonists induce specific anti-inflammatory and anticancer effects via a molecular mechanism that entails modulation of the Wnt and the NF-κB signal transduction pathways. Currently, A3AR agonists are being developed for the treatment of inflammatory diseases including rheumatoid arthritis and psoriasis; ophthalmic diseases such as dry eye syndrome and glaucoma; liver diseases such as hepatocellular carcinoma and hepatitis. PMID:22033198

  18. Swertisin, a C-glucosylflavone, ameliorates scopolamine-induced memory impairment in mice with its adenosine A1 receptor antagonistic property.

    PubMed

    Lee, Hyung Eun; Jeon, Se Jin; Ryu, Byeol; Park, Se Jin; Ko, Sang Yoon; Lee, Younghwan; Kim, Eunji; Lee, Sunhee; Kim, Haneul; Jang, Dae Sik; Ryu, Jong Hoon

    2016-06-01

    Swertisin, a C-glucosylflavone isolated from Swertia japonica, has been known to have anti-inflammatory or antidiabetic activities. Until yet, however, its cognitive function is not investigated. In the present study, we endeavored to elucidate the effects of swertisin on cholinergic blockade-induced memory impairment. Swertisin (5 or 10mg/kg, p.o.) significantly ameliorated scopolamine-induced cognitive impairment in the several behavioral tasks. Also, single administration of swertisin (10mg/kg, p.o.) in normal naïve mice enhanced the latency time in the passive avoidance task. In addition, the ameliorating effect of swertisin on scopolamine-induced memory impairment was significantly antagonized by a sub-effective dose of N6-cyclopentyladenosine (CPA, 0.1mg/kg, i.p). The adenosine A1 receptor antagonistic property of swertisin was confirmed by receptor binding assay. Furthermore, the administration of swertisin significantly increased the phosphorylation levels of hippocampal or cortical protein kinase A (PKA, 5 or 10mg/kg) and CREB (10mg/kg), and co-administration of CPA (0.1mg/kg, i.p) blocked the increased phosphorylated levels of PKA and CREB in the both cortex and hippocampus. Taken together, these results indicate that the memory-ameliorating effects of swertisin may be, in part, mediated through the adenosinergic neurotransmitter system, and that swertisin may be useful for the treatment of cognitive dysfunction observed in several diseases such as Alzheimer's disease. PMID:26996316

  19. Cyclic, nonequilibrium models of glucocorticoid antagonism: role of activation, nuclear binding and receptor recycling.

    PubMed

    Munck, A; Holbrook, N J

    1988-10-01

    Quantitative models that have been proposed to date to explain mechanisms of glucocorticoid antagonism have generally been of the equilibrium type, involving hypothetical allosteric equilibria between active and inactive states of the receptor or the steroid-receptor complex. We describe here the agonist-antagonist relationships predicted by a nonequilibrium cyclic model that we have recently devised to account for the kinetic behavior of glucocorticoid-receptor complexes in intact rat thymus cells. This model simulates quantitatively most kinetic and steady state results that have been obtained so far. It postulates the existence of only well-established receptor species, and its kinetic parameters can in principle be determined by receptor measurements with intact cells. To calculate the steady state agonist-antagonist properties it is assumed that biological activity is proportional to the total amount of nuclear-bound complex, whether formed by agonist or antagonist. The agonist activity of a steroid is determined by the steady state ratio of nuclear-bound to total complexes it forms. This ratio varies from 0 for a pure antagonist to 1 for a pure agonist. It turns out to be independent of agonist and antagonist concentrations, and a function only of the rate constants for the reactions of the complexes formed by a steroid. Analysis of the dependence of the ratio on each rate constant shows quantitatively how each reaction in the cyclic model--activation of the nonactivated complex, nuclear binding of the activated complexes, and dissociation and recycling of activated and nuclear-bound complexes--affects antagonist properties. Steady state interactions of agonists with antagonists are found to be determined by equations that are identical to those for competition in simple equilibrium systems. Predicted dose-response relations agree qualitatively with experimentally observed relations. They are similar to those predicted by two-state allosteric models, although

  20. Adenosine is required for sustained inflammasome activation via the A2A receptor and the HIF-1α pathway

    NASA Astrophysics Data System (ADS)

    Ouyang, Xinshou; Ghani, Ayaz; Malik, Ahsan; Wilder, Tuere; Colegio, Oscar Rene; Flavell, Richard Anthony; Cronstein, Bruce Neil; Mehal, Wajahat Zafar

    2013-12-01

    Inflammasome pathways are important in chronic diseases; however, it is not known how the signalling is sustained after initiation. Inflammasome activation is dependent on stimuli such as lipopolysaccharide (LPS) and ATP that provide two distinct signals resulting in rapid production of interleukin (IL)-1β, with the lack of response to repeat stimulation. Here we report that adenosine is a key regulator of inflammasome activity, increasing the duration of the inflammatory response via the A2A receptor. Adenosine does not replace signals provided by stimuli such as LPS or ATP but sustains inflammasome activity via a cAMP/PKA/CREB/HIF-1α pathway. In the setting of the lack of IL-1β responses after previous exposure to LPS, adenosine can supersede this tolerogenic state and drive IL-1β production. These data reveal that inflammasome activity is sustained, after initial activation, by A2A receptor-mediated signalling.

  1. Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer

    PubMed Central

    Bonaventura, Jordi; Navarro, Gemma; Casadó-Anguera, Verònica; Azdad, Karima; Rea, William; Moreno, Estefanía; Brugarolas, Marc; Mallol, Josefa; Canela, Enric I.; Lluís, Carme; Cortés, Antoni; Volkow, Nora D.; Schiffmann, Serge N.; Ferré, Sergi; Casadó, Vicent

    2015-01-01

    Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain. PMID:26100888

  2. Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer.

    PubMed

    Bonaventura, Jordi; Navarro, Gemma; Casadó-Anguera, Verònica; Azdad, Karima; Rea, William; Moreno, Estefanía; Brugarolas, Marc; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Cortés, Antoni; Volkow, Nora D; Schiffmann, Serge N; Ferré, Sergi; Casadó, Vicent

    2015-07-01

    Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain. PMID:26100888

  3. Altered thermoregulation via sensitization of A1 adenosine receptors in dietary-restricted rats

    PubMed Central

    Jinka, Tulasi R.; Carlson, Zachary A.; Moore, Jeanette T.

    2010-01-01

    Rationale Evidence links longevity to dietary restriction (DR). A decrease in body temperature (Tb) is thought to contribute to enhanced longevity because lower Tb reduces oxidative metabolism and oxidative stress. It is as yet unclear how DR decreases Tb. Objective Here, we test the hypothesis that prolonged DR decreases Tb by sensitizing adenosine A1 receptors (A1AR) and adenosine-induced cooling. Methods and results Sprague–Dawley rats were dietary restricted using an every-other-day feeding protocol. Rats were fed every other day for 27 days and then administered the A1AR agonist, N6-cyclohexyladenosine (CHA; 0.5 mg/kg, i.p.). Respiratory rate (RR) and subcutaneous Tb measured using IPTT-300 transponders were monitored every day and after drug administration. DR animals displayed lower RR on day 20 and lower Tb on day 22 compared to animals fed ad libitum and displayed a larger response to CHA. In all cases, RR declined before Tb. Contrary to previous reports, a higher dose of CHA (5 mg/kg, i.p.) was lethal in both dietary groups. We next tested the hypothesis that sensitization to the effects of CHA was due to increased surface expression of A1AR within the hypothalamus. We report that the abundance of A1AR in the membrane fraction increases in hypothalamus, but not cortex of DR rats. Conclusion These results suggest that every-other-day feeding lowers Tb via sensitization of thermoregulatory effects of endogenous adenosine by increasing surface expression of A1AR. Discussion Evidence that diet can modulate purinergic signaling has implications for the treatment of stroke, brain injury, epilepsy, and aging. PMID:20186398

  4. A2B Adenosine Receptor Agonist Improves Erectile Function in Diabetic Rats.

    PubMed

    Wen, Jiaming; Wang, Bohan; Du, Chuanjun; Xu, Gang; Zhang, Zhewei; Li, Yi; Zhang, Nan

    2015-01-01

    Diabetes is an important risk factor for erectile dysfunction (ED). Recent studies have indicated that A2B adenosine receptor (ADORA2B) signaling is essential for penile erection. Thus, we hypothesize that diabetic ED may be attributed to impaired A2B adenosine signaling. To test this hypothesis, we generated diabetic rats by injecting streptozocin as animal model. After 12 weeks, immunohistochemistry staining was used to localize the expression of ADORA2B. Western Blot and quantitative PCR were employed to determine ADORA2B expression level. Intracavernosal pressure (ICP) measurement was used to evaluate erectile function. Diabetic rats received a single intravenous injection of BAY 60-6583, an ADORA2B agonist, or vehicle solution, at 60 min before the ICP measurement. The results showed that ADORA2B expressed in the nerve bundle, smooth muscle, and endothelium in penile tissue of control mice. Western Blot and quantitative PCR results indicated that the expression levels of ADORA2B protein and mRNA were significantly reduced in penile tissues of diabetic rats. Functional studies showed that the erectile response induced by electrical stimulation was remarkably decreased in diabetic rats, compared with age-matched control rats. However, at 60 min after BAY 60-6583 treatment, the erectile function was improved in diabetic rats, suggesting that enhancement of ADORA2B signaling may improve erectile function in diabetic ED. This preclinical study has revealed a previously unrecognized therapeutic possibility of BAY 60-6583 as an effective and mechanism-based drug to treat diabetic ED. In conclusion, we propose that impaired A2B adenosine signaling is one of the pathological mechanisms of diabetic ED. PMID:26447087

  5. Adenosine and the adenosine A2A receptor agonist, CGS21680, upregulate CD39 and CD73 expression through E2F-1 and CREB in regulatory T cells isolated from septic mice.

    PubMed

    Bao, Rui; Shui, Xianqi; Hou, Jiong; Li, Jinbao; Deng, Xiaoming; Zhu, Xiaoyan; Yang, Tao

    2016-09-01

    The number of regulatory T cells (Treg cells) and the expression of ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1; also known as CD39) and 5'-ectonucleotidase (NT5E; also known as CD73) on the Treg cell surface are increased during sepsis. In this study, to determine the factors leading to the high expression of CD39 and CD73, and the regulation of the CD39/CD73/adenosine pathway in Treg cells under septic conditions, we constructed a mouse model of sepsis and separated the Treg cells using a flow cytometer. The Treg cells isolated from the peritoneal lavage and splenocytes of the mice were treated with adenosine or the specific adenosine A2A receptor agonist, CGS21680, and were transfected with specific siRNA targeting E2F transcription factor 1 (E2F-1) or cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), which are predicted transcription regulatory factors of CD39 or CD73. The regulatory relationships among these factors were then determined by western blot analysis and dual-luciferase reporter assay. In addition, changes in adenosine metabolism were measured in the treated cells. The results revealed that adenosine and CGS21680 significantly upregulated CD39 and CD73 expression (P<0.01). E2F-1 and CREB induced CD39 and CD73 expression, and were upregulated by adenosine and CGS21680. Adenosine triphosphate (ATP) hydrolysis and adenosine generation were inhibited by the knockdown of E2F-1 or CREB, and were accelerated in the presence of CGS21680. Based on these results, it can be inferred that adenosine, the adenosine A2A receptor agonist, E2F-1 and CREB are the possible factors contributing to the high expression of CD39 and CD73 on the Treg cell surface during sepsis. Adenosine and its A2A receptor agonist served as the signal transducer factors of the CD39/CD73/adenosine pathway, accelerating adenosine generation. Our study may benefit further research on adenosine metabolism for the treatment of sepsis

  6. Sleep fragmentation impairs ventilatory long-term facilitation via adenosine A1 receptors

    PubMed Central

    McGuire, Michelle; Tartar, Jaime L; Cao, Ying; McCarley, Robert W; White, David P; Strecker, Robert E; Ling, Liming

    2008-01-01

    Sleep fragmentation (SF), a primary feature of obstructive sleep apnoea (OSA), impairs hippocampal long-term potentiation and causes cognitive/attention deficits. However, its influence upon respiratory control has hardly been studied. This study examined the effect of SF on ventilatory long-term facilitation (LTF, a persistent augmentation of respiratory activity after episodic hypoxia) and the hypoxic ventilatory response (HVR), and investigated the role of adenosine A1 receptors in these SF effects in conscious adult male Sprague–Dawley rats. SF, confirmed by sleep architecture recordings, was achieved by periodic, forced locomotion in a rotating drum (30 s rotation/90 s stop for 24 h). LTF, elicited by five episodes of 5 min poikilocapnic hypoxia (10% O2) with 5 min intervals, was measured by plethysmography. Resting ventilation and metabolic rate were unchanged, HVR was reduced (150.6 ± 3.5%versus 110.4 ± 12.3%) and LTF was eliminated (22.6 ± 0.5%versus−0.1 ± 1.3%) shortly after 24 h SF. The SF-induced impairments were SF duration dependent, and completely reversible as HVR (< 24 h) and LTF (< 48 h) returned spontaneously to their pre-SF values. The SF-impaired HVR was improved (130.3 ± 4.2%) and SF-eliminated LTF was restored (19.6 ± 0.9%) by systemic injection of the adenosine A1 receptor antagonist 8-CPT (2.5 mg kg−1) ∼30 min before LTF elicitation. Both HVR and LTF were also similarly impaired by 24 h total sleep deprivation or 24 h repeated cage tapping-induced SF, but not by a 24 h locomotion control protocol for SF. Collectively, these data suggest that: (1) 24 h SF impairs LTF and poikilocapnic HVR; (2) these impairments require A1 receptors; and (3) SF of OSA may exacerbate OSA via impaired ventilatory control mechanisms. PMID:18787037

  7. Reduced striatal adenosine A2A receptor levels define a molecular subgroup in schizophrenia.

    PubMed

    Villar-Menéndez, Izaskun; Díaz-Sánchez, Sara; Blanch, Marta; Albasanz, José Luis; Pereira-Veiga, Thais; Monje, Alfonso; Planchat, Luis Maria; Ferrer, Isidre; Martín, Mairena; Barrachina, Marta

    2014-04-01

    Schizophrenia (SZ) is a mental disorder of unknown origin. Some scientific evidence seems to indicate that SZ is not a single disease entity, since there are patient groups with clear symptomatic, course and biomarker differences. SZ is characterized by a hyperdopaminergic state related to high dopamine D2 receptor activity. It has also been proposed that there is a hypoadenosynergic state. Adenosine is a nucleoside widely distributed in the organism with neuromodulative and neuroprotective activity in the central nervous system. In the brain, the most abundant adenosine receptors are A1R and A2AR. In the present report, we characterize the presence of both receptors in human postmortem putamens of patients suffering SZ with real time TaqMan PCR, western blotting and radioligand binding assay. We show that A1R levels remain unchanged with respect to age-matched controls, whereas nearly fifty percent of patients have reduced A2AR, at the transcriptional and translational levels. Moreover, we describe how DNA methylation plays a role in the pathological A2AR levels with the bisulfite-sequencing technique. In fact, an increase in 5-methylcytosine percentage in the 5' UTR region of ADORA2A was found in those SZ patients with reduced A2AR levels. Interestingly, there was a relationship between the A2A/β-actin ratio and motor disturbances as assessed with some items of the PANSS, AIMS and SAS scales. Therefore, there may be a subgroup of SZ patients with reduced striatal A2AR levels accompanied by an altered motor phenotype. PMID:24433848

  8. Antagonism of protease-activated receptor 2 protects against experimental colitis.

    PubMed

    Lohman, Rink-Jan; Cotterell, Adam J; Suen, Jacky; Liu, Ligong; Do, Anh T; Vesey, David A; Fairlie, David P

    2012-02-01

    Many trypsin-like serine proteases such as β-tryptase are involved in the pathogenesis of colitis and inflammatory bowel diseases. Inhibitors of individual proteases show limited efficacy in treating such conditions, but also probably disrupt digestive and defensive functions of proteases. Here, we investigate whether masking their common target, protease-activated receptor 2 (PAR2), is an effective therapeutic strategy for treating acute and chronic experimental colitis in rats. A novel PAR2 antagonist (5-isoxazoyl-Cha-Ile-spiro[indene-1,4'-piperidine]; GB88) was evaluated for the blockade of intracellular calcium release in colonocytes and anti-inflammatory activity in acute (PAR2 agonist-induced) versus chronic [2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced] models of colitis in Wistar rats. Disease progression (disease activity index, weight loss, and mortality) and postmortem colonic histopathology (inflammation, bowel wall thickness, and myeloperoxidase) were measured. PAR2 and tryptase colocalization were investigated by using immunohistochemistry. GB88 was a more potent antagonist of PAR2 activation in colonocytes than another reported compound, N¹-3-methylbutyryl-N⁴-6-aminohexanoyl-piperazine (ENMD-1068) (IC₅₀ 8 μM versus 5 mM). Acute colonic inflammation induced in rats by the PAR2 agonist SLIGRL-NH₂ was inhibited by oral administration of GB88 (10 mg/kg) with markedly reduced edema, mucin depletion, PAR2 receptor internalization, and mastocytosis. Chronic TNBS-induced colitis in rats was ameliorated by GB88 (10 mg/kg/day p.o.), which reduced mortality and pathology (including colon obstruction, ulceration, wall thickness, and myeloperoxidase release) more effectively than the clinically used drug sulfasalazine (100 mg/kg/day p.o.). These disease-modifying properties for the PAR2 antagonist in both acute and chronic experimental colitis strongly support a pathogenic role for PAR2 and PAR2-activating proteases and therapeutic potential for

  9. Prenatal NMDA Receptor Antagonism Impaired Proliferation of Neuronal Progenitor, Leading to Fewer Glutamatergic Neurons in the Prefrontal Cortex

    PubMed Central

    Toriumi, Kazuya; Mouri, Akihiro; Narusawa, Shiho; Aoyama, Yuki; Ikawa, Natsumi; Lu, Lingling; Nagai, Taku; Mamiya, Takayoshi; Kim, Hyoung-Chun; Nabeshima, Toshitaka

    2012-01-01

    N-methyl--aspartate (NMDA) receptor is a glutamate receptor which has an important role on mammalian brain development. We have reported that prenatal treatment with phencyclidine (PCP), a NMDA receptor antagonist, induces long-lasting behavioral deficits and neurochemical changes. However, the mechanism by which the prenatal antagonism of NMDA receptor affects neurodevelopment, resulting in behavioral deficits, has remained unclear. Here, we report that prenatal NMDA receptor antagonism impaired the proliferation of neuronal progenitors, leading to a decrease in the progenitor pool in the ventricular and the subventricular zone. Furthermore, using a PCR array focused on neurogenesis and neuronal stem cells, we evaluated changes in gene expression causing the impairment of neuronal progenitor proliferation and found aberrant gene expression, such as Notch2 and Ntn1, in prenatal PCP-treated mice. Consequently, the density of glutamatergic neurons in the prefrontal cortex was decreased, probably resulting in glutamatergic hypofunction. Prenatal PCP-treated mice displayed behavioral deficits in cognitive memory and sensorimotor gating until adulthood. These findings suggest that NMDA receptors regulate the proliferation and maturation of progenitor cells for glutamatergic neuron during neurodevelopment, probably via the regulation of gene expression. PMID:22257896

  10. Novel arylsulfonamide derivatives with 5-HT₆/5-HT₇ receptor antagonism targeting behavioral and psychological symptoms of dementia.

    PubMed

    Kołaczkowski, Marcin; Marcinkowska, Monika; Bucki, Adam; Pawłowski, Maciej; Mitka, Katarzyna; Jaśkowska, Jolanta; Kowalski, Piotr; Kazek, Grzegorz; Siwek, Agata; Wasik, Anna; Wesołowska, Anna; Mierzejewski, Paweł; Bienkowski, Przemyslaw

    2014-06-12

    In order to target behavioral and psychological symptoms of dementia (BPSD), we used molecular modeling-assisted design to obtain novel multifunctional arylsulfonamide derivatives that potently antagonize 5-HT(6/7/2A) and D2 receptors, without interacting with M1 receptors and hERG channels. In vitro studies confirmed their antagonism of 5-HT(7/2A) and D2 receptors and weak interactions with key antitargets (M1R and hERG) associated with side effects. Marked 5-HT6 receptor affinities were also observed, notably for 6-fluoro-3-(piperidin-4-yl)-1,2-benzoxazole derivatives connected by a 3-4 unit alkyl linker with mono- or bicyclic, lipophilic arylsulfonamide moieties. N-[4-[4-(6-Fluoro-1,2-benzoxazol-3-yl)piperidin-1-yl]butyl]benzothiophene-2-sulfonamide (72) was characterized in vitro on 14 targets and antitargets. It displayed dual blockade of 5-HT6 and D2 receptors and negligible interactions at hERG and M1 receptors. Unlike reference antipsychotics, 72 displayed marked antipsychotic and antidepressant activity in rats after oral administration, in the absence of cognitive or motor impairment. This profile is particularly attractive when targeting a fragile, elderly BPSD patient population. PMID:24805037

  11. Chronic sleep restriction induces long-lasting changes in adenosine and noradrenaline receptor density in the rat brain

    PubMed Central

    WEISSHAUPT, ANGELA; WEDEKIND, FRANZISKA; KROLL, TINA; MCCARLEY, ROBERT W.

    2015-01-01

    SUMMARY Although chronic sleep restriction frequently produces long-lasting behavioural and physiological impairments in humans, the underlying neural mechanisms are unknown. Here we used a rat model of chronic sleep restriction to investigate the role of brain adenosine and noradrenaline systems, known to regulate sleep and wakefulness, respectively. The density of adenosine A1 and A2a receptors and β-adrenergic receptors before, during and following 5 days of sleep restriction was assessed with autoradiography. Rats (n = 48) were sleep-deprived for 18 h day–1 for 5 consecutive days (SR1–SR5), followed by 3 unrestricted recovery sleep days (R1–R3). Brains were collected at the beginning of the light period, which was immediately after the end of sleep deprivation on sleep restriction days. Chronic sleep restriction increased adenosine A1 receptor density significantly in nine of the 13 brain areas analysed with elevations also observed on R3 (+18 to +32%). In contrast, chronic sleep restriction reduced adenosine A2a receptor density significantly in one of the three brain areas analysed (olfactory tubercle which declined 26–31% from SR1 to R1). A decrease in b-adrenergic receptors density was seen in substantia innominata and ventral pallidum which remained reduced on R3, but no changes were found in the anterior cingulate cortex. These data suggest that chronic sleep restriction can induce long-term changes in the brain adenosine and noradrenaline receptors, which may underlie the long-lasting neurocognitive impairments observed in chronic sleep restriction. PMID:25900125

  12. Orexin Receptor Antagonism Improves Sleep and Reduces Seizures in Kcna1-null Mice

    PubMed Central

    Roundtree, Harrison M.; Simeone, Timothy A.; Johnson, Chaz; Matthews, Stephanie A.; Samson, Kaeli K.; Simeone, Kristina A.

    2016-01-01

    other epilepsy models. Citation: Roundtree HM, Simeone TA, Johnson C, Matthews SA, Samson KK, Simeone KA. Orexin receptor antagonism improves sleep and reduces seizures in Kcna1-null mice. SLEEP 2016;39(2):357–368. PMID:26446112

  13. Recent developments in adenosine receptor ligands and their potential as novel drugs☆

    PubMed Central

    Müller, Christa E.; Jacobson, Kenneth A.

    2012-01-01

    Medicinal chemical approaches have been applied to all four of the adenosine receptor (AR) subtypes (A1, A2A, A2B, and A3) to create selective agonists and antagonists for each. The most recent class of selective AR ligands to be reported is the class of A2BAR agonists. The availability of these selective ligands has facilitated research on therapeutic applications of modulating the ARs and in some cases has provided clinical candidates. Prodrug approaches have been developed which improve the bioavailability of the drugs, reduce side-effects, and/or may lead to site-selective effects. The A2A agonist regadenoson (Lexiscan®), a diagnostic drug for myocardial perfusion imaging, is the first selective AR agonist to be approved. Other selective agonists and antagonists are or were undergoing clinical trials for a broad range of indications, including capadenoson and tecadenoson (A1 agonists) for atrial fibrillation, or paroxysmal supraventricular tachycardia, respectively, apadenoson and binodenoson (A2A agonists) for myocardial perfusion imaging, preladenant (A2A antagonist) for the treatment of Parkinson’s disease, and CF101 and CF102 (A3 agonists) for inflammatory diseases and cancer, respectively. This article is part of a Special Issue entitled: “Adenosine Receptors”. PMID:21185259

  14. Orally Active Adenosine A1 Receptor Agonists with Antinociceptive Effects in Mice

    PubMed Central

    Korboukh, Ilia; Hull-Ryde, Emily A.; Rittiner, Joseph E.; Randhawa, Amarjit S.; Coleman, Jennifer; Fitzpatrick, Brendan J.; Setola, Vincent; Janzen, William P.; Frye, Stephen V.; Zylka, Mark J.; Jin, Jian

    2012-01-01

    Adenosine A1 receptor (A1AR) agonists have antinociceptive effects in multiple preclinical models of acute and chronic pain. Although numerous A1AR agonists have been developed, clinical applications of these agents have been hampered by their cardiovascular side effects. Herein we report a series of novel A1AR agonists, some of which are structurally related to adenosine 5′-monophosphate (5′-AMP), a naturally occurring nucleotide that itself activates A1AR. These novel compounds potently activate A1AR in several orthogonal in vitro assays and are subtype selective for A1AR over A2AAR, A2BAR, and A3AR. Among them, UNC32A (3a) is orally active and has dose-dependent antinociceptive effects in wild-type mice. The antinociceptive effects of 3a were completely abolished in A1AR knockout mice, revealing a strict dependence on A1AR for activity. The apparent lack of cardiovascular side effects when administered orally and high affinity (Ki of 36 nM for the human A1AR) make this compound potentially suitable as a therapeutic. PMID:22738238

  15. Involvement of adenosine A2A receptors in depression and anxiety.

    PubMed

    Yamada, Koji; Kobayashi, Minoru; Kanda, Tomoyuki

    2014-01-01

    When administered to normal healthy patients, a nonselective adenosine A1/A2A antagonist, caffeine, tended to improve anxiety and depression at low doses and to exacerbate anxiety at high doses. Caffeine also appears to enhance anxiety-related symptoms in patients with panic disorder, and A2A receptor-deficient mice have been reported to exhibit higher anxiety-like behaviors, as well as a lower incidence of depression-like behaviors. Some selective A2A antagonists were reported to ameliorate anxiety-like behaviors in rodents, while others did not affect these behaviors. In addition, most A2A antagonists showed inhibitory effects on depression-like behaviors. The mechanisms underlying the relationship between A2A receptor antagonists and anxiety and depression remain unclear at the present time, although many studies have produced hypotheses. Given that a selective A2A receptor antagonist has recently become available for use in humans, research on the role of A2A receptors in the treatment of mental illness should progress in the near future. PMID:25175973

  16. Role of brainstem adenosine A1 receptors in the cardiovascular response to hypothalamic defence area stimulation in the anaesthetized rat.

    PubMed Central

    St Lambert, J. H.; Dashwood, M. R.; Spyer, K. M.

    1996-01-01

    1. The role of centrally located adenosine A1 receptors in the cardiovascular changes associated with the hypothalamic defence response has been investigated by in vitro autoradiography and the intraventricular application of an A1 receptor antagonist. 2. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), a highly selective adenosine A1 antagonist and its vehicle, ethanol, were administered directly into the posterior portion of the fourth ventricle of alpha-chloralose anaesthetized, paralysed and artificially ventilated rats. 3. DPCPX (0.01 to 0.3 mg kg-1) caused a dose-dependent decrease in the magnitude of the evoked pressor response (from -13 to -23 mmHg) elicited on hypothalamic defence area stimulation at a dose 10 fold lower than that required to produce an equivalent effect following systemic administration whilst ethanol, the vehicle, had no effect. 4. In vitro autoradiography revealed a heterogeneous distribution of adenosine A1 binding sites in the lower brainstem of rats. Image analysis showed the ventrolateral medulla to have the highest density of A1 receptors. Intermediate levels of binding were seen in caudal regions of the nucleus tractus solitarii and the hypoglossal nucleus. 5. These data imply that a proportion of the cardiovascular response to hypothalamic defence area stimulation are produced by the activation of adenosine A1 receptors localized close to the surface of, or adjacent to, the fourth ventricle in the immediate vicinity of the injection site. PMID:8789379

  17. Synthesis and pharmacological characterization of novel xanthine carboxylate amides as A2A adenosine receptor ligands exhibiting bronchospasmolytic activity.

    PubMed

    Yadav, Rakesh; Bansal, Ranju; Rohilla, Suman; Kachler, Sonja; Klotz, Karl-Norbert

    2016-04-01

    The carboxylate amides of 8-phenyl-1,3-dimethylxanthine described herein represent a new series of selective ligands of the adenosine A2A receptors exhibiting bronchospasmolytic activity. The effects of location of 8-phenyl substitutions on the adenosine receptor (AR) binding affinities of the newly synthesized xanthines have also been studied. The compounds displayed moderate to potent binding affinities toward various adenosine receptor subtypes when evaluated through radioligand binding studies. However, most of the compounds showed the maximum affinity for the A2A subtype, some with high selectivity versus all other subtypes. Xanthine carboxylate amide 13b with a diethylaminoethylamino moiety at the para-position of the 8-phenylxanthine scaffold was identified as the most potent A2A adenosine receptor ligand with Ki=0.06μM. Similarly potent and highly A2A-selective are the isovanillin derivatives 16a and 16d. In addition, the newly synthesized xanthine derivatives showed good in vivo bronchospasmolytic activity when tested in guinea pigs. PMID:26851736

  18. Localization of the A{sub 3} adenosine receptor gene (ADORA3) to human chromosome 1p

    SciTech Connect

    Monitto, C.L.; Levitt, R.C.; Holroyd, K.J.

    1995-04-10

    Adenosine modulates important physiologic functions involving the cardiovascular system, brain, kidneys, lungs, GI tract, and immune system. To date four adenosine receptors have been identified: A{sub 1}, A{sub 2a}, A{sub 2b}, and A{sub 3}. Activation of these receptors results in inhibition (A{sub 1} and A{sub 3}) or stimulation (A{sub 2a} and A{sub 2b}) of intracellular adenyl cyclase activity, stimulation of K{sup +} flux, inhibition of Ca{sup 2+} flux, and modulation of inositol phospholipid turnover. A{sub 3} receptors have been identified and sequenced in the testes, brain, lung, liver, kidney, and heart of various species, including the rat, mouse, and human. A{sub 3} receptor activation is responsible for release of inflammatory mediators from mast cells, which can cause allergic bronchoconstriction. In addition, they can produce systemic vasodilation and locomotor depression via activation of A{sub 3} receptors in the brain. Given the potential importance of A{sub 3} receptor activity in the pathogenesis of pulmonary, cardiovascular, and central nervous system disease states, we set out to localize the human A{sub 3} adenosine receptor gene (ADORA3). 9 refs., 1 fig.

  19. Carbamate substituted 2-amino-4,6-diphenylpyrimidines as adenosine receptor antagonists.

    PubMed

    Robinson, Sarel J; Petzer, Jacobus P; Rousseau, Amanda L; Terre'Blanche, Gisella; Petzer, Anél; Lourens, Anna C U

    2016-02-01

    A novel series of carbamate substituted 2-amino-4,6-diphenylpyrimidines was evaluated as potential dual adenosine A1 and A2A receptor antagonists. The majority of the synthesised compounds exhibited promising dual affinities, with A1Ki values ranging from 0.175 to 10.7 nM and A2AKi values ranging from 1.58 to 451 nM. The in vivo activity illustrated for 3-(2-amino-6-phenylpyrimidin-4-yl)phenyl morpholine-4-carboxylate (4c) is indicative of the potential of these compounds as therapeutic agents in the treatment of Parkinson's disease, although physicochemical properties may require optimisation. PMID:26776359

  20. Role of Adenosine Receptor(s) in the Control of Vascular Tone in the Mouse Pudendal Artery.

    PubMed

    Labazi, Hicham; Tilley, Stephen L; Ledent, Catherine; Mustafa, S Jamal

    2016-03-01

    Activation of adenosine receptors (ARs) has been implicated in the modulation of renal and cardiovascular systems, as well as erectile functions. Recent studies suggest that adenosine-mediated regulation of erectile function is mainly mediated through A2BAR activation. However, no studies have been conducted to determine the contribution of AR subtype in the regulation of the vascular tone of the pudendal artery (PA), the major artery supplying and controlling blood flow to the penis. Our aim was to characterize the contribution of AR subtypes and identify signaling mechanisms involved in adenosine-mediated vascular tone regulation in the PA. We used a DMT wire myograph for muscle tension measurements in isolated PAs from wild-type, A2AAR knockout, A2BAR knockout, and A2A/A2BAR double-knockout mice. Real-time reverse transcription-polymerase chain reaction was used to determine the expression of the AR subtypes. Data from our pharmacologic and genetic approaches suggest that AR activation-mediated vasodilation in the PA is mediated by both the A2AAR and A2BAR, whereas neither the A1AR nor A3AR play a role in vascular tone regulation of the PA. In addition, we showed that A2AAR- and A2BAR-mediated vasorelaxation requires activation of nitric oxide and potassium channels; however, only the A2AAR-mediated response requires protein kinase A activation. Our data are complemented by mRNA expression showing the expression of all AR subtypes with the exception of the A3AR. AR signaling in the PA may play an important role in mediating erection and represent a promising therapeutic option for the treatment of erectile dysfunction. PMID:26718241

  1. The role of the A(2A) adenosine receptor subtype in functional hyperaemia in the hindlimb of anaesthetized cats.

    PubMed Central

    Poucher, S M

    1996-01-01

    1. The present study was designed to investigate the contribution of the A(2A) adenosine receptor subtype in the functional hyperaemia response during muscle contraction. 2. In cats anaesthetized with sodium pentobarbitone and breathing spontaneously following tracheotomy, the left sciatic and femoral nerves were electrically stimulated at 3 Hz for 20 min to induce muscle contraction, and hindlimb blood flow was measured with a flow probe. The contribution of the A(2A) adenosine receptor subtype was assessed using ZM 241385, a potent and selective A(2A) adenosine receptor antagonist. 3. In a control group, the muscle isometric tension measured in the extensor digitorum longus-tibialis anterior muscle group was 6.64 +/- 0.66 kg (100 g muscle mass)(-1) and hindlimb vascular conductance was 0.22 +/- 0.03 ml mmHg(-1)(kg body mass)(-1) at 20 min of contraction. Administration of vehicle did not affect these parameters upon a second contraction period: 6.31 +/- 0.61 kg (100 g muscle mass)(-1) and 0.23 +/- 0.03 ml mmHg(-1) (kg body mass)(-1), respectively. Total hindlimb conductance during contraction was unaffected (5.5 +/- 3.7% decrease). 4. ZM 241385 (1.0 mg kg(-1)) did not alter the amount of force produced by the muscle at 20 min of contraction. Hindlimb conductance response was reduced by 27.1 +/- 4.8% following the A(2A) selective adenosine receptor antagonist, similar to that observed with the non-selective antagonist 8-phenyltheophylline. 5. These results show that adenosine acting at the A(2A) subtype receptor can contribute up to 30% of the functional hyperaemia response in the hindlimb of anaesthetized cats. PMID:9019545

  2. Understanding the implications of dissolved organic carbon when assessing antagonism in vitro: An example with an estrogen receptor assay.

    PubMed

    Neale, Peta A; Escher, Beate I; Leusch, Frederic D L

    2015-09-01

    Both estrogenic and anti-estrogenic activity has been observed in water samples. Some studies have suggested that dissolved organic carbon (DOC), which can be co-extracted during sample enrichment, contributes to the apparent antagonistic effect. DOC has a high sorption capacity for the estrogen receptor (ER) agonist 17β-estradiol, which may reduce the available 17β-estradiol concentration in the antagonist testing mode and potentially lead to apparent antagonism. The aim of the study was to determine the influence of DOC when assessing antagonism in an ER reporter gene assay. The presence of DOC shifted the 17β-estradiol concentration-effect curve to higher concentrations, increasing the nominal EC50 value by up to 0.3 log units. However, this shift was within the usual variability associated with repeated measurements of concentration-effect curves. This shift was not due to DOC being an antagonist itself or interfering with fluorescence measurements, but was due to DOC reducing the bioavailability of 17β-estradiol. This was demonstrated by modelling the DOC sorption corrected 17β-estradiol concentration using experimental DOC-water partition coefficients (KDOC). While the shift in the 17β-estradiol concentration-effect curve was minor, sorption of 17β-estradiol to DOC can have an impact when assessing antagonism. At the EC50 agonist concentration, both modelled and experimental results showed that DOC at concentrations similar to that co-extracted in water samples caused suppression of the agonist at levels that would be classified as antagonism. The suppression was less pronounced at the EC80 agonist concentration, hence this is recommended when assessing antagonism of DOC rich samples, such as surface water and wastewater. PMID:25978675

  3. Adenosine A2A Receptor Activation Prevents Wear Particle-Induced Osteolysis

    PubMed Central

    Mediero, Aránzazu; Frenkel, Sally R.; Wilder, Tuere; He, Wenjie; Mazumder, Amitabha; Cronstein, Bruce N.

    2012-01-01

    Prosthesis loosening, associated with wear-particle–induced inflammation and osteoclast-mediated bone destruction, is a common cause for joint implant failure, leading to revision surgery. Adenosine A2A receptors (A2AR) mediate potent anti-inflammatory effects in many tissues and prevent osteoclast differentiation. We tested the hypothesis that an A2AR agonist could reduce osteoclast-mediated bone resorption in a murine calvaria model of wear-particle–induced bone resorption. C57Bl/6 and A2A knockout (A2ARKO) mice received ultrahigh-molecular weight polyethylene particles (UHMWPE) and were treated daily with either saline or the A2AR agonist CGS21680. After 2 weeks, micro-computed tomography of calvaria demonstrated that CGS21680 reduced particle-induced bone pitting and porosity in a dose-dependent manner, increasing cortical bone and bone volume compared to control mice. Histological examination demonstrated diminished inflammation after treatment with CGS21680. In A2AKO mice, CGS21680 did not affect osteoclast-mediated bone resorption or inflammation. Levels of bone-resorption markers receptor activator of nuclear factor-kB (RANK), RANK ligand (RANKL), cathepsin K, CD163, and osteopontin were reduced following CGS21680 treatment, together with a reduction in osteoclasts. Secretion of interleukin 1β (IL-1β) and TNFα was significantly decreased, whereas IL-10 was markedly increased in bone by CGS21680. These results in mice suggest that site-specific delivery of an adenosine A2AR agonist could enhance implant survival, delaying or eliminating the need for revision arthroplastic surgery. PMID:22623741

  4. NMDA Receptor Agonism and Antagonism within the Amygdaloid Central Nucleus Suppresses Pain Affect: Differential Contribution of the Ventrolateral Periaqueductal Gray

    PubMed Central

    Spuz, Catherine A.; Tomaszycki, Michelle L.; Borszcz, George S.

    2015-01-01

    The amygdala contributes to the generation of pain affect and the amygdaloid central nucleus (CeA) receives nociceptive input that is mediated by glutamatergic neurotransmission. The present study compared the contribution of N-methyl-D-aspartate (NMDA) receptor agonism and antagonism in CeA to generation of the affective response of rats to an acute noxious stimulus. Vocalizations that occur following a brief tail shock (vocalization afterdischarges) are a validated rodent model of pain affect, and were preferentially suppressed, in a dose dependent manner, by bilateral injection into CeA of NMDA (.1 µg, .25 µg, .5 µg, or 1 µg/side), or the NMDA receptor antagonist D-2-amino-5-phosphonovalerate (AP5, 1 µg, 2 µg, or 4 µg/side). Vocalizations that occur during tail shock were suppressed to a lesser degree, whereas, spinal motor reflexes (tail flick and hind limb movements) were unaffected by injection of NMDA or AP5 into CeA. Injection of NMDA, but not AP5, into CeA increased c-Fos immunoreactivity in the ventrolateral periaqueductal gray (vlPAG), and unilateral injection of the µ-opiate receptor antagonist H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP, 0.25 µg) into vlPAG prevented the antinociception generated by injection of NMDA into CeA. These findings demonstrate that although NMDA receptor agonism and antagonism in CeA produce similar suppression of pain behaviors they do so via different neurobiological mechanisms. Perspective The amygdala contributes to production of the emotional dimension of pain. NMDA receptor agonism and antagonism within the central nucleus of the amygdala suppressed rats’ emotional response to acute painful stimulation. Understanding the neurobiology underlying emotional responses to pain will provide insights into new treatments for pain and its associated affective disorders. PMID:25261341

  5. Progress in the discovery of selective, high affinity A(2B) adenosine receptor antagonists as clinical candidates.

    PubMed

    Kalla, Rao V; Zablocki, Jeff

    2009-03-01

    The selective, high affinity A(2B) adenosine receptor (AdoR) antagonists that were synthesized by several research groups should aid in determining the role of the A(2B) AdoR in inflammatory diseases like asthma or rheumatoid arthritis (RA) and angiogenic diseases like diabetic retinopathy or cancer. CV Therapeutics scientists discovered the selective, high affinity A(2B) AdoR antagonist 10, a 8-(4-pyrazolyl)-xanthine derivative [CVT-6883, K(i)(hA(2B)) = 22 nM; K(i)(hA(1)) = 1,940 nM; K(i)(hA(2A)) = 3,280; and K(i)(hA(3)) = 1,070 nM] that has favorable pharmacokinetic (PK) properties (t (1/2) = 4 h and F > 35% rat). Compound 10 demonstrated functional antagonism at the A(2B) AdoR (K(B) = 6 nM) and efficacy in a mouse model of asthma. In two phase 1 clinical trials, CVT-6883 was found to be safe, well tolerated, and suitable for once daily dosing. A second compound 20, 8-(5-pyrazolyl)-xanthine, has been nominated for development from Baraldi's group in conjunction with King Pharmaceuticals that has favorable A(2B) AdoR affinity and selectivity [K(i)(hA(2B)) = 5.5 nM; K(i)(hA(1)) > 1,000 nM; K(i)(hA(2A)) > 1,000; and K(i)(hA(3)) > 1,000 nM], and it has been demonstrated to be a functional antagonist. A third compound 32, a 2-aminopyrimidine, from the Almirall group has high A(2B) AdoR affinity and selectivity [K(i)(hA(2B)) = 17 nM; K(i)(hA(1)) > 1,000 nM; K(i)(hA(2A)) > 2,500; and K(i)(hA(3)) > 1,000 nM], and 32 has been moved into preclinical safety testing. Since three highly selective, high affinity A(2B) AdoR antagonists have been nominated for development with 10 (CVT-6883) being the furthest along in the development process, the role of the A(2B) AdoR in various disease states will soon be established. PMID:18568423

  6. A1-, A2A- and A3-subtype adenosine receptors modulate intraocular pressure in the mouse

    PubMed Central

    Avila, Marcel Y; Stone, Richard A; Civan, Mortimer M

    2001-01-01

    Despite the potential importance of the mouse in studying the pharmacology of aqueous dynamics, measurement of intraocular pressure (IOP) in its very small eye has been problematic. Utilizing a novel servo-null electrophysiologic approach recently applied to the mouse, we have identified a diversity of adenosine-receptor mechanisms in modulating IOP in this species. We report the first evidence that A3 receptors increase IOP in any species, and verify in the mouse reports with larger mammals that A1 receptors lower and A2A receptors increase IOP. PMID:11564641

  7. A/sub 1/ and A/sub 2/ adenosine receptor regulation of erythropoietin production

    SciTech Connect

    Ueno, M.; Brookins, J.; Beckman, B.; Fisher, J.W.

    1988-01-01

    The effects of adenosine (ADE) and ADE agonists on erythropoietin (Ep) production were determined using percent (%) /sup 59/Fe incorporation in red cells of exhypoxic polycythemic mice. The hemisulfate salt of ADE produced a significant increase in % /sup 59/Fe incorporation in response to hypoxia in concentrations of 400 to 1600 nmol/kg/day. 5'-N-ethyl-carboxamideadenosine (NECA), a selective A/sub 2/ receptor agonist, increased radioiron incorporation in a dose-dependent manner. In contrast, N/sup 6/-cyclohexyladenosine (CHA), a selective A/sub 1/ receptor agonist, did not affect radioiron incorporation in concentrations up to 1600 nmol/kg/day. Albuterol, a beta 2-adrenergic agonist, enhanced % /sup 59/Fe incorporation in polycythemic mice and low doses of CHA, which were not effective alone on % /sup 59/Fe incorporation in polycythemic mice exposed to hypoxia, inhibited the enhancement in radioiron induced by albuterol plus hypoxia. Theophylline, a well-known antagonist of ADE receptors, blocked the ADE and NECA enhancement in radioiron incorporation at a dose of theophylline alone which produced only a slight enhancement of % /sup 59/Fe incorporation.

  8. Ethanol-induced increase in portal blood flow: Role of acetate and A sub 1 - and A sub 2 -adenosine receptors

    SciTech Connect

    Carmichael, F.J.; Saldivia, V.; Varghese, G.A.; Israel, Y.; Orrego, H. Univ. of Toronto, Ontario )

    1988-10-01

    The increase in portal blood flow induced by ethanol appears to be adenosine mediated. Acetate, which is released by the liver during ethanol metabolism, is known to increase adenosine levels in tissues and in blood. The effects of acetate on portal blood flow were investigated in rats using the microsphere technique. The intravenous infusion of acetate resulted in vasodilation of the preportal vasculature and in a dose-dependent increase in portal blood flow. This acetate-induced increase in portal blood flow was suppressed by the adenosine receptor blocker, 8-phenyltheophylline. Using the A{sub 1}-adenosine receptor agonist N-6-cyclohexyl adenosine and the A{sub 2}-agonist 5{prime}-N-ethylcarboxamido adenosine, we demonstrate that the effect of adenosine on the preportal vasculature is mediated by the A{sub 2}-subtype of adenosine receptors. In conclusion, these data support the hypothesis that the increase in portal blood flow after ethanol administration results from a preportal vasodilatory effect of adenosine formed from acetate metabolism in extrahepatic tissues.

  9. Glucocorticoid receptor antagonism as a novel therapy for triple-negative breast cancer

    PubMed Central

    Skor, Maxwell N.; Wonder, Erin L.; Kocherginsky, Masha; Goyal, Anju; Hall, Ben A.; Cai, Yi; Conzen, Suzanne D.

    2013-01-01

    Purpose: Triple-negative breast cancer (TNBC) accounts for 10-20% of newly diagnosed invasive breast cancer. Finding effective targets for chemotherapy-resistant TNBC has proven difficult in part because of TNBC’s molecular heterogeneity. We have previously reported that, likely because of GR’s anti-apoptotic activity in ER-negative breast epithelial and cancer cells, high glucocorticoid receptor (GR) expression/activity in early-stage TNBC significantly correlates with chemotherapy-resistance and increased recurrence. We hypothesized that pre-treatment with mifepristone, a (GR)-antagonist, would potentiate the efficacy of chemotherapy in GR+ TNBC by inhibiting GR’s anti-apoptotic signaling pathways and increasing the cytotoxic efficiency of chemotherapy. Experimental Design: TNBC cell apoptosis was examined in the context of physiological glucocorticoid concentrations, chemotherapy, and/or pharmacologic concentrations of mifepristone. We used high-throughput live microscopy with continuous recording to measure apoptotic cells stained with a fluorescent dye, and Western analysis to detect caspase-3 and PARP cleavage. The effect of mifepristone on GR-mediated gene expression was also measured. TNBC xenograft studies were performed in female severe combined immunodeficient (SCID) mice and tumors were measured following treatment with vehicle, paclitaxel or mifepristone/paclitaxel. Results: We found that although mifepristone treatment alone had no significant effect on TNBC cell viability or clonogenicity in the absence of chemotherapy, the addition of mifepristone to dexamethasone/paclitaxel treatment significantly increased cytotoxicity and caspase-3/PARP cleavage. Mifepristone also antagonized GR-induced SGK1 and MKP1/DUSP1 gene expression, while significantly augmenting paclitaxel-induced GR+ MDA-MB-231 xenograft tumor shrinkage in vivo. Conclusions: These results suggest that mifepristone pre-treatment could be a useful strategy for increasing tumor cell

  10. Farnesoid X receptor antagonizes JNK signaling pathway in liver carcinogenesis by activating SOD3.

    PubMed

    Wang, Yan-Dong; Chen, Wei-Dong; Li, Cunbao; Guo, Cong; Li, Yanyan; Qi, Hui; Shen, Hailing; Kong, Jing; Long, Xuecheng; Yuan, Frank; Wang, Xichun; Huang, Wendong

    2015-02-01

    The farnesoid X receptor (FXR) is a key metabolic and homeostatic regulator in the liver. In the present work, we identify a novel role of FXR in antagonizing c-Jun N-terminal kinase (JNK) signaling pathway in liver carcinogenesis by activating superoxide dismutase 3 (SOD3) transcription. Compared with wild-type mouse liver, FXR(-/-) mouse liver showed elevated JNK phosphorylation. JNK1 deletion suppressed the increase of diethylnitrosamine-induced tumor number in FXR(-/-) mice. These results suggest that JNK1 plays a key role in chemical-induced liver carcinogenesis in FXR(-/-) mice. We found that ligand-activated FXR was able to alleviate H₂O₂or tetradecanoylphorbol acetate-induced JNK phosphorylation in human hepatoblastoma (HepG2) cells or mouse primary hepatocytes. FXR ligand decreased H₂O₂-induced reactive oxygen species (ROS) levels in wild-type but not FXR(-/-) mouse hepatocytes. FXR knockdown abolished the inhibition of 3-[2-[2-chloro-4-[[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl]-Benzoic acid (GW4064) on JNK phosphorylation and ROS production induced by H₂O₂in HepG2 cells. The gene expression of SOD3, an antioxidant defense enzyme, was increased by FXR activation in vitro and in vivo. An FXR-responsive element, inverted repeat separated by 1 nucleotide in SOD3 promoter, was identified by a combination of transcriptional reporter assays, EMSAs, and chromatin immunoprecipitation assays, which indicated that SOD3 could be a direct FXR target gene. SOD3 knockdown abolished the inhibition of GW4064 on JNK phosphorylation induced by H₂O₂in HepG2 cells. In summary, FXR may regulate SOD3 expression to suppress ROS production, resulting in decreasing JNK activity. These results suggest that FXR, as a novel JNK suppressor, may be an attractive therapeutic target for liver cancer treatment. PMID:25496033

  11. Homeostatic action of adenosine A3 and A1 receptor agonists on proliferation of hematopoietic precursor cells.

    PubMed

    Hofer, Michal; Pospísil, Milan; Znojil, Vladimír; Holá, Jirina; Streitová, Denisa; Vacek, Antonín

    2008-07-01

    Two adenosine receptor agonists, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and N6-cyclopentyladenosine (CPA), which selectively activate adenosine A3 and A1 receptors, respectively, were tested for their ability to influence proliferation of granulocytic and erythroid cells in femoral bone marrow of mice using morphological criteria. Agonists were given intraperitoneally to mice in repeated isomolar doses of 200 nmol/kg. Three variants of experiments were performed to investigate the action of the agonists under normal resting state of mice and in phases of cell depletion and subsequent regeneration after treatment with the cytotoxic drug 5-fluorouracil. In the case of granulopoiesis, IB-MECA 1) increased by a moderate but significant level proliferation of cells under normal resting state; 2) strongly increased proliferation of cells in the cell depletion phase; but 3) did not influence cell proliferation in the regeneration phase. CPA did not influence cell proliferation under normal resting state and in the cell depletion phase, but strongly suppressed the overshooting cell proliferation in the regeneration phase. The stimulatory effect of IB-MECA on cell proliferation of erythroid cells was observed only when this agonist was administered during the cell depletion phase. CPA did not modulate erythroid proliferation in any of the functional states investigated, probably due to the lower demand for cell production as compared with granulopoiesis. The results indicate opposite effects of the two adenosine receptor agonists on proliferation of hematopoietic cells and suggest the plasticity and homeostatic role of the adenosine receptor expression. PMID:18445770

  12. Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS) 2, 3 and 4 in Astrocyte-Like Cells

    PubMed Central

    Eusemann, Till Nicolas; Willmroth, Frank; Fiebich, Bernd; Biber, Knut; van Calker, Dietrich

    2015-01-01

    The “regulators of g-protein signalling” (RGS) comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells) and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists. PMID:26263491

  13. Adenosine A1 receptors heterodimerize with β1- and β2-adrenergic receptors creating novel receptor complexes with altered G protein coupling and signaling.

    PubMed

    Chandrasekera, P Charukeshi; Wan, Tina C; Gizewski, Elizabeth T; Auchampach, John A; Lasley, Robert D

    2013-04-01

    G protein coupled receptors play crucial roles in mediating cellular responses to external stimuli, and increasing evidence suggests that they function as multiple units comprising homo/heterodimers and hetero-oligomers. Adenosine and β-adrenergic receptors are co-expressed in numerous tissues and mediate important cellular responses to the autocoid adenosine and sympathetic stimulation, respectively. The present study was undertaken to examine whether adenosine A1ARs heterodimerize with β1- and/or β2-adrenergic receptors (β1R and β2R), and whether such interactions lead to functional consequences. Co-immunoprecipitation and co-localization studies with differentially epitope-tagged A1, β1, and β2 receptors transiently co-expressed in HEK-293 cells indicate that A1AR forms constitutive heterodimers with both β1R and β2R. This heterodimerization significantly influenced orthosteric ligand binding affinity of both β1R and β2R without altering ligand binding properties of A1AR. Receptor-mediated ERK1/2 phosphorylation significantly increased in cells expressing A1AR/β1R and A1AR/β2R heteromers. β-Receptor-mediated cAMP production was not altered in A1AR/β1R expressing cells, but was significantly reduced in the A1AR/β2R cells. The inhibitory effect of the A1AR on cAMP production was abrogated in both A1AR/β1R and A1AR/β2R expressing cells in response to the A1AR agonist CCPA. Co-immunoprecipitation studies conducted with human heart tissue lysates indicate that endogenous A1AR, β1R, and β2R also form heterodimers. Taken together, our data suggest that heterodimerization between A1 and β receptors leads to altered receptor pharmacology, functional coupling, and intracellular signaling pathways. Unique and differential receptor cross-talk between these two important receptor families may offer the opportunity to fine-tune crucial signaling responses and development of more specific therapeutic interventions. PMID:23291003

  14. A2B adenosine receptor dampens hypoxia-induced vascular leak

    PubMed Central

    Eckle, Tobias; Faigle, Marion; Grenz, Almut; Laucher, Stefanie; Thompson, Linda F.

    2008-01-01

    Extracellular adenosine has been implicated in adaptation to hypoxia and previous studies demonstrated a central role in vascular responses. Here, we examined the contribution of individual adenosine receptors (ARs: A1AR/A2AAR/A2BAR/A3AR) to vascular leak induced by hypoxia. Initial profiling studies revealed that siRNA-mediated repression of the A2BAR selectively increased endothelial leak in response to hypoxia in vitro. In parallel, vascular permeability was significantly increased in vascular organs of A2BAR−/−-mice subjected to ambient hypoxia (8% oxygen, 4 hours; eg, lung: 2.1 ± 0.12-fold increase). By contrast, hypoxia-induced vascular leak was not accentuated in A1AR−/−-, A2AAR−/−-, or A3AR−/−-deficient mice, suggesting a degree of specificity for the A2BAR. Further studies in wild type mice revealed that the selective A2BAR antagonist PSB1115 resulted in profound increases in hypoxia-associated vascular leakage while A2BAR agonist (BAY60-6583 [2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)-. phenyl]pyridin-2-ylsulfanyl]acetamide]) treatment was associated with almost complete reversal of hypoxia-induced vascular leakage (eg, lung: 2.0 ± 0.21-fold reduction). Studies in bone marrow chimeric A2BAR mice suggested a predominant role of vascular A2BARs in this response, while hypoxia-associated increases in tissue neutrophils were, at least in part, mediated by A2BAR expressing hematopoietic cells. Taken together, these studies provide pharmacologic and genetic evidence for vascular A2BAR signaling as central control point of hypoxia-associated vascular leak. PMID:18056839

  15. Emotional instability but intact spatial cognition in adenosine receptor 1 knock out mice.

    PubMed

    Lang, Undine E; Lang, Florian; Richter, Kerstin; Vallon, Volker; Lipp, Hans-Peter; Schnermann, Jürgen; Wolfer, David P

    2003-10-17

    Several lines of evidence point to the involvement of adenosine in the regulation of important central mechanisms such as cognition, arousal, aggression and anxiety. In order to elucidate the involvement of the adenosine A1 receptor (A1AR) in spatial learning and the control of exploratory behaviour, we assessed A1AR knockout mice (A1AR-/-) and their wild-type littermates (A1AR+/+) in a place navigation task in the water maze and in a battery of forced and free exploration tests. In the water maze, A1AR-/- mice showed normal escape latencies and were indistinguishable from controls with respect to measures of spatial performance during both training and probe trial. But despite normal performance they showed increased wall hugging, most prominently after the relocation of the goal platform for reversal training. Quantitative analysis of strategy choices indicated that wall hugging was increased mainly at the expense of chaining and passive floating, whereas the frequency of trials characterised as direct swims or focal searching was normal in A1AR-/- mice. These results indicate intact spatial cognition, but mildly altered emotional reactions to the water maze environment. In line with this interpretation, A1AR-/- mice showed normal levels and patterns of activity, but a mild increase of some measures of anxiety in our battery of forced and free exploration paradigms. These results are in line with findings published using a genetically similar line, but demonstrate that the magnitude of the changes and the range of affected behavioural measures may vary considerably depending on the environmental conditions during testing. PMID:14529816

  16. Signaling through the A2B Adenosine Receptor Dampens Endotoxin-Induced Acute Lung Injury

    PubMed Central

    Schingnitz, Ulrich; Hartman, Katherine; MacManus, Christopher F.; Eckle, Tobias; Zug, Stephanie; Colgan, Sean P.; Eltzschig, Holger K.

    2010-01-01

    Sepsis and septic acute lung injury are among the leading causes for morbidity and mortality of critical illness. Extracellular adenosine is a signaling molecule implicated in the cellular adaptation to hypoxia, ischemia or inflammation. Therefore, we pursued the role of the A2B adenosine receptor (A2BAR) as potential therapeutic target in endotoxin-induced acute lung injury. We gained initial insight from in vitro studies of cultured endothelia or epithelia exposed to inflammatory mediators showing time-dependent induction of the A2BAR (up to 12.9±3.4-fold, p<0.05). Similarly, murine studies of endotoxin-induced lung injury identified an almost 4.6-fold induction of A2BAR transcript and corresponding protein induction with LPS-exposure. Studies utilizing A2BAR promoter constructs and RNA-protection assays indicated that A2BAR induction involved mRNA stability. Functional studies of LPS-induced lung injury revealed that pharmacological inhibition or genetic deletion of the A2BAR was associated with dramatic increases in lung inflammation and histologic tissue injury. Studies of A2BAR-bone marrow chimeric mice suggested pulmonary A2BAR signaling in lung protection. Finally, studies with a specific A2BAR agonist (BAY 60-6583) demonstrated attenuation of lung inflammation and pulmonary edema in wild-type but not in gene-targeted mice for the A2BAR. These studies suggest the A2BAR as potential therapeutic target in the treatment of endotoxin-induced forms of acute lung injury. PMID:20348420

  17. Striatal Pre- and Postsynaptic Profile of Adenosine A2A Receptor Antagonists

    PubMed Central

    Quiroz, César; Beaumont, Vahri; Goldberg, Steven R.; Lluís, Carme; Cortés, Antoni; Franco, Rafael; Casadó, Vicent; Canela, Enric I.; Ferré, Sergi

    2011-01-01

    Striatal adenosine A2A receptors (A2ARs) are highly expressed in medium spiny neurons (MSNs) of the indirect efferent pathway, where they heteromerize with dopamine D2 receptors (D2Rs). A2ARs are also localized presynaptically in cortico-striatal glutamatergic terminals contacting MSNs of the direct efferent pathway, where they heteromerize with adenosine A1 receptors (A1Rs). It has been hypothesized that postsynaptic A2AR antagonists should be useful in Parkinson's disease, while presynaptic A2AR antagonists could be beneficial in dyskinetic disorders, such as Huntington's disease, obsessive-compulsive disorders and drug addiction. The aim or this work was to determine whether selective A2AR antagonists may be subdivided according to a preferential pre- versus postsynaptic mechanism of action. The potency at blocking the motor output and striatal glutamate release induced by cortical electrical stimulation and the potency at inducing locomotor activation were used as in vivo measures of pre- and postsynaptic activities, respectively. SCH-442416 and KW-6002 showed a significant preferential pre- and postsynaptic profile, respectively, while the other tested compounds (MSX-2, SCH-420814, ZM-241385 and SCH-58261) showed no clear preference. Radioligand-binding experiments were performed in cells expressing A2AR-D2R and A1R-A2AR heteromers to determine possible differences in the affinity of these compounds for different A2AR heteromers. Heteromerization played a key role in the presynaptic profile of SCH-442416, since it bound with much less affinity to A2AR when co-expressed with D2R than with A1R. KW-6002 showed the best relative affinity for A2AR co-expressed with D2R than co-expressed with A1R, which can at least partially explain the postsynaptic profile of this compound. Also, the in vitro pharmacological profile of MSX-2, SCH-420814, ZM-241385 and SCH-58261 was is in accordance with their mixed pre- and postsynaptic profile. On the basis of their preferential

  18. Computational study of the binding modes of caffeine to the adenosine A2A receptor.

    PubMed

    Liu, Yuli; Burger, Steven K; Ayers, Paul W; Vöhringer-Martinez, Esteban

    2011-12-01

    Using the recently solved crystal structure of the human adenosine A(2A) receptor, we applied MM/PBSA to compare the binding modes of caffeine with those of the high-affinity selective antagonist ZM241385. MD simulations were performed in the environment of the lipid membrane bilayer. Four low-energy binding modes of caffeine-A(2A) were found, all of which had similar energies. Assuming an equal contribution of each binding mode of caffeine, the computed binding free energy difference between caffeine and ZM241385 is -2.4 kcal/mol, which compares favorably with the experimental value, -3.6 kcal/mol. The configurational entropy contribution of -0.9 kcal/mol from multiple binding modes of caffeine helps explain how a small molecule like caffeine can compete with a significantly larger molecule, ZM241385, which can form many more interactions with the receptor. We also performed residue-wise energy decomposition and found that Phe168, Leu249, and Ile274 contribute most significantly to the binding modes of caffeine and ZM241385. PMID:21970461

  19. A2A adenosine receptor deletion is protective in a mouse model of Tauopathy.

    PubMed

    Laurent, C; Burnouf, S; Ferry, B; Batalha, V L; Coelho, J E; Baqi, Y; Malik, E; Mariciniak, E; Parrot, S; Van der Jeugd, A; Faivre, E; Flaten, V; Ledent, C; D'Hooge, R; Sergeant, N; Hamdane, M; Humez, S; Müller, C E; Lopes, L V; Buée, L; Blum, D

    2016-01-01

    Consumption of caffeine, a non-selective adenosine A2A receptor (A2AR) antagonist, reduces the risk of developing Alzheimer's disease (AD) in humans and mitigates both amyloid and Tau burden in transgenic mouse models. However, the impact of selective A2AR blockade on the progressive development of AD-related lesions and associated memory impairments has not been investigated. In the present study, we removed the gene encoding A2AR from THY-Tau22 mice and analysed the subsequent effects on both pathological (Tau phosphorylation and aggregation, neuro-inflammation) and functional impairments (spatial learning and memory, hippocampal plasticity, neurotransmitter profile). We found that deleting A2ARs protect from Tau pathology-induced deficits in terms of spatial memory and hippocampal long-term depression. These effects were concomitant with a normalization of the hippocampal glutamate/gamma-amino butyric acid ratio, together with a global reduction in neuro-inflammatory markers and a decrease in Tau hyperphosphorylation. Additionally, oral therapy using a specific A2AR antagonist (MSX-3) significantly improved memory and reduced Tau hyperphosphorylation in THY-Tau22 mice. By showing that A2AR genetic or pharmacological blockade improves the pathological phenotype in a Tau transgenic mouse model, the present data highlight A2A receptors as important molecular targets to consider against AD and Tauopathies. PMID:25450226

  20. Adenosine A1 receptors mediate the intracisternal injection of orexin-induced antinociceptive action against colonic distension in conscious rats.

    PubMed

    Okumura, Toshikatsu; Nozu, Tsukasa; Kumei, Shima; Takakusaki, Kaoru; Miyagishi, Saori; Ohhira, Masumi

    2016-03-15

    We have recently demonstrated that orexin acts centrally through the brain orexin 1 receptors to induce an antinociceptive action against colonic distension in conscious rats. Adenosine signaling is capable of inducing an antinociceptive action against somatic pain; however, the association between changes in the adenosinergic system and visceral pain perception has not been investigated. In the present study, we hypothesized that the adenosinergic system may be involved in visceral nociception, and thus, adenosine signaling may mediate orexin-induced visceral antinociception. Visceral sensation was evaluated based on the colonic distension-induced abdominal withdrawal reflex (AWR) in conscious rats. Subcutaneous (0.04-0.2mg/rat) or intracisternal (0.8-4μg/rat) injection of N(6)-cyclopentyladenosine (CPA), an adenosine A1 receptor (A1R) agonist, increased the threshold volume of colonic distension-induced AWR in a dose-dependent manner, thereby suggesting that CPA acts centrally in the brain to induce an antinociceptive action against colonic distension. Pretreatment with theophylline, an adenosine antagonist, or 1,3-dipropyl-8-cyclopentylxanthine, an A1R antagonist, subcutaneously injected potently blocked the centrally injected CPA- or orexin-A-induced antinociceptive action against colonic distension. These results suggest that adenosinergic signaling via A1Rs in the brain induces visceral antinociception and that adenosinergic signaling is involved in the central orexin-induced antinociceptive action against colonic distension. PMID:26944127

  1. Anti-nociceptive properties of the xanthine oxidase inhibitor allopurinol in mice: role of A1 adenosine receptors

    PubMed Central

    Schmidt, AP; Böhmer, AE; Antunes, C; Schallenberger, C; Porciúncula, LO; Elisabetsky, E; Lara, DR; Souza, DO

    2009-01-01

    Background and purpose Allopurinol is a potent inhibitor of the enzyme xanthine oxidase, used primarily in the treatment of hyperuricemia and gout. It is well known that purines exert multiple effects on pain transmission. We hypothesized that the inhibition of xanthine oxidase by allopurinol, thereby reducing purine degradation, could be a valid strategy to enhance purinergic activity. The aim of this study was to investigate the anti-nociceptive profile of allopurinol on chemical and thermal pain models in mice. Experimental approach Mice received an intraperitoneal (i.p.) injection of vehicle (Tween 10%) or allopurinol (10–400 mg kg−1). Anti-nociceptive effects were measured with intraplantar capsaicin, intraplantar glutamate, tail-flick or hot-plate tests. Key results Allopurinol presented dose-dependent anti-nociceptive effects in all models. The opioid antagonist naloxone did not affect these anti-nociceptive effects. The non-selective adenosine-receptor antagonist caffeine and the selective A1 adenosine-receptor antagonist, DPCPX, but not the selective A2A adenosine-receptor antagonist, SCH58261, completely prevented allopurinol-induced anti-nociception. No obvious motor deficits were produced by allopurinol, at doses up to 200 mg kg−1. Allopurinol also caused an increase in cerebrospinal fluid levels of purines, including the nucleosides adenosine and guanosine, and decreased cerebrospinal fluid concentration of uric acid. Conclusions and implications Allopurinol-induced anti-nociception may be related to adenosine accumulation. Allopurinol is an old and extensively used compound and seems to be well tolerated with no obvious central nervous system toxic effects at high doses. This drug may be useful to treat pain syndromes in humans. PMID:19133997

  2. Antagonism of Nav channels and α1-adrenergic receptors contributes to vascular smooth muscle effects of ranolazine.

    PubMed

    Virsolvy, Anne; Farah, Charlotte; Pertuit, Nolwenn; Kong, Lingyan; Lacampagne, Alain; Reboul, Cyril; Aimond, Franck; Richard, Sylvain

    2015-01-01

    Ranolazine is a recently developed drug used for the treatment of patients with chronic stable angina. It is a selective inhibitor of the persistent cardiac Na(+) current (INa), and is known to reduce the Na(+)-dependent Ca(2+) overload that occurs in cardiomyocytes during ischemia. Vascular effects of ranolazine, such as vasorelaxation,have been reported and may involve multiple pathways. As voltage-gated Na(+) channels (Nav) present in arteries play a role in contraction, we hypothesized that ranolazine could target these channels. We studied the effects of ranolazine in vitro on cultured aortic smooth muscle cells (SMC) and ex vivo on rat aortas in conditions known to specifically activate or promote INa. We observed that in the presence of the Nav channel agonist veratridine, ranolazine inhibited INa and intracellular Ca(2+) calcium increase in SMC, and arterial vasoconstriction. In arterial SMC, ranolazine inhibited the activity of tetrodotoxin-sensitive voltage-gated Nav channels and thus antagonized contraction promoted by low KCl depolarization. Furthermore, the vasorelaxant effects of ranolazine, also observed in human arteries and independent of the endothelium, involved antagonization of the α1-adrenergic receptor. Combined α1-adrenergic antagonization and inhibition of SMCs Nav channels could be involved in the vascular effects of ranolazine. PMID:26655634

  3. Antagonism of Nav channels and α1-adrenergic receptors contributes to vascular smooth muscle effects of ranolazine

    PubMed Central

    Virsolvy, Anne; Farah, Charlotte; Pertuit, Nolwenn; Kong, Lingyan; Lacampagne, Alain; Reboul, Cyril; Aimond, Franck; Richard, Sylvain

    2015-01-01

    Ranolazine is a recently developed drug used for the treatment of patients with chronic stable angina. It is a selective inhibitor of the persistent cardiac Na+ current (INa), and is known to reduce the Na+-dependent Ca2+ overload that occurs in cardiomyocytes during ischemia. Vascular effects of ranolazine, such as vasorelaxation,have been reported and may involve multiple pathways. As voltage-gated Na+ channels (Nav) present in arteries play a role in contraction, we hypothesized that ranolazine could target these channels. We studied the effects of ranolazine in vitro on cultured aortic smooth muscle cells (SMC) and ex vivo on rat aortas in conditions known to specifically activate or promote INa. We observed that in the presence of the Nav channel agonist veratridine, ranolazine inhibited INa and intracellular Ca2+ calcium increase in SMC, and arterial vasoconstriction. In arterial SMC, ranolazine inhibited the activity of tetrodotoxin-sensitive voltage-gated Nav channels and thus antagonized contraction promoted by low KCl depolarization. Furthermore, the vasorelaxant effects of ranolazine, also observed in human arteries and independent of the endothelium, involved antagonization of the α1-adrenergic receptor. Combined α1-adrenergic antagonization and inhibition of SMCs Nav channels could be involved in the vascular effects of ranolazine. PMID:26655634

  4. Adenosine A(1), A(2a), A(2b), and A(3) receptors in hematopoiesis. 1. Expression of receptor mRNA in four mouse hematopoietic precursor cells.

    PubMed

    Streitová, D; Sefc, L; Savvulidi, F; Pospísil, M; Holá, J; Hofer, M

    2010-01-01

    Four mouse bone marrow or thymus cell populations, namely granulopoietic/monocytopoietic, erythropoietic, B-lymphopoietic, and T-lymphopoietic precursor cells have been assayed by RT-PCR technique for the presence and relative amounts of adenosine A(1), A(2a), A(2b), and A(3) receptor mRNA. It has been found that (i) all four populations studied express all four adenosine receptor subtypes, (ii) the A(1), receptor is the least expressed in all populations studied, (iii) the A(3) receptor is markedly expressed in the populations of granulopoietic/monocytopoietic and erythropoietic cells, (iv) the A(2a) receptor is markedly expressed in the populations of B-lymphopoietic and T-lymphopoietic cells, and v) the A(2b) receptor does not predominate in any of the precursor cells studied. Our data offer a new possibility for the assessment of the readiness of these cells to respond, by receptor-mediated mechanisms, to adenosine or its analogs present in the tissues as a result of endogenous processes and/or following their administration. PMID:19249907

  5. Insulin Restores Gestational Diabetes Mellitus–Reduced Adenosine Transport Involving Differential Expression of Insulin Receptor Isoforms in Human Umbilical Vein Endothelium

    PubMed Central

    Westermeier, Francisco; Salomón, Carlos; González, Marcelo; Puebla, Carlos; Guzmán-Gutiérrez, Enrique; Cifuentes, Fredi; Leiva, Andrea; Casanello, Paola; Sobrevia, Luis

    2011-01-01

    OBJECTIVE To determine whether insulin reverses gestational diabetes mellitus (GDM)–reduced expression and activity of human equilibrative nucleoside transporters 1 (hENT1) in human umbilical vein endothelium cells (HUVECs). RESEARCH DESIGN AND METHODS Primary cultured HUVECs from full-term normal (n = 44) and diet-treated GDM (n = 44) pregnancies were used. Insulin effect was assayed on hENT1 expression (protein, mRNA, SLC29A1 promoter activity) and activity (initial rates of adenosine transport) as well as endothelial nitric oxide (NO) synthase activity (serine1177 phosphorylation, l-citrulline formation). Adenosine concentration in culture medium and umbilical vein blood (high-performance liquid chromatography) as well as insulin receptor A and B expression (quantitative PCR) were determined. Reactivity of umbilical vein rings to adenosine and insulin was assayed by wire myography. Experiments were in the absence or presence of l-NG-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) or ZM-241385 (an A2A-adenosine receptor antagonist). RESULTS Umbilical vein blood adenosine concentration was higher, and the adenosine- and insulin-induced NO/endothelium-dependent umbilical vein relaxation was lower in GDM. Cells from GDM exhibited increased insulin receptor A isoform expression in addition to the reported NO–dependent inhibition of hENT1-adenosine transport and SLC29A1 reporter repression, and increased extracellular concentration of adenosine and NO synthase activity. Insulin reversed all these parameters to values in normal pregnancies, an effect blocked by ZM-241385 and l-NAME. CONCLUSIONS GDM and normal pregnancy HUVEC phenotypes are differentially responsive to insulin, a phenomenon where insulin acts as protecting factor for endothelial dysfunction characteristic of this syndrome. Abnormal adenosine plasma levels, and potentially A2A-adenosine receptors and insulin receptor A, will play crucial roles in this phenomenon in GDM. PMID:21515851

  6. The Cross-Species A3 Adenosine-Receptor Antagonist MRS 1292 Inhibits Adenosine-Triggered Human Nonpigmented Ciliary Epithelial Cell Fluid Release and Reduces Mouse Intraocular Pressure

    PubMed Central

    Yang, Hui; Avila, Marcel Y.; Peterson-Yantorno, Kim; Coca-Prados, Miguel; Stone, Richard A.; Jacobson, Kenneth A.; Civan, Mortimer M.

    2012-01-01

    Purpose Antagonists to A3 adenosine receptors (ARs) lower mouse intraocular pressure (IOP), but extension to humans is limited by species variability. We tested whether the specific A3AR antagonist MRS 1292, designed to cross species, mimicks the effects of other A3AR antagonists on cultured human nonpigmented ciliary epithelial (NPE) cells and mouse IOP. Methods NPE cell volume was monitored by electronic cell sorting. Mouse IOP was measured with the Servo-Null Micropipette System. Results Adenosine triggered A3AR-mediated shrinkage of human NPE cells. Shrinkage was blocked by MRS 1292 (IC50 = 42 ± 11 nM, p < 0.01) and by another A3AR antagonist effective in this system, MRS 1191. Topical application of the A3AR agonist IB-MECA increased mouse IOP. MRS 1292 reduced IOP by 4.0 ± 0.8 mmHg at 25-μM droplet concentration (n = 10, p < 0.005). Conclusions MRS 1292 inhibits A3AR-mediated shrinkage of human NPE cells and reduces mouse IOP, consistent with its putative action as a cross-species A3 antagonist. PMID:16146920

  7. Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling

    PubMed Central

    Mundell, SJ; Matharu, A-L; Nisar, S; Palmer, TM; Benovic, JL; Kelly, E

    2010-01-01

    Background and purpose: We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A2B adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5′-(N-ethylcarboxamido)-adenosine. Experimental approach: The trafficking of the wild type A2B adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy. Key results: The wild type A2B adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln325-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu330-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln325-stop, Ser326-stop and Phe328-stop receptors. Following internalization, the wild type A2B adenosine receptor recycled rapidly to the cell surface, whereas the Gln325-stop receptor did not recycle. Conclusions and implications: Deletion of the COOH-terminus of the A2B adenosine receptor beyond Leu330 switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A2B adenosine receptor following prolonged agonist addition. PMID:20128803

  8. Influence of metabotropic glutamate receptor agonists on the inhibitory effects of adenosine A1 receptor activation in the rat hippocampus.

    PubMed

    de Mendonça, A; Ribeiro, J A

    1997-08-01

    1. Glutamate and other amino acids are the main excitatory neurotransmitters in many brain regions, including the hippocampus, by activating ion channel-coupled glutamate receptors, as well as metabotropic receptors linked to G proteins and second messenger systems. Several conditions which promote the release of glutamate, like frequency stimulation and hypoxia, also lead to an increase in the extracellular levels of the important neuromodulator, adenosine. We studied whether the activation of different subgroups of metabotropic glutamate receptors (mGluR) could modify the known inhibitory effects of a selective adenosine A1 receptor agonist on synaptic transmission in the hippocampus. The experiments were performed on hippocampal slices taken from young (12-14 days old) rats. Stimulation was delivered to the Schaffer collateral/commissural fibres, and evoked field excitatory postsynaptic potentials (fe. p.s.p.) recorded extracellularly from the stratum radiatum in the CAI area. 2. The concentration-response curve for the inhibitory effects of the selective adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA; 2-50 nM), on the fe.p.s.p. slope (EC50 = 12.5 (9.2-17.3; 95% confidence intervals)) was displaced to the right by the group I mGluR selective agonist, (R,S)-3,5-dihydroxyphenylglycine (DPHG; 10 microM) (EC50 = 27.2 (21.4-34.5) nM, n = 4). The attenuation of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope by DHPG (10 microM) was blocked in the presence of the mGluR antagonist (which blocks group I and II mGluR), (R,S)-alpha-methyl-4-carboxyphenylglycine (MCPG; 500 microM). DHPG (10 microM) itself had an inhibitory effect of 20.1 +/- 1.9% (n = 4) on the fe.p.s.p. slope. 3. The concentration-response curves for the inhibitory effects of CPA (2-20 nM) on the fe.p.s.p. slope were not modified either in the presence of the group II mGluR selective agonist, (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I; 1 microM), or in the presence of

  9. Characterization of the binding of a novel nonxanthine adenosine antagonist radioligand, ( sup 3 H)CGS 15943, to multiple affinity states of the adenosine A1 receptor in the rat cortex

    SciTech Connect

    Jarvis, M.F.; Williams, M.; Do, U.H.; Sills, M.A. )

    1991-01-01

    The triazoloquinazoline CGS 15943 is the first reported nonxanthine adenosine antagonist that has high affinity for brain adenosine receptors. In the present study, the binding of (3H) CGS 15943 to recognition sites in rat cortical membranes was characterized. Saturation experiments revealed that (3H)CGS 15943 labeled a single class of recognition sites with high affinity and limited capacity. Competition studies revealed that the binding of (3H)CGS 15943 was consistent with the labeling of brain adenosine A1 receptors. Adenosine agonists inhibited 1 nM (3H)CGS 15943 binding with the following order of activity N6-cyclopentyladenosine (IC50 = 15 nM) greater than 2-chloroadenosine greater than (R)-N6-phenylisopropyladenosine greater than 5'-N6-ethylcarboxamidoadenosine greater than (S)N6-phenylisopropyladenosine greater than CGS 21680 greater than CV 1808 (IC50 greater than 10,000 nM). The potency order for adenosine antagonists was CGS 15943 (IC50 = 5 nM) greater than 8-phenyltheophylline greater than 1,3-dipropyl-8-(4-amino-2-chloro)phenylxanthine greater than 1,3-diethyl-8-phenylxanthine greater than theophylline = caffeine (IC50 greater than 10,000 nM). Antagonist inhibition curves were steep and best described by a one-site binding model. In contrast, adenosine A1 agonist competition curves were shallow, as indicated by Hill coefficients less than unity. Computer analysis revealed that these inhibition curves were best described by a two-site binding model. Agonist competition curves generated in the presence of 1 mM GTP resulted in a rightward shift and steepening of the inhibition-concentration curves, whereas antagonist binding was not altered in the presence of GTP. The complex binding interactions found with adenosine agonists indicate that (3H)CGS 15943 labels both high and low affinity components of the adenosine A1 receptor in the rat cortex.

  10. Key Modulatory Role of Presynaptic Adenosine A2A Receptors in Cortical Neurotransmission to the Striatal Direct Pathway

    PubMed Central

    Quiroz, César; Luján, Rafael; Uchigashima, Motokazu; Simoes, Ana Patrícia; Lerner, Talia N.; Borycz, Janusz; Kachroo, Anil; Canas, Paula M.; Orru, Marco; Schwarzschild, Michael A.; Rosin, Diane L.; Kreitzer, Anatol C.; Cunha, Rodrigo A.; Watanabe, Masahiko; Ferré, Sergi

    2010-01-01

    Basal ganglia processing results from a balanced activation of direct and indirect striatal efferent pathways, which are controlled by dopamine D1 and D2 receptors, respectively. Adenosine A2A receptors are considered novel anti-parkinsonian targets, based on their selective postsynaptic localization in the indirect pathway, where they modulate D2 receptor function. The present study provides evidence for the existence of an additional functionally significant segregation of A2A receptors at the presynaptic level. Using integrated anatomical, electrophysiological and biochemical approaches, we demonstrate that presynaptic A2A receptors are preferentially localized in cortical glutamatergic terminals that contact striatal neurons of the direct pathway, where they exert a selective modulation of cortico-striatal neurotransmission. Presynaptic striatal A2A receptors could provide a new target for the treatment of neuropsychiatric disorders. PMID:19936569

  11. Inflammatory Lung Injury After Cardiopulmonary Bypass is Attenuated by Adenosine A2A Receptor Activation

    PubMed Central

    Lisle, Turner C; Gazoni, Leo M; Fernandez, Lucas G; Sharma, Ashish K; Bellizzi, Andrew M; Schifflett, Grant D; Laubach, Victor E; Kron, Irving L

    2008-01-01

    Objectives Cardiopulmonary bypass has been shown to exert an inflammatory response within the lung, often resulting in postoperative pulmonary dysfunction. Several studies have shown that adenosine A2A receptor (A2AR) activation attenuates lung ischemia-reperfusion injury, however the effect of A2AR activation on cardiopulmonary bypass-induced lung injury has not been studied. We hypothesized that specific A2AR activation by ATL313 would attenuate inflammatory lung injury following cardiopulmonary bypass. Methods Adult male Sprague-Dawley rats were randomly divided into three groups: 1) SHAM group (underwent cannulation+heparinization only); 2) CONTROL group (underwent 90-minutes of normothermic cardiopulmonary bypass with normal whole-blood priming solution; 3) ATL group (underwent 90-minutes of normothermic cardiopulmonary bypass with ATL313 added to the normal priming solution). Results There was significantly less pulmonary edema and lung injury in the ATL group compared to the CONTROL group. The ATL group had significant reductions in bronchoalveolar lavage interleukin-1, interleukin-6, interferon-γ and myeloperoxidase levels compared to the CONTROL group. Similarly, lung tissue interleukin-6, tumor necrosis factor-α, and interferon-γ were significantly decreased in the ATL group compared to the CONTROL group. There was no significant difference between the SHAM and ATL groups in the amount of pulmonary edema, lung injury, or levels of pro-inflammatory cytokines. Conclusions The addition of a potent A2AR agonist to the normal priming solution prior to the initiation of CPB significantly protects the lung from the inflammatory effects of CPB and reduces the amount of lung injury. A2AR agonists could represent a new therapeutic strategy for reducing the potentially devastating consequences of the inflammatory response associated with CPB. Ultra-mini Abstract Pharmacologic activation of the adenosine A2A receptor during cardiopulmonary bypass resulted in

  12. Allosteric mechanisms within the adenosine A2A-dopamine D2 receptor heterotetramer.

    PubMed

    Ferré, Sergi; Bonaventura, Jordi; Tomasi, Dardo; Navarro, Gemma; Moreno, Estefanía; Cortés, Antonio; Lluís, Carme; Casadó, Vicent; Volkow, Nora D

    2016-05-01

    The structure constituted by a G protein coupled receptor (GPCR) homodimer and a G protein provides a main functional unit and oligomeric entities can be viewed as multiples of dimers. For GPCR heteromers, experimental evidence supports a tetrameric structure, comprised of two different homodimers, each able to signal with its preferred G protein. GPCR homomers and heteromers can act as the conduit of allosteric interactions between orthosteric ligands. The well-known agonist/agonist allosteric interaction in the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer, by which A2AR agonists decrease the affinity of D2R agonists, gave the first rationale for the use of A2AR antagonists in Parkinson's disease. We review new pharmacological findings that can be explained in the frame of a tetrameric structure of the A2AR-D2R heteromer: first, ligand-independent allosteric modulations by the D2R that result in changes of the binding properties of A2AR ligands; second, differential modulation of the intrinsic efficacy of D2R ligands for G protein-dependent and independent signaling; third, the canonical antagonistic Gs-Gi interaction within the frame of the heteromer; and fourth, the ability of A2AR antagonists, including caffeine, to also exert the same allosteric modulations of D2R ligands than A2AR agonists, while A2AR agonists and antagonists counteract each other's effects. These findings can have important clinical implications when evaluating the use of A2AR antagonists. They also call for the need of monitoring caffeine intake when evaluating the effect of D2R ligands, when used as therapeutic agents in neuropsychiatric disorders or as probes in imaging studies. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'. PMID:26051403

  13. Adenosine A2A receptor-mediated control of pilocarpine-induced tremulous jaw movements is Parkinson's disease-associated GPR37 receptor-dependent.

    PubMed

    Gandía, Jorge; Morató, Xavier; Stagljar, Igor; Fernández-Dueñas, Víctor; Ciruela, Francisco

    2015-07-15

    GPR37, also known as parkin associated endothelin-like receptor (Pael-R), is an orphan GPCR that aggregates intracellularly in a juvenile form of Parkinson's disease. However, little is known about the function of this orphan receptor. Here, using a model for parkisonian tremor, the pilocarpine-induced tremulous jaw movements (TJMs), we show that the deletion of GPR37 attenuated the TJMs in response to this cholinomimetic. Interestingly, the control that adenosine A2A receptor exerted over TJMs was lost in the absence of GPR37, thus pointing to a pivotal role of this orphan receptor in the adenosinergic control of parkinsonian tremor. PMID:25862943

  14. Adenosine receptor activation is responsible for prolonged depression of synaptic transmission after spreading depolarization in brain slices.

    PubMed

    Lindquist, B E; Shuttleworth, C W

    2012-10-25

    Spreading depolarization (SD) is a slowly propagating, coordinated depolarization of brain tissue, which is followed by a transient (5-10min) depression of synaptic activity. The mechanisms for synaptic depression after SD are incompletely understood. We examined the relative contributions of action potential failure and adenosine receptor activation to the suppression of evoked synaptic activity in murine brain slices. Focal micro-injection of potassium chloride (KCl) was used to induce SD and synaptic potentials were evoked by electrical stimulation of Schaffer collateral inputs to hippocampal area Cornu Ammonis area 1 (CA1). SD was accompanied by loss of both presynaptic action potentials (as assessed from fiber volleys) and field excitatory postsynaptic potentials (fEPSPs). Fiber volleys recovered rapidly upon neutralization of the extracellular direct current (DC) potential, whereas fEPSPs underwent a secondary suppression phase lasting several minutes. Paired-pulse ratio was elevated during the secondary suppression period, consistent with a presynaptic mechanism of synaptic depression. A transient increase in extracellular adenosine concentration was detected during the period of secondary suppression. Antagonists of adenosine A1 receptors (8-cyclopentyl-1,3-dipropylxanthine [DPCPX] or 8-cyclopentyl-1,3-dimethylxanthine [8-CPT]) greatly accelerated fEPSP recovery and abolished increases in paired-pulse ratio normally observed after SD. The duration of fEPSP suppression was correlated with both the duration of the DC shift and the area of tissue depolarized, consistent with the model that adenosine accumulates in proportion to the metabolic burden of SD. These results suggest that in brain slices, the duration of the DC shift approximately defined the period of action potential failure, but the secondary depression of evoked responses was in large part due to endogenous adenosine accumulation after SD. PMID:22864185

  15. Study of an adenosine A1 receptor agonist on trigeminally evoked dural blood vessel dilation in the anaesthetized rat.

    PubMed

    Honey, A C; Bland-Ward, P A; Connor, H E; Feniuk, W; Humphrey, P P A

    2002-05-01

    The purpose of this study was to use intravital microscopy to determine the effect of a selective adenosine A1 receptor agonist, GR79236 (1, 3 and 10 microg/kg i.v.), on neurogenic dural blood vessel dilation in anaesthetized rats. Vasodilation was evoked either by electrical stimulation of perivascular trigeminal nerves or by intravenous CGRP. GR79236 (1-10 microg/kg i.v.) caused a dose-dependent inhibition of neurogenic vasodilation, but had no significant effect on dural vasodilation caused by CGRP. GR79236 (1-3 microg/kg i.v.) had no effect on basal dural vessel diameter, but caused transient dose-dependant bradycardia and hypotension. Bradycardia was more prolonged following 10 microg/kg i.v. GR79236. Pre-treatment with the adenosine A1 receptor antagonist DPCPX (1 mg/kg i.v.) prevented the inhibitory effect of GR79236 (10 microg/kg i.v.) on neurogenic vasodilation as well as GR79236-induced bradycardia and hypotension. These data suggest that the inhibition of neurogenic vasodilation by GR79236 is mediated via the activation of prejunctional adenosine A1 receptors. Provided the systemic cardiovascular effects could be limited, such a mechanism may offer a novel approach to migraine therapy. PMID:12100087

  16. Cardiovascular protection and antioxidant activity of the extracts from the mycelia of Cordyceps sinensis act partially via adenosine receptors.

    PubMed

    Yan, Xiao-Feng; Zhang, Zhong-Miao; Yao, Hong-Yi; Guan, Yan; Zhu, Jian-Ping; Zhang, Lin-Hui; Jia, Yong-Liang; Wang, Ru-Wei

    2013-11-01

    Mycelia of cultured Cordyceps sinensis (CS) is one of the most common substitutes for natural CS and was approved for arrhythmia in China. However, the role of CS in ameliorating injury during ischemia-reperfusion (I/R) is still unclear. We examined effects of extracts from CS on I/R and investigated the possible mechanisms. Post-ischemic coronary perfusion pressure, ventricular function, and coronary flow were measured using the Langendorff mouse heart model. Oxidative stress of cardiac homogenates was performed using an ELISA. Our results indicate that CS affords cardioprotection possibly through enhanced adenosine receptor activation. Cardioprotection was demonstrated by reduced post-ischemic diastolic dysfunction and improved recovery of pressure development and coronary flow. Treatment with CS largely abrogates oxidative stress and damage in glucose- or pyruvate-perfused hearts. Importantly, observed reductions in oxidative stress [glutathione disulfide (GSSG)]/[GSSG + glutathione] and [malondialdehyde (MDA)]/[superoxide dismutase + MDA] ratios as well as the resultant damage upon CS treatment correlate with functional markers of post-ischemic myocardial outcome. These effects of CS were partially blocked by 8-ρ-sulfophenyltheophylline, an adenosine receptor antagonist. Our results demonstrate a suppressive role of CS in ischemic contracture. Meanwhile, the results also suggest pre-ischemic adenosine receptor activation may be involved in reducing contracture in hearts pretreated with CS. PMID:23192916

  17. Influence of metabotropic glutamate receptor agonists on the inhibitory effects of adenosine A1 receptor activation in the rat hippocampus

    PubMed Central

    de Mendonça, Alexandre; Ribeiro, J A

    1997-01-01

    Glutamate and other amino acids are the main excitatory neurotransmitters in many brain regions, including the hippocampus, by activating ion channel-coupled glutamate receptors, as well as metabotropic receptors linked to G proteins and second messenger systems. Several conditions which promote the release of glutamate, like frequency stimulation and hypoxia, also lead to an increase in the extracellular levels of the important neuromodulator, adenosine. We studied whether the activation of different subgroups of metabotropic glutamate receptors (mGluR) could modify the known inhibitory effects of a selective adenosine A1 receptor agonist on synaptic transmission in the hippocampus. The experiments were performed on hippocampal slices taken from young (12–14 days old) rats. Stimulation was delivered to the Schaffer collateral/commissural fibres, and evoked field excitatory postsynaptic potentials (fe.p.s.p.) recorded extracellularly from the stratum radiatum in the CA1 area. The concentration-response curve for the inhibitory effects of the selective adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA; 2–50 nM), on the fe.p.s.p. slope (EC50=12.5 (9.2–17.3; 95% confidence intervals)) was displaced to the right by the group I mGluR selective agonist, (R,S)-3,5-dihydroxyphenylglycine (DPHG; 10 μM) (EC50=27.2 (21.4–34.5) nM, n=4). The attenuation of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope by DHPG (10 μM) was blocked in the presence of the mGluR antagonist (which blocks group I and II mGluR), (R,S)-α-methyl-4-carboxyphenylglycine (MCPG; 500 μM). DHPG (10 μM) itself had an inhibitory effect of 20.1±1.9% (n=4) on the fe.p.s.p. slope. The concentration-response curves for the inhibitory effects of CPA (2–20 nM) on the fe.p.s.p. slope were not modified either in the presence of the group II mGluR selective agonist, (2S,3S,4S)-α-(carboxycyclopropyl)glycine (L-CCG-I; 1 μM), or in the presence of the non

  18. Increased desensitization of dopamine D₂ receptor-mediated response in the ventral tegmental area in the absence of adenosine A(2A) receptors.

    PubMed

    Al-Hasani, R; Foster, J D; Metaxas, A; Ledent, C; Hourani, S M O; Kitchen, I; Chen, Y

    2011-09-01

    G-protein coupled receptors interact to provide additional regulatory mechanisms for neurotransmitter signaling. Adenosine A(2A) receptors are expressed at a high density in striatal neurons, where they closely interact with dopamine D₂ receptors and modulate effects of dopamine and responses to psychostimulants. A(2A) receptors are expressed at much lower densities in other forebrain neurons but play a more prominent yet opposing role to striatal receptors in response to psychostimulants in mice. It is, therefore, possible that A(2A) receptors expressed at low levels elsewhere in the brain may also regulate neurotransmitter systems and modulate neuronal functions. Dopamine D₂ receptors play an important role in autoinhibition of neuronal firing in dopamine neurons of the ventral tegmental area (VTA) and dopamine release in other brain areas. Here, we examined the effect of A(2A) receptor deletion on D₂ receptor-mediated inhibition of neuronal firing in dopamine neurons in the VTA. Spontaneous activity of dopamine neurons was recorded in midbrain slices, and concentration-dependent effects of the dopamine D₂ receptor agonist, quinpirole, was compared between wild-type and A(2A) knockout mice. The potency of quinpirole applied in single concentrations and the expression of D₂ receptors were not altered in the VTA of the knockout mice. However, quinpirole applied in stepwise escalating concentrations caused significantly reduced maximal inhibition in A(2A) knockout mice, indicating an enhanced agonist-induced desensitization of D₂ receptors in the absence of A(2A) receptors. The A(2A) receptor agonist, CGS21680, did not exert any effect on dopamine neuron firing or response to quinpirole, revealing a novel non-pharmacological interaction between adenosine A(2A) receptors and dopaminergic neurotransmission in midbrain dopamine neurons. Altered D₂ receptor desensitization may result in changes in dopamine neuron firing rate and pattern and dopamine

  19. Adenosine-Activated Nanochannels Inspired by G-Protein-Coupled Receptors.

    PubMed

    Li, Pei; Kong, Xiang-Yu; Xie, Ganhua; Xiao, Kai; Zhang, Zhen; Wen, Liping; Jiang, Lei

    2016-04-01

    A bioinspired adenosine activated nanodevice is demonstrated in which the conformations of the designed aptamer change and cause signal transmission according to the emergence of adenosine. This bioinspired system exhibits very high response ratios (activated/nonactivated ratio up to 614) and excellent stability and reversibility, and shows promising applications in the fields of biosensors, pharmaceutica, and healthcare systems. PMID:26915491

  20. A2A adenosine receptor regulates the human blood brain barrier permeability

    PubMed Central

    Kim, Do-Geun; Bynoe, Margaret S.

    2015-01-01

    The blood brain barrier (BBB) symbolically represents the gateway to the central nervous system. It is a single layer of specialized endothelial cells that coats the central nervous system (CNS) vasculature and physically separates the brain environment from the blood constituents, to maintain the homeostasis of the CNS. However, this protective measure is a hindrance to the delivery of therapeutics to treat neurological diseases. Here, we show that activation of A2A adenosine receptor (AR) with an FDA-approved agonist potently permeabilizes an in vitro primary human brain endothelial barrier (hBBB) to the passage of chemotherapeutic drugs and T cells. T cell migration under AR signaling occurs primarily by paracellular transendothelial route. Permeabilization of the hBBB is rapid, time-dependent and reversible and is mediated by morphological changes in actin-cytoskeletal reorganization induced by RhoA signaling and a potent down-regulation of Claudin-5 and VE-Cadherin. Moreover, the kinetics of BBB permeability in mice closely overlaps with the permeability kinetics of the hBBB. These data suggest that activation of A2A AR is an endogenous mechanism that may be used for CNS drug delivery in human. PMID:25262373

  1. A1 adenosine receptor deficiency or inhibition reduces atherosclerotic lesions in apolipoprotein E deficient mice

    PubMed Central

    Teng, Bunyen; Smith, Jonathan D.; Rosenfeld, Michael E.; Robinet, Peggy; Davis, Mary E.; Morrison, R. Ray; Mustafa, S. Jamal

    2014-01-01

    Aims The goal of this study was to determine whether the A1 adenosine receptor (AR) plays a role in atherosclerosis development and to explore its potential mechanisms. Methods and results Double knockout (DKO) mice, deficient in the genes encoding A1 AR and apolipoprotein E (apoE), demonstrated reduced atherosclerotic lesions in aortic arch (en face), aortic root, and innominate arteries when compared with apoE-deficient mice (APOE-KO) of the same age. Treating APOE-KO with an A1 AR antagonist (DPCPX) also led to a concentration-dependent reduction in lesions. The total plasma cholesterol and triglyceride levels were not different between DKO and APOE-KO; however, higher triglyceride was observed in DKO fed a high-fat diet. DKO also had higher body weights than APOE-KO. Plasma cytokine concentrations (IL-5, IL-6, and IL-13) were significantly lower in DKO. Proliferating cell nuclear antigen expression was also significantly reduced in the aorta from DKO. Despite smaller lesions in DKO, the composition of the innominate artery lesion and cholesterol loading and efflux from bone marrow-derived macrophages of DKO were not different from APOE-KO. Conclusion The A1 AR may play a role in the development of atherosclerosis, possibly due to its pro-inflammatory and mitogenic properties. PMID:24525840

  2. Structure-Activity Analysis of Biased Agonism at the Human Adenosine A3 Receptor

    PubMed Central

    Baltos, Jo-Anne; Paoletta, Silvia; Nguyen, Anh T. N.; Gregory, Karen J.; Tosh, Dilip K.; Christopoulos, Arthur; Jacobson, Kenneth A.

    2016-01-01

    Biased agonism at G protein–coupled receptors (GPCRs) has significant implications for current drug discovery, but molecular determinants that govern ligand bias remain largely unknown. The adenosine A3 GPCR (A3AR) is a potential therapeutic target for various conditions, including cancer, inflammation, and ischemia, but for which biased agonism remains largely unexplored. We now report the generation of bias “fingerprints” for prototypical ribose containing A3AR agonists and rigidified (N)-methanocarba 5′-N-methyluronamide nucleoside derivatives with regard to their ability to mediate different signaling pathways. Relative to the reference prototypical agonist IB-MECA, (N)-methanocarba 5′-N-methyluronamide nucleoside derivatives with significant N6 or C2 modifications, including elongated aryl-ethynyl groups, exhibited biased agonism. Significant positive correlation was observed between the C2 substituent length (in Å) and bias toward cell survival. Molecular modeling suggests that extended C2 substituents on (N)-methanocarba 5′-N-methyluronamide nucleosides promote a progressive outward shift of the A3AR transmembrane domain 2, which may contribute to the subset of A3AR conformations stabilized on biased agonist binding. PMID:27136943

  3. Structure-Activity Analysis of Biased Agonism at the Human Adenosine A3 Receptor.

    PubMed

    Baltos, Jo-Anne; Paoletta, Silvia; Nguyen, Anh T N; Gregory, Karen J; Tosh, Dilip K; Christopoulos, Arthur; Jacobson, Kenneth A; May, Lauren T

    2016-07-01

    Biased agonism at G protein-coupled receptors (GPCRs) has significant implications for current drug discovery, but molecular determinants that govern ligand bias remain largely unknown. The adenosine A3 GPCR (A3AR) is a potential therapeutic target for various conditions, including cancer, inflammation, and ischemia, but for which biased agonism remains largely unexplored. We now report the generation of bias "fingerprints" for prototypical ribose containing A3AR agonists and rigidified (N)-methanocarba 5'-N-methyluronamide nucleoside derivatives with regard to their ability to mediate different signaling pathways. Relative to the reference prototypical agonist IB-MECA, (N)-methanocarba 5'-N-methyluronamide nucleoside derivatives with significant N(6) or C2 modifications, including elongated aryl-ethynyl groups, exhibited biased agonism. Significant positive correlation was observed between the C2 substituent length (in Å) and bias toward cell survival. Molecular modeling suggests that extended C2 substituents on (N)-methanocarba 5'-N-methyluronamide nucleosides promote a progressive outward shift of the A3AR transmembrane domain 2, which may contribute to the subset of A3AR conformations stabilized on biased agonist binding. PMID:27136943

  4. A2B adenosine receptor activity is reduced in neutrophils from patients with systemic sclerosis

    PubMed Central

    Bazzichi, Laura; Trincavelli, Letizia; Rossi, Alessandra; De Feo, Francesca; Lucacchini, Antonio; Bombardieri, Stefano; Martini, Claudia

    2005-01-01

    We conducted the present study to investigate protein expression and functioning of A2A and A2B adenosine receptors (ARs) in neutrophils of patients affected by systemic sclerosis (SSc). The presence of A2A and A2B ARs was assessed by immunoblotting using specific antibodies. Equilibrium A2A and A2B ARs binding parameters were evaluated by radioligand binding assay. Functional studies were conducted to investigate coupling of the A2B AR to the adenylyl cyclase pathway. This is the first report of the use of Western blot analysis to confirm the presence of A2A and A2B ARs in human neutrophils. No significant changes in A2A AR binding parameters or expression levels were detected between SSc patients and healthy control individuals. A significant decrease (65%) in the maximum density of A2B AR binding sites occurred in SSc neutrophils, whereas no changes in the affinity constant values were found. Moreover, a decrease in A2B AR mediated adenylyl cyclase activity was observed in patients with SSc. Our findings demonstrate the occurrence of selective alterations in A2B AR density and signalling in SSc. PMID:15743465

  5. The A2B adenosine receptor modulates pulmonary hypertension associated with interstitial lung disease.

    PubMed

    Karmouty-Quintana, Harry; Zhong, Hongyan; Acero, Luis; Weng, Tingting; Melicoff, Ernestina; West, James D; Hemnes, Anna; Grenz, Almut; Eltzschig, Holger K; Blackwell, Timothy S; Xia, Yang; Johnston, Richard A; Zeng, Dewan; Belardinelli, Luiz; Blackburn, Michael R

    2012-06-01

    Development of pulmonary hypertension is a common and deadly complication of interstitial lung disease. Little is known regarding the cellular and molecular mechanisms that lead to pulmonary hypertension in patients with interstitial lung disease, and effective treatment options are lacking. The purpose of this study was to examine the adenosine 2B receptor (A(2B)R) as a regulator of vascular remodeling and pulmonary hypertension secondary to pulmonary fibrosis. To accomplish this, cellular and molecular changes in vascular remodeling were monitored in mice exposed to bleomycin in conjunction with genetic removal of the A(2B)R or treatment with the A(2B)R antagonist GS-6201. Results demonstrated that GS-6201 treatment or genetic removal of the A(2B)R attenuated vascular remodeling and hypertension in our model. Furthermore, direct A(2B)R activation on vascular cells promoted interleukin-6 and endothelin-1 release. These studies identify a novel mechanism of disease progression to pulmonary hypertension and support the development of A(2B)R antagonists for the treatment of pulmonary hypertension secondary to interstitial lung disease. PMID:22415303

  6. IFN-γ Prevents Adenosine Receptor (A2bR) Upregulation To Sustain the Macrophage Activation Response.

    PubMed

    Cohen, Heather B; Ward, Amanda; Hamidzadeh, Kajal; Ravid, Katya; Mosser, David M

    2015-10-15

    The priming of macrophages with IFN-γ prior to TLR stimulation results in enhanced and prolonged inflammatory cytokine production. In this study, we demonstrate that, following TLR stimulation, macrophages upregulate the adenosine 2b receptor (A2bR) to enhance their sensitivity to immunosuppressive extracellular adenosine. This upregulation of A2bR leads to the induction of macrophages with an immunoregulatory phenotype and the downregulation of inflammation. IFN-γ priming of macrophages selectively prevents the induction of the A2bR in macrophages to mitigate sensitivity to adenosine and to prevent this regulatory transition. IFN-γ-mediated A2bR blockade leads to a prolonged production of TNF-α and IL-12 in response to TLR ligation. The pharmacologic inhibition or the genetic deletion of the A2bR results in a hyperinflammatory response to TLR ligation, similar to IFN-γ treatment of macrophages. Conversely, the overexpression of A2bR on macrophages blunts the IFN-γ effects and promotes the development of immunoregulatory macrophages. Thus, we propose a novel mechanism whereby IFN-γ contributes to host defense by desensitizing macrophages to the immunoregulatory effects of adenosine. This mechanism overcomes the transient nature of TLR activation, and prolongs the antimicrobial state of the classically activated macrophage. This study may offer promising new targets to improve the clinical outcome of inflammatory diseases in which macrophage activation is dysregulated. PMID:26355158

  7. Sedation and histamine H1-receptor antagonism: studies in man with the enantiomers of chlorpheniramine and dimethindene.

    PubMed Central

    Nicholson, A. N.; Pascoe, P. A.; Turner, C.; Ganellin, C. R.; Greengrass, P. M.; Casy, A. F.; Mercer, A. D.

    1991-01-01

    1. The effects of 10 mg (+)- and (-)-chlorpheniramine and 5 mg (+)- and (-)-dimethindene on daytime sleep latencies, digit symbol substitution and subjective assessments of mood and well-being were studied in 6 healthy young adult humans. Each subject also took 5 mg triprolidine hydrochloride as an active control and two placebos. 2. Daytime sleep latencies were reduced with triprolidine, (+)-chlorpheniramine and (-)-dimethindene, and subjects also reported that they felt more sleepy after (+)-chlorpheniramine and (-)-dimethindene. Performance on digit symbol substitution was impaired with (+)-chlorpheniramine. 3. Changes in measures with (-)-chlorpheniramine and (+)-dimethindene were not different from changes with placebo. 4. In the present study, changes in measures of drowsiness and performance were limited to the enantiomers with high affinity for the histamine H1-receptor. These findings strongly suggest that sedation can arise from H1-receptor antagonism alone, and provide further support for the belief that the histaminergic system is concerned with the regulation of alertness in man. PMID:1686208

  8. The Role of cGMP on Adenosine A1 Receptor-mediated Inhibition of Synaptic Transmission at the Hippocampus

    PubMed Central

    Pinto, Isa; Serpa, André; Sebastião, Ana M.; Cascalheira, José F.

    2016-01-01

    Both adenosine A1 receptor and cGMP inhibit synaptic transmission at the hippocampus and recently it was found that A1 receptor increased cGMP levels in hippocampus, but the role of cGMP on A1 receptor-mediated inhibition of synaptic transmission remains to be established. In the present work we investigated if blocking the NOS/sGC/cGMP/PKG pathway using nitric oxide synthase (NOS), protein kinase G (PKG), and soluble guanylyl cyclase (sGC) inhibitors modify the A1 receptor effect on synaptic transmission. Neurotransmission was evaluated by measuring the slope of field excitatory postsynaptic potentials (fEPSPs) evoked by electrical stimulation at hippocampal slices. N6-cyclopentyladenosine (CPA, 15 nM), a selective A1 receptor agonist, reversibly decreased the fEPSPs by 54 ± 5%. Incubation of the slices with an inhibitor of NOS (L-NAME, 200 μM) decreased the CPA effect on fEPSPs by 57 ± 9% in female rats. In males, ODQ (10 μM), an sGC inhibitor, decreased the CPA inhibitory effect on fEPSPs by 23 ± 6%, but only when adenosine deaminase (ADA,1 U/ml) was present; similar results were found in females, where ODQ decreased CPA-induced inhibition of fEPSP slope by 23 ± 7%. In male rats, the presence of the PKG inhibitor (KT5823, 1 nM) decreased the CPA effect by 45.0 ± 9%; similar results were obtained in females, where KT5823 caused a 32 ± 9% decrease on the CPA effect. In conclusion, the results suggest that the inhibitory action of adenosine A1 receptors on synaptic transmission at hippocampus is, in part, mediated by the NOS/sGC/cGMP/PKG pathway. PMID:27148059

  9. Adenosine Receptor Stimulation by Polydeoxyribonucleotide Improves Tissue Repair and Symptomology in Experimental Colitis.

    PubMed

    Pallio, Giovanni; Bitto, Alessandra; Pizzino, Gabriele; Galfo, Federica; Irrera, Natasha; Squadrito, Francesco; Squadrito, Giovanni; Pallio, Socrate; Anastasi, Giuseppe P; Cutroneo, Giuseppina; Macrì, Antonio; Altavilla, Domenica

    2016-01-01

    Activation of the adenosine receptor pathway has been demonstrated to be effective in improving tissue remodeling and blunting the inflammatory response. Active colitis is characterized by an intense inflammatory reaction resulting in extensive tissue damage. Symptomatic improvement requires both control of the inflammatory process and repair and remodeling of damaged tissues. We investigated the ability of an A2A receptor agonist, polydeoxyribonucleotide (PDRN), to restore tissue structural integrity in two experimental colitis models using male Sprague-Dawley rats. In the first model, colitis was induced with a single intra-colonic instillation of dinitrobenzenesulfonic acid (DNBS), 25 mg diluted in 0.8 ml 50% ethanol. After 6 h, animals were randomized to receive either PDRN (8 mg/kg/i.p.), or PDRN + the A2A antagonist [3,7-dimethyl-1-propargylxanthine (DMPX); 10 mg/kg/i.p.], or vehicle (0.8 ml saline solution) daily. In the second model, dextran sulfate sodium (DSS) was dissolved in drinking water at a concentration of 8%. Control animals received standard drinking water. After 24 h animals were randomized to receive PDRN or PDRN+DMPX as described above. Rats were sacrificed 7 days after receiving DNBS or 5 days after DSS. In both experimental models of colitis, PDRN ameliorated the clinical symptoms and weight loss associated with disease as well as promoted the histological repair of damaged tissues. Moreover, PDRN reduced expression of inflammatory cytokines, myeloperoxidase activity, and malondialdehyde. All these effects were abolished by the concomitant administration of the A2A antagonist DMPX. Our study suggests that PDRN may represent a promising treatment for improving tissue repair during inflammatory bowel diseases. PMID:27601997

  10. Adenosine Receptor Stimulation by Polydeoxyribonucleotide Improves Tissue Repair and Symptomology in Experimental Colitis

    PubMed Central

    Pallio, Giovanni; Bitto, Alessandra; Pizzino, Gabriele; Galfo, Federica; Irrera, Natasha; Squadrito, Francesco; Squadrito, Giovanni; Pallio, Socrate; Anastasi, Giuseppe P.; Cutroneo, Giuseppina; Macrì, Antonio; Altavilla, Domenica

    2016-01-01

    Activation of the adenosine receptor pathway has been demonstrated to be effective in improving tissue remodeling and blunting the inflammatory response. Active colitis is characterized by an intense inflammatory reaction resulting in extensive tissue damage. Symptomatic improvement requires both control of the inflammatory process and repair and remodeling of damaged tissues. We investigated the ability of an A2A receptor agonist, polydeoxyribonucleotide (PDRN), to restore tissue structural integrity in two experimental colitis models using male Sprague-Dawley rats. In the first model, colitis was induced with a single intra-colonic instillation of dinitrobenzenesulfonic acid (DNBS), 25 mg diluted in 0.8 ml 50% ethanol. After 6 h, animals were randomized to receive either PDRN (8 mg/kg/i.p.), or PDRN + the A2A antagonist [3,7-dimethyl-1-propargylxanthine (DMPX); 10 mg/kg/i.p.], or vehicle (0.8 ml saline solution) daily. In the second model, dextran sulfate sodium (DSS) was dissolved in drinking water at a concentration of 8%. Control animals received standard drinking water. After 24 h animals were randomized to receive PDRN or PDRN+DMPX as described above. Rats were sacrificed 7 days after receiving DNBS or 5 days after DSS. In both experimental models of colitis, PDRN ameliorated the clinical symptoms and weight loss associated with disease as well as promoted the histological repair of damaged tissues. Moreover, PDRN reduced expression of inflammatory cytokines, myeloperoxidase activity, and malondialdehyde. All these effects were abolished by the concomitant administration of the A2A antagonist DMPX. Our study suggests that PDRN may represent a promising treatment for improving tissue repair during inflammatory bowel diseases. PMID:27601997

  11. Mercaptoacetate blocks fatty acid-induced GLP-1 secretion in male rats by directly antagonizing GPR40 fatty acid receptors.

    PubMed

    Li, Ai-Jun; Wang, Qing; Dinh, Thu T; Simasko, Steve M; Ritter, Sue

    2016-04-15

    Mercaptoacetate (MA) is an orexigenic agent reported to block fatty acid (FA) oxidation. Recently, however, we reported evidence from isolated nodose ganglion neurons that MA antagonizes the G protein-coupled long- and medium-chain FA receptor GPR40. GPR40 mediates FA-induced secretion of the satietogenic incretin peptide glucagon-like peptide 1 (GLP-1), by enteroendocrine L cells, as well as FA-induced enhancement of glucose-stimulated insulin secretion. Our results in cultured nodose neurons suggest that MA would also block GPR40 in enteroendocrine cells controlling GLP-1 secretion. If so, this would suggest an alternative mechanism by which MA increases food intake. We tested the hypothesis that MA blocks FA-induced GLP-1 secretion in vitro using cultured STC-1 cells (a murine enteroendocrine cell line) and in vivo in adult male rats. In vitro, MA blocked the increase in both cytosolic Ca(2+)and GLP-1 release stimulated by FAs and also reduced (but less effectively) the response of STC-1 cells to grifolic acid, a partial agonist of the GPR120 FA receptor. In vivo, MA reduced GLP-1 secretion following olive oil gavage while also increasing glucose and decreasing insulin levels. The carnitine palmatoyltransferase 1 antagonist etomoxir did not alter these responses. Results indicate that MA's actions, including its orexigenic effect, are mediated by GPR40 (and possibly GPR120) receptor antagonism and not by blockade of fat oxidation, as previously believed. Analysis of MA's interaction with GPR40 may facilitate understanding of the multiple functions of this receptor and the manner in which FAs participate in the control of hunger and satiety. PMID:26791830

  12. New functional activity of aripiprazole revealed: Robust antagonism of D2 dopamine receptor-stimulated Gβγ signaling.

    PubMed

    Brust, Tarsis F; Hayes, Michael P; Roman, David L; Watts, Val J

    2015-01-01

    The dopamine D2 receptor (DRD2) is a G protein-coupled receptor (GPCR) that is generally considered to be a primary target in the treatment of schizophrenia. First generation antipsychotic drugs (e.g. haloperidol) are antagonists of the DRD2, while second generation antipsychotic drugs (e.g. olanzapine) antagonize DRD2 and 5HT2A receptors. Notably, both these classes of drugs may cause side effects associated with D2 receptor antagonism (e.g. hyperprolactemia and extrapyramidal symptoms). The novel, "third generation" antipsychotic drug, aripiprazole is also used to treat schizophrenia, with the remarkable advantage that its tendency to cause extrapyramidal symptoms is minimal. Aripiprazole is considered a partial agonist of the DRD2, but it also has partial agonist/antagonist activity for other GPCRs. Further, aripiprazole has been reported to have a unique activity profile in functional assays with the DRD2. In the present study the molecular pharmacology of aripiprazole was further examined in HEK cell models stably expressing the DRD2 and specific isoforms of adenylyl cyclase to assess functional responses of Gα and Gβγ subunits. Additional studies examined the activity of aripiprazole in DRD2-mediated heterologous sensitization of adenylyl cyclase and cell-based dynamic mass redistribution (DMR). Aripiprazole displayed a unique functional profile for modulation of G proteins, being a partial agonist for Gαi/o and a robust antagonist for Gβγ signaling. Additionally, aripiprazole was a weak partial agonist for both heterologous sensitization and dynamic mass redistribution. PMID:25449598

  13. Treatment with Adenosine Receptor Agonist Ameliorates Pain Induced by Acute and Chronic Inflammation.

    PubMed

    Montes, Guilherme Carneiro; Hammes, Nathalia; da Rocha, Miguel Divino; Montagnoli, Tadeu Lima; Fraga, Carlos Alberto Manssour; Barreiro, Eliezer J; Sudo, Roberto Takashi; Zapata-Sudo, Gisele

    2016-08-01

    Rheumatoid arthritis is an inflammatory autoimmune condition, and tumor necrosis factor-α (TNF-α) plays an important role in its pathophysiology. In vitro, (E)-N'-(3,4-dimethoxybenzylidene)-N-methylbenzohydrazide (LASSBio-1359) has exhibited anti-TNF-α properties, and in vivo these effects are mediated via activation of adenosine receptor. This work investigates the antinociceptive action of LASSBio-1359 in murine models of acute and chronic inflammatory pain. Male mice received an intraperitoneal injection of LASSBio-1359 and then were evaluated in formalin- and carrageenan-induced paw edema assays. Complete Freund's adjuvant (CFA) was used to induce a mouse model of monoarthritis. These mice were treated with LASSBio-1359 by oral gavage to evaluate thermal and mechanical hyperalgesia. TNF-α and inducible nitric oxide synthase (iNOS) expression as well as histologic features were analyzed. The time of reactivity to formalin in the neurogenic phase was reduced from 56.3 ± 6.0 seconds to 32.7 ± 2.2 seconds and 23.8 ± 2.6 seconds after treatment with LASSBio-1359 at doses of 10 mg/kg and 20 mg/kg, respectively. A reversal of the antinociceptive action of LASSBio-1359 was observed in the inflammatory phase after treatment with ZM 241385 [4-(2-[7-amino-2-(2-furly)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol], an adenosine A2A antagonist. Carrageenan-induced thermal and mechanical hyperalgesia were reduced after treatment with LASSBio-1359. Similarly, CFA-induced thermal and mechanical hyperalgesia were reduced after treatment with LASSBio-1359 (25 and 50 mg/kg). Levels of TNF-α and iNOS expression increased in the monoarthritis model and were normalized in animals treated with LASSBio-1359, which was also associated with beneficial effects in the histologic analysis. These results suggest that LASSBio-1359 represents an alternative treatment of monoarthritis. PMID:27194479

  14. Mass spectrometry-based ligand binding assays on adenosine A1 and A2A receptors.

    PubMed

    Massink, A; Holzheimer, M; Hölscher, A; Louvel, J; Guo, D; Spijksma, G; Hankemeier, T; IJzerman, A P

    2015-12-01

    Conventional methods to measure ligand-receptor binding parameters typically require radiolabeled ligands as probes. Despite the robustness of radioligand binding assays, they carry inherent disadvantages in terms of safety precautions, expensive synthesis, special lab requirements, and waste disposal. Mass spectrometry (MS) is a method that can selectively detect ligands without the need of a label. The sensitivity of MS equipment increases progressively, and currently, it is possible to detect low ligand quantities that are usually found in ligand binding assays. We developed a label-free MS ligand binding (MS binding) assay on the adenosine A(1) and A(2A) receptors (A(1)AR and A(2A)AR), which are well-characterized members of the class A G protein-coupled receptor (GPCR) family. Radioligand binding assays for both receptors are well established, and ample data is available to compare and evaluate the performance of an MS binding assay. 1,3-Dipropyl-8-cyclopentyl-xanthine (DPCPX) and 4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol (ZM-241,385) are high-affinity ligands selective for the A(1)AR and A(2A)AR, respectively. To proof the feasibility of MS binding on the A(1)AR and A(2A)AR, we first developed an MS detection method for unlabeled DPCPX and ZM-241,385. To serve as internal standards, both compounds were also deuterium-labeled. Subsequently, we investigated whether the two unlabeled compounds could substitute for their radiolabeled counterparts as marker ligands in binding experiments, including saturation, displacement, dissociation, and competition association assays. Furthermore, we investigated the accuracy of these assays if the use of internal standards was excluded. The results demonstrate the feasibility of the MS binding assay, even in the absence of a deuterium-labeled internal standard, and provide great promise for the further development of label-free assays based on MS for other GPCRs. PMID

  15. Adenosine A(1), A(2a), A(2b), and A(3) receptors in hematopoiesis. 2. Expression of receptor mRNA in resting and lipopolysaccharide-activated mouse RAW 264.7 macrophages.

    PubMed

    Streitová, D; Hofer, M; Holá, J; Vacek, A; Pospísil, M

    2010-01-01

    Expression of mRNA for adenosine receptor subtypes A(1), A(2a), A(2b), and A(3) in normal and lipopolysaccharide (LPS)-activated murine RAW 264.7 macrophages has been investigated using the method of quantitative real-time polymerase chain reaction. The results have shown a very low, unquantifiable expression of adenosine A(1) receptor mRNA in both normal and LPS-activated macrophages. The other three adenosine receptor mRNAs have been found to be expressed at various but always quantifiable levels. Activation of the macrophages by LPS induced upregulation of the expression of adenosine receptor A(2a) and A(2b) mRNA, whereas the expression of adenosine receptor A(3) mRNA was downregulated. Unstimulated macrophages exhibited a high expression of the A(2b) adenosine receptor mRNA. The findings are discussed from the point of view of the antiinflammatory and hematopoiesis-stimulating roles of the adenosine receptor signaling. PMID:19249906

  16. NK1 receptor activation in rat rostral ventrolateral medulla selectively attenuates somato-sympathetic reflex while antagonism attenuates sympathetic chemoreflex.

    PubMed

    Makeham, John M; Goodchild, Ann K; Pilowsky, Paul M

    2005-06-01

    The effects of activation and blockade of the neurokinin 1 (NK1) receptor in the rostral ventrolateral medulla (RVLM) on arterial blood pressure (ABP), splanchnic sympathetic nerve activity (sSNA), phrenic nerve activity, the somato-sympathetic reflex, baroreflex, and chemoreflex were studied in urethane-anesthetized and artificially ventilated Sprague-Dawley rats. Bilateral microinjection of either the stable substance P analog (pGlu5, MePhe8, Sar9)SP(5-11) (DiMe-SP) or the highly selective NK1 agonist [Sar9, Met (O(2))11]SP into the RVLM resulted in an increase in ABP, sSNA, and heart rate and an abolition of phrenic nerve activity. The effects of [Sar9, Met (O(2))11]SP were blocked by the selective nonpeptide NK1 receptor antagonist WIN 51708. NK1 receptor activation also dramatically attenuated the somato-sympathetic reflex elicited by tibial nerve stimulation, while leaving the baroreflex and chemoreflex unaffected. This effect was again blocked by WIN 51708. NK1 receptor antagonism in the RVLM, with WIN 51708 significantly attenuated the sympathoexcitatory response to hypoxia but had no effect on baseline respiratory function. Our findings suggest that substance P and the NK1 receptor play a significant role in the cardiorespiratory reflexes integrated within the RVLM. PMID:15731401

  17. Genetic blockade of adenosine A2A receptors induces cognitive impairments and anatomical changes related to psychotic symptoms in mice.

    PubMed

    Moscoso-Castro, Maria; Gracia-Rubio, Irene; Ciruela, Francisco; Valverde, Olga

    2016-07-01

    Schizophrenia is a chronic severe mental disorder with a presumed neurodevelopmental origin, and no effective treatment. Schizophrenia is a multifactorial disease with genetic, environmental and neurochemical etiology. The main theories on the pathophysiology of this disorder include alterations in dopaminergic and glutamatergic neurotransmission in limbic and cortical areas of the brain. Early hypotheses also suggested that nucleoside adenosine is a putative affected neurotransmitter system, and clinical evidence suggests that adenosine adjuvants improve treatment outcomes, especially in poorly responsive patients. Hence, it is important to elucidate the role of the neuromodulator adenosine in the pathophysiology of schizophrenia. A2A adenosine receptor (A2AR) subtypes are expressed in brain areas controlling motivational responses and cognition, including striatum, and in lower levels in hippocampus and cerebral cortex. The aim of this study was to characterize A2AR knockout (KO) mice with complete and specific inactivation of A2AR, as an animal model for schizophrenia. We performed behavioral, anatomical and neurochemical studies to assess psychotic-like symptoms in adult male and female KO and wild-type (WT) littermates. Our results show impairments in inhibitory responses and sensory gating in A2AR KO animals. Hyperlocomotion induced by d-amphetamine and MK-801 was reduced in KO animals when compared to WT littermates. Moreover, A2AR KO animals show motor disturbances, social and cognitive alterations. Finally, behavioral impairments were associated with enlargement of brain lateral ventricles and decreased BDNF levels in the hippocampus. These data highlight the role of adenosine in the pathophysiology of schizophrenia and provide new possibilities for the therapeutic management of schizophrenia. PMID:27133030

  18. Controlling the Dissociation of Ligands from the Adenosine A2A Receptor through Modulation of Salt Bridge Strength.

    PubMed

    Segala, Elena; Guo, Dong; Cheng, Robert K Y; Bortolato, Andrea; Deflorian, Francesca; Doré, Andrew S; Errey, James C; Heitman, Laura H; IJzerman, Adriaan P; Marshall, Fiona H; Cooke, Robert M

    2016-07-14

    The association and dissociation kinetics of ligands binding to proteins vary considerably, but the mechanisms behind this variability are poorly understood, limiting their utilization for drug discovery. This is particularly so for G protein-coupled receptors (GPCRs) where high resolution structural information is only beginning to emerge. Engineering the human A2A adenosine receptor has allowed structures to be solved in complex with the reference compound ZM241385 and four related ligands at high resolution. Differences between the structures are limited, with the most pronounced being the interaction of each ligand with a salt bridge on the extracellular side of the receptor. Mutagenesis experiments confirm the role of this salt bridge in controlling the dissociation kinetics of the ligands from the receptor, while molecular dynamics simulations demonstrate the ability of ligands to modulate salt bridge stability. These results shed light on a structural determinant of ligand dissociation kinetics and identify a means by which this property may be optimized. PMID:27312113

  19. Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein-2

    PubMed Central

    Mediero, Aránzazu; Wilder, Tuere; Perez-Aso, Miguel; Cronstein, Bruce N.

    2015-01-01

    Promoting bone regeneration and repair of bone defects is a need that has not been well met to date. We have previously found that adenosine, acting via A2A receptors (A2AR) promotes wound healing and inhibits inflammatory osteolysis and hypothesized that A2AR might be a novel target to promote bone regeneration. Therefore, we determined whether direct A2AR stimulation or increasing endogenous adenosine concentrations via purine transport blockade with dipyridamole regulates bone formation. We determined whether coverage of a 3 mm trephine defect in a mouse skull with a collagen scaffold soaked in saline, bone morphogenetic protein-2 (BMP-2; 200 ng), 1 μM CGS21680 (A2AR agonist, EC50 = 160 nM), or 1 μM dipyridamole (EC50 = 32 nM) promoted bone regeneration. Microcomputed tomography examination demonstrated that CGS21680 and dipyridamole markedly enhanced bone regeneration as well as BMP-2 8 wk after surgery (60 ± 2%, 79 ± 2%, and 75 ± 1% bone regeneration, respectively, vs. 32 ± 2% in control, P < 0.001). Blockade by a selective A2AR antagonist (ZM241385, 1 μM) or deletion of A2AR abrogated the effect of CGS21680 and dipyridamole on bone regeneration. Both CGS21680 and dipyridamole treatment increased alkaline phosphatase-positive osteoblasts and diminished tartrate resistance acid phosphatase-positive osteoclasts in the defects. In vivo imaging with a fluorescent dye for new bone formation revealed a strong fluorescent signal in treated animals that was equivalent to BMP-2. In conclusion, stimulation of A2AR by specific agonists or by increasing endogenous adenosine levels stimulates new bone formation as well as BMP-2 and represents a novel approach to stimulating bone regeneration.—Mediero, A., Wilder, T., Perez-Aso, M., Cronstein, B. N. Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein-2. PMID:25573752

  20. Design, Synthesis, and Biological Evaluation of Novel Nonsteroidal Farnesoid X Receptor (FXR) Antagonists: Molecular Basis of FXR Antagonism.

    PubMed

    Huang, Huang; Si, Pei; Wang, Lei; Xu, Yong; Xu, Xin; Zhu, Jin; Jiang, Hualiang; Li, Weihua; Chen, Lili; Li, Jian

    2015-07-01

    Farnesoid X receptor (FXR) plays an important role in the regulation of cholesterol, lipid, and glucose metabolism. Recently, several studies on the molecular basis of FXR antagonism have been reported. However, none of these studies employs an FXR antagonist with nonsteroidal scaffold. On the basis of our previously reported FXR antagonist with a trisubstituted isoxazole scaffold, a novel nonsteroidal FXR ligand was designed and used as a lead for structural modification. In total, 39 new trisubstituted isoxazole derivatives were designed and synthesized, which led to pharmacological profiles ranging from agonist to antagonist toward FXR. Notably, compound 5s (4'-[(3-{[3-(2-chlorophenyl)-5-(2-thienyl)isoxazol-4-yl]methoxy}-1H-pyrazol-1-yl)methyl]biphenyl-2-carboxylic acid), containing a thienyl-substituted isoxazole ring, displayed the best antagonistic activity against FXR with good cellular potency (IC50 =12.2 ± 0.2 μM). Eventually, this compound was used as a probe in a molecular dynamics simulation assay. Our results allowed us to propose an essential molecular basis for FXR antagonism, which is consistent with a previously reported antagonistic mechanism; furthermore, E467 on H12 was found to be a hot-spot residue and may be important for the future design of nonsteroidal antagonists of FXR. PMID:25982493

  1. Modulation of dopamine-mediated facilitation at the neuromuscular junction of Wistar rats: A role for adenosine A1/A2A receptors and P2 purinoceptors.

    PubMed

    Elnozahi, Neveen A; AlQot, Hadir E; Mohy El-Din, Mahmoud M; Bistawroos, Azza E; Abou Zeit-Har, Mohamed S

    2016-06-21

    This study aims to understand how dopamine and the neuromodulators, adenosine and adenosine triphosphate (ATP) modulate neuromuscular transmission. Adenosine and ATP are well-recognized for their regulatory effects on dopamine in the central nervous system. However, if similar interactions occur at the neuromuscular junction is unknown. We hypothesize that the activation of adenosine A1/A2A and/or P2 purinoceptors may influence the action of dopamine on neuromuscular transmission. Using the rat phrenic nerve hemi-diaphragm, we assessed the influence of dopamine, adenosine and ATP on the height of nerve-evoked muscle twitches. We investigated how the selective blockade of adenosine A1 receptors (2.5nM DPCPX), adenosine A2A receptors (50nM CSC) and P2 purinoceptors (100μM suramin) modified the effects of dopamine. Dopamine alone increased indirect muscle contractions while adenosine and ATP either enhanced or depressed nerve-evoked muscle twitches in a concentration-dependent manner. The facilitatory effects of 256μM dopamine were significantly reduced to 29.62±2.79% or 53.69±5.45% in the presence of DPCPX or CSC, respectively, relative to 70.03±1.57% with dopamine alone. Alternatively, the action of 256μM dopamine was potentiated from 70.03±1.57, in the absence of suramin, to 86.83±4.36%, in the presence of suramin. It can be concluded that the activation of adenosine A1 and A2A receptors and P2 purinoceptors potentially play a central role in the regulation of dopamine effects at the neuromuscular junction. Clinically this study offers new insights for the indirect manipulation of neuromuscular transmission for the treatment of disorders characterized by motor dysfunction. PMID:27060487

  2. 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

  3. Multi-Inhibitory Effects of A2A Adenosine Receptor Signaling on Neutrophil Adhesion Under Flow.

    PubMed

    Yago, Tadayuki; Tsukamoto, Hiroki; Liu, Zhenghui; Wang, Ying; Thompson, Linda F; McEver, Rodger P

    2015-10-15

    A2A adenosine receptor (A2AAR) signaling negatively regulates inflammatory responses in many disease models, but the detailed mechanisms remain unclear. We used the selective A2AAR agonist, ATL313, to examine how A2AAR signaling affects human and murine neutrophil adhesion under flow. Treating neutrophils with ATL313 inhibited selectin-induced, β2 integrin-dependent slow rolling and chemokine-induced, β2 integrin-dependent arrest on ICAM-1. ATL313 inhibited selectin-induced β2 integrin extension, which supports slow rolling, and chemokine-induced hybrid domain "swing-out," which supports arrest. Furthermore, ATL313 inhibited integrin outside-in signaling as revealed by reduced neutrophil superoxide production and spreading on immobilized anti-β2 integrin Ab. ATL313 suppressed selectin-triggered activation of Src family kinases (SFKs) and p38 MAPK, chemokine-triggered activation of Ras-related protein 1, and β2 integrin-triggered activation of SFKs and Vav cytoskeletal regulatory proteins. ATL313 activated protein kinase A and its substrate C-terminal Src kinase, an inhibitor of SFKs. Treating neutrophils with a protein kinase A inhibitor blocked the actions of ATL313. In vivo, ATL313-treated neutrophils rolled faster and arrested much less frequently in postcapillary venules of the murine cremaster muscle after TNF-α challenge. Furthermore, ATL313 markedly suppressed neutrophil migration into the peritoneum challenged with thioglycollate. ATL313 did not affect A2AAR-deficient neutrophils, confirming its specificity. Our findings provide new insights into the anti-inflammatory mechanisms of A2AAR signaling and the potential utility of A2AAR agonists in inflammatory diseases. PMID:26355151

  4. Molecular mechanism of allosteric modulation at GPCRs: insight from a binding kinetics study at the human A1 adenosine receptor

    PubMed Central

    Guo, Dong; Venhorst, Suzanne N; Massink, Arnault; van Veldhoven, Jacobus P D; Vauquelin, Georges; IJzerman, Adriaan P; Heitman, Laura H

    2014-01-01

    Background and Purpose Many GPCRs can be allosterically modulated by small-molecule ligands. This modulation is best understood in terms of the kinetics of the ligand–receptor interaction. However, many current kinetic assays require at least the (radio)labelling of the orthosteric ligand, which is impractical for studying a range of ligands. Here, we describe the application of a so-called competition association assay at the adenosine A1 receptor for this purpose. Experimental Approach We used a competition association assay to examine the binding kinetics of several unlabelled orthosteric agonists of the A1 receptor in the absence or presence of two allosteric modulators. We also tested three bitopic ligands, in which an orthosteric and an allosteric pharmacophore were covalently linked with different spacer lengths. The relevance of the competition association assay for the binding kinetics of the bitopic ligands was also explored by analysing simulated data. Key Results The binding kinetics of an unlabelled orthosteric ligand were affected by the addition of an allosteric modulator and such effects were probe- and concentration-dependent. Covalently linking the orthosteric and allosteric pharmacophores into one bitopic molecule had a substantial effect on the overall on- or off-rate. Conclusion and Implications The competition association assay is a useful tool for exploring the allosteric modulation of the human adenosine A1 receptor. This assay may have general applicability to study allosteric modulation at other GPCRs as well. PMID:25040887

  5. Adenosine A1 Receptor-Mediated Endocytosis of AMPA Receptors Contributes to Impairments in Long-Term Potentiation (LTP) in the Middle-Aged Rat Hippocampus.

    PubMed

    Chen, Zhicheng; Stockwell, Jocelyn; Cayabyab, Francisco S

    2016-05-01

    Aging causes multiple changes in the mammalian brain, including changes in synaptic signaling. Previous reports have shown increased extracellular adenosine in the aging brain, and we recently reported that activation of adenosine A1 receptors (A1Rs) induces AMPA receptor (AMPAR) internalization in rat hippocampus. This study investigated whether aging-related changes in the rat hippocampus include altered surface expression of adenosine A1 and A2A receptors, and whether these changes correspond to changes in AMPAR surface expression and altered synaptic plasticity. We found reduced A1R surface expression in middle-aged rat hippocampus, and also reduced GluA1 and GluA2 AMPAR subunit surface expression. Using a chemically-induced LTP (cLTP) experimental protocol, we recorded fEPSPs in young (1 month old) and middle-aged (7-12 month old) rat hippocampal slices. There were significant impairments in cLTP in middle-aged slices, suggesting impaired synaptic plasticity. Since we previously showed that the A1R agonist N(6)-cyclopentyladenosine (CPA) reduced both A1Rs and GluA2/GluA1 AMPARs, we hypothesized that the observed impaired synaptic plasticity in middle-aged brains is regulated by A1R-mediated AMPAR internalization by clathrin-mediated endocytosis. Following cLTP, we found a significant increase in GluA1 and GluA2 surface expression in young rats, which was blunted in middle-aged brains or in young brains pretreated with CPA. Blocking A1Rs with 8-cyclopentyl-1,3-dipropylxanthine or AMPAR endocytosis with either Tat-GluA2-3Y peptide or dynasore (dynamin inhibitor) similarly enhanced AMPAR surface expression following cLTP. These data suggest that age-dependent alteration in adenosine receptor expression contributes to increased AMPAR endocytosis and impaired synaptic plasticity in aged brains. PMID:26700433

  6. Up regulation of A2B adenosine receptor on monocytes are crucially required for immune pathogenicity in Indian patients exposed to Leishmania donovani.

    PubMed

    Vijayamahantesh; Amit, Ajay; Kumar, Santosh; Dikhit, Manas R; Jha, Pravin K; Singh, Ashish K; Sinha, Kislay K; Pandey, Krishna; Das, V N R; Das, Pradeep; Bimal, Sanjiva

    2016-03-01

    Adenosine, an endogenous purine nucleoside is one such extracellular signalling molecule whose role in regulation of anti-inflammatory cytokines and immune pathogenicity in visceral leishmaniasis is not fully understood. Here, we investigated the relationship between Leishmania donovani infection and expression of A2B receptor on monocytes in VL patients in their pre and post treatment stage. We also investigated the molecular mechanisms influencing the interaction between immunopathogenicity and infection by exposing Leishmania donovani pulsed macrophages to Adenosine. A direct correlation of up-regulated A2B expression on monocytes with increased parasite load was also observed. Our results also suggested that A2B receptor activation is critically required for the stimulatory effect of adenosine on IL-10 production and suppression of nitric oxide release. The stimulatory effect of adenosine on Leishmania donovani induced IL-10 production required ERK1/2 activation and is p-38 MAPK independent. PMID:26748211

  7. [The role of adenosine Al receptors and mitochondrial K+ATP channels in the mechanism of increasing the resistance to acute hypoxia in the combined effects of hypoxia and hypercapnia].

    PubMed

    Tregub, P P; Kulikov, V P; Stepanova, L A; Zabrodina, A S; Nagibaeva, M E

    2014-01-01

    We studied the role of the role of mitoK+ATp channels and Al-adenosine receptor in the mechanism of increasing the resistance to acute hypoxia after hypoxic, hypercapnic and hypercapnic-hypoxic preconditioning. It is shown that mitochondrial ATP-sensitive potassium channels and Al-adenosine receptors, an important mechanism of preconditioning have a high value to increase the resistance to acute hypoxia/ischemia in the combined effect of hypoxia and hypercapnia. However, with regard to the adenosine receptor, this mechanism is realized without the participation hypercapnic component, which apparently starts neuroprotection without activation of the adenosine Al receptors. PMID:25980226

  8. Optogenetic activation of intracellular adenosine A2A receptor signaling in the hippocampus is sufficient to trigger CREB phosphorylation and impair memory.

    PubMed

    Li, P; Rial, D; Canas, P M; Yoo, J-H; Li, W; Zhou, X; Wang, Y; van Westen, G J P; Payen, M-P; Augusto, E; Gonçalves, N; Tomé, A R; Li, Z; Wu, Z; Hou, X; Zhou, Y; IJzerman, A P; PIJzerman, Ad; Boyden, E S; Cunha, R A; Qu, J; Chen, J-F

    2015-11-01

    Human and animal studies have converged to suggest that caffeine consumption prevents memory deficits in aging and Alzheimer's disease through the antagonism of adenosine A2A receptors (A2ARs). To test if A2AR activation in the hippocampus is actually sufficient to impair memory function and to begin elucidating the intracellular pathways operated by A2AR, we have developed a chimeric rhodopsin-A2AR protein (optoA2AR), which retains the extracellular and transmembrane domains of rhodopsin (conferring light responsiveness and eliminating adenosine-binding pockets) fused to the intracellular loop of A2AR to confer specific A2AR signaling. The specificity of the optoA2AR signaling was confirmed by light-induced selective enhancement of cAMP and phospho-mitogen-activated protein kinase (p-MAPK) (but not cGMP) levels in human embryonic kidney 293 (HEK293) cells, which was abolished by a point mutation at the C terminal of A2AR. Supporting its physiological relevance, optoA2AR activation and the A2AR agonist CGS21680 produced similar activation of cAMP and p-MAPK signaling in HEK293 cells, of p-MAPK in the nucleus accumbens and of c-Fos/phosphorylated-CREB (p-CREB) in the hippocampus, and similarly enhanced long-term potentiation in the hippocampus. Remarkably, optoA2AR activation triggered a preferential p-CREB signaling in the hippocampus and impaired spatial memory performance, while optoA2AR activation in the nucleus accumbens triggered MAPK signaling and modulated locomotor activity. This shows that the recruitment of intracellular A2AR signaling in the hippocampus is sufficient to trigger memory dysfunction. Furthermore, the demonstration that the biased A2AR signaling and functions depend on intracellular A2AR loops prompts the possibility of targeting the intracellular A2AR-interacting partners to selectively control different neuropsychiatric behaviors. PMID:25687775

  9. Comparative pharmacokinetics and pharmacodynamics of platelet adenosine diphosphate receptor antagonists and their clinical implications.

    PubMed

    Floyd, Christopher N; Passacquale, Gabriella; Ferro, Albert

    2012-07-01

    Over the last two decades or more, anti-platelet therapy has become established as a cornerstone in the treatment of patients with ischaemic cardiovascular disease, since such drugs effectively reduce arterial thrombotic events. The original agent used in this context was aspirin (acetylsalicylic acid) but, with the advent of adenosine diphosphate (ADP) receptor antagonists, the use of dual anti-platelet therapy has resulted in further improvement in cardiovascular outcomes when compared with aspirin alone. The first group of platelet ADP receptor antagonists to be developed was the thienopyridine class, which comprise inactive pro-drugs that require in vivo metabolism to their active metabolites before exerting their inhibitory effect on the P2Y(12) receptor. Clopidogrel has been the principal ADP receptor antagonist in use over the past decade, but is limited by variability in its in vivo inhibition of platelet aggregation (IPA). The pharmacokinetics of clopidogrel are unpredictable due to their vulnerability to multiple independent factors including genetic polymorphisms. Expression of the 3435T/T genetic variant encoding the MDR1 gene for the P-glycoprotein efflux transporter results in a significantly reduced maximum drug concentration and area under the plasma concentration-time curve as intestinal absorption of clopidogrel is reduced; and the expression of the mutant *2 allele of CYP2C19 results in similar pharmacokinetic effects as the two cytochrome P450 (CYP)-mediated steps required for the production of the active metabolite of clopidogrel are impaired. These variable pharmacokinetics lead to erratic pharmacodynamics and cannot reliably be overcome with increased dosing. Both prasugrel, a third-generation thienopyridine, and ticagrelor, a cyto-pentyl-triazolo-pyrimidine, have more predictable pharmacokinetics and enhanced pharmacodynamics than clopidogrel. Neither appears to be affected by the same genetic polymorphisms as clopidogrel; prasugrel requires

  10. Medicinal chemistry of adenosine, P2Y and P2X receptors.

    PubMed

    Jacobson, Kenneth A; Müller, Christa E

    2016-05-01

    Pharmacological tool compounds are now available to define action at the adenosine (ARs), P2Y and P2X receptors. We present a selection of the most commonly used agents to study purines in the nervous system. Some of these compounds, including A1 and A3 AR agonists, P2Y1R and P2Y12R antagonists, and P2X3, P2X4 and P2X7 antagonists, are potentially of clinical use in treatment of disorders of the nervous system, such as chronic pain, neurodegeneration and brain injury. Agonists of the A2AAR and P2Y2R are already used clinically, P2Y12R antagonists are widely used antithrombotics and an antagonist of the A2AAR is approved in Japan for treating Parkinson's disease. The selectivity defined for some of the previously introduced compounds has been revised with updated pharmacological characterization, for example, various AR agonists and antagonists were deemed A1AR or A3AR selective based on human data, but species differences indicated a reduction in selectivity ratios in other species. Also, many of the P2R ligands still lack bioavailability due to charged groups or hydrolytic (either enzymatic or chemical) instability. X-ray crystallographic structures of AR and P2YRs have shifted the mode of ligand discovery to structure-based approaches rather than previous empirical approaches. The X-ray structures can be utilized either for in silico screening of chemically diverse libraries for the discovery of novel ligands or for enhancement of the properties of known ligands by chemical modification. Although X-ray structures of the zebrafish P2X4R have been reported, there is scant structural information about ligand recognition in these trimeric ion channels. In summary, there are definitive, selective agonists and antagonists for all of the ARs and some of the P2YRs; while the pharmacochemistry of P2XRs is still in nascent stages. The therapeutic potential of selectively modulating these receptors is continuing to gain interest in such fields as cancer, inflammation, pain

  11. Adenosine A1 Receptors in Mouse Pontine Reticular Formation Modulate Nociception Only in the Presence of Systemic Leptin

    PubMed Central

    Watson, Sarah L.; Watson, Christopher J.; Baghdoyan, Helen A.; Lydic, Ralph

    2014-01-01

    Human obesity is associated with increased leptin levels and pain, but the specific brain regions and neurochemical mechanisms underlying this association remain poorly understood. This study used adult male C57BL/6J (B6, n = 14) mice and leptin-deficient, obese B6.Cg-Lepob/J (obese, n = 10) mice to evaluate the hypothesis that nociception is altered by systemic leptin levels and by adenosine A1 receptors in the pontine reticular formation. Nociception was quantified as paw withdrawal latency (PWL) in s after onset of a thermal stimulus. PWL was converted to percent maximum possible effect (%MPE). After obtaining baseline PWL measures, the pontine reticular formation was microinjected with saline (control), three concentrations of the adenosine A1 receptor agonist N6-p-sulfophenyladenosine (SPA), or super-active mouse leptin receptor antagonist (SMLA) followed by SPA 15 min later, and PWL was again quantified. In obese, leptin-deficient mice, nociception was quantified before and during leptin replacement via subcutaneous osmotic pumps. SPA was administered into the pontine reticular formation of leptin-replaced mice and PWL testing was repeated. During baseline (before vehicle or SPA administration), PWL was significantly (p = 0.0013) lower in leptin-replaced obese mice than in B6 mice. Microinjecting SPA into the pontine reticular formation of B6 mice caused a significant (p = 0.0003) concentration-dependent increase in %MPE. SPA also significantly (p < 0.05) increased %MPE in B6 mice and in leptin-replaced obese mice, but not in leptin-deficient obese mice. Microinjection of the mouse super-active leptin antagonist (SMLA) into the pontine reticular formation before SPA did not alter PWL. The results show for the first time that pontine reticular formation administration of the adenosine A1 receptor agonist SPA produced antinociception only in the presence of systemic leptin. The concentration-response data support the interpretation that adenosine A1 receptors

  12. Adenosine inhibition of gamma-aminobutyric acid release from slices of rat cerebral cortex.

    PubMed Central

    Hollins, C.; Stone, T. W.

    1980-01-01

    1 The effect of purine compounds on the potassium-evoked release of 14C-labelled gamma-aminobutyric acid (GABA) has been studied in 400 micrometers slices of rat cerebral cortex in vitro. 2 Adenosine and adenosine 5' monophosphate (AMP) inhibited the release of GABA at 10(-5) to 10(-3) M. Adenosine triphosphate (ATP) produced a significant inhibition of release only at 10(-3) M. 3 Theophylline 10(-4) or 10(-3) M reduced the inhibitory effect of adenosine, but did not change basal release of GABA. 4 Dipyridamole 10(-5) M itself reduced evoked GABA release, but did not prevent the inhibitory effect of adenosine, implying that adenosine was acting at an extracellularly directed receptor. 5 Calcium removal or antagonism by verapamil reduced the evoked release of GABA, but adenosine did not produce any further reduction of the calcium-independent release. This may indicate that the inhibitory effect of adenosine on GABA release results from interference with calcium influx or availability within the terminals. PMID:7378648

  13. GABAA receptor antagonism at the hypoglossal motor nucleus increases genioglossus muscle activity in NREM but not REM sleep.

    PubMed

    Morrison, Janna L; Sood, Sandeep; Liu, Hattie; Park, Eileen; Nolan, Philip; Horner, Richard L

    2003-04-15

    The pharyngeal muscles, such as the genioglossus (GG) muscle of the tongue, are important for effective lung ventilation since they maintain an open airspace. Rapid-eye-movement (REM) sleep, however, recruits powerful neural mechanisms that can abolish GG activity, even during strong reflex respiratory stimulation by elevated CO2. In vitro studies have demonstrated the presence of GABAA receptors on hypoglossal motoneurons, and these and other data have led to the speculation that GABAA mechanisms may contribute to the suppression of hypoglossal motor outflow to the GG muscle in REM sleep. We have developed an animal model that allows us to chronically manipulate neurotransmission at the hypoglossal motor nucleus using microdialysis across natural sleep-wake states in rats. The present study tests the hypothesis that microdialysis perfusion of the GABAA receptor antagonist bicuculline into the hypoglossal motor nucleus will prevent the suppression of GG muscle activity in REM sleep during both room-air and CO2-stimulated breathing. Ten rats were implanted with electroencephalogram and neck muscle electrodes to record sleep-wake states, and GG and diaphragm electrodes for respiratory muscle recording. Microdialysis probes were implanted into the hypoglossal motor nucleus for perfusion of artificial cerebrospinal fluid (ACSF) or 100 microM bicuculline during room-air and CO2-stimulated breathing (7 % inspired CO2). GABAA receptor antagonism at the hypoglossal motor nucleus increased respiratory-related GG activity during both room-air (P = 0.01) and CO2-stimulated breathing (P = 0.007), indicating a background inhibitory GABA tone. However, the effects of bicuculline on GG activity depended on the prevailing sleep-wake state (P < 0.005), with bicuculline increasing GG activity in non-REM (NREM) sleep and wakefulness both in room air and hypercapnia (P < 0.01), but GG activity was effectively abolished in those REM periods without phasic twitches in the GG muscle

  14. Thromboxane-A(2)/prostaglandin-H(2) receptors Characterization and antagonism.

    PubMed

    Morinelli, T A; Halushka, P V

    1991-01-01

    Thromboxane A(2) (TXA(2)) is a potent vasoconstrictor and platelet aggregator whose synthesis is increased in a variety of cardiovascular diseases. TXA(2) receptor antagonists have been used to (a) establish a pathophysiologic role for TXA(2) in a variety of cardiovascular diseases, (b) subtype platelet and vascular receptors, (c) elucidate structural characteristics of the receptor, and (d) aid in its purification. However, much still remains to be learned about the structure and function of TXA(2) receptors. PMID:21239318

  15. Antagonism of Lateral Amygdala Alpha1-Adrenergic Receptors Facilitates Fear Conditioning and Long-Term Potentiation

    ERIC Educational Resources Information Center

    Lazzaro, Stephanie C.; Hou, Mian; Cunha, Catarina; LeDoux, Joseph E.; Cain, Christopher K.

    2010-01-01

    Norepinephrine receptors have been studied in emotion, memory, and attention. However, the role of alpha1-adrenergic receptors in fear conditioning, a major model of emotional learning, is poorly understood. We examined the effect of terazosin, an alpha1-adrenergic receptor antagonist, on cued fear conditioning. Systemic or intra-lateral amygdala…

  16. Presynaptic Adenosine Receptor-Mediated Regulation of Diverse Thalamocortical Short-Term Plasticity in the Mouse Whisker Pathway.

    PubMed

    Ferrati, Giovanni; Martini, Francisco J; Maravall, Miguel

    2016-01-01

    Short-term synaptic plasticity (STP) sets the sensitivity of a synapse to incoming activity and determines the temporal patterns that it best transmits. In "driver" thalamocortical (TC) synaptic populations, STP is dominated by depression during stimulation from rest. However, during ongoing stimulation, lemniscal TC connections onto layer 4 neurons in mouse barrel cortex express variable STP. Each synapse responds to input trains with a distinct pattern of depression or facilitation around its mean steady-state response. As a result, in common with other synaptic populations, lemniscal TC synapses express diverse rather than uniform dynamics, allowing for a rich representation of temporally varying stimuli. Here, we show that this STP diversity is regulated presynaptically. Presynaptic adenosine receptors of the A1R type, but not kainate receptors (KARs), modulate STP behavior. Blocking the receptors does not eliminate diversity, indicating that diversity is related to heterogeneous expression of multiple mechanisms in the pathway from presynaptic calcium influx to neurotransmitter release. PMID:26941610

  17. Highlights in basic autonomic neurosciences: Central adenosine A1 receptor – The key to a hypometabolic state and therapeutic hypothermia?

    PubMed Central

    Tupone, D.; Madden, C.J.; Morrison, S.F.

    2016-01-01

    The positive outcome that hypothermia contributes to brain and cardiac protection following ischemia has stimulated research in the development of pharmacological approaches to induce a hypothermic/hypometabolic state. Here we review three papers to highlight the role of the adenosine 1 receptor (A1AR) as a potential mediator and physiological regulator of a hypothermic state in both hibernating and non-hibernating mammals. We would like to emphasize the importance of comparative studies between hibernating and non-hibernating species that could lead to important discoveries on the mechanisms inducing hibernation and how they might be translated to induce a clinically useful hypothermic state. PMID:23465354

  18. Dual Allosteric Effect in Glycine/NMDA Receptor Antagonism: A Comparative QSAR Approach

    PubMed Central

    Sharma, Manish; Gupta, Vipin B.

    2010-01-01

    A comparative Hansch type QSAR study was conducted using multiple regression analysis on various sets of quinoxalines, quinoxalin-4-ones, quinazoline-2-carboxylates, 4-hydroxyquinolin-2(1H)-ones, 2-carboxytetrahydroquinolines, phenyl-hydroxy-quinolones, nitroquinolones and 4-substituted-3-phenylquinolin-2(1H)-ones as selective glycine/NMDA site antagonists. Ten statistically validated equations were developed, which indicated the importance of CMR, Verloop’s sterimol L1 and ClogP parameters in contributing towards biological activity. Interestingly, normal and inverse parabolic relationships were found with CMR in different series, indicating a dual allosteric binding mode in glycine/NMDA antagonism. Equations reveal an optimum CMR of 10 ± 10% is required for good potency of antagonists. Other equations indicate the presence of anionic functionality at 4-position of quinoline/quinolone ring system is not absolutely required for effective binding. The observations are laterally validated and in accordance with previous studies.

  19. β-Nicotinamide adenine dinucleotide acts at prejunctional adenosine A1 receptors to suppress inhibitory musculomotor neurotransmission in guinea pig colon and human jejunum.

    PubMed

    Wang, Guo-Du; Wang, Xi-Yu; Liu, Sumei; Xia, Yun; Zou, Fei; Qu, Meihua; Needleman, Bradley J; Mikami, Dean J; Wood, Jackie D

    2015-06-01

    Intracellular microelectrodes were used to record neurogenic inhibitory junction potentials in the intestinal circular muscle coat. Electrical field stimulation was used to stimulate intramural neurons and evoke contraction of the smooth musculature. Exposure to β-nicotinamide adenine dinucleotide (β-NAD) did not alter smooth muscle membrane potential in guinea pig colon or human jejunum. ATP, ADP, β-NAD, and adenosine, as well as the purinergic P2Y1 receptor antagonists MRS 2179 and MRS 2500 and the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine, each suppressed inhibitory junction potentials in guinea pig and human preparations. β-NAD suppressed contractile force of twitch-like contractions evoked by electrical field stimulation in guinea pig and human preparations. P2Y1 receptor antagonists did not reverse this action. Stimulation of adenosine A1 receptors with 2-chloro-N6-cyclopentyladenosine suppressed the force of twitch contractions evoked by electrical field stimulation in like manner to the action of β-NAD. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine suppressed the inhibitory action of β-NAD on the force of electrically evoked contractions. The results do not support an inhibitory neurotransmitter role for β-NAD at intestinal neuromuscular junctions. The data suggest that β-NAD is a ligand for the adenosine A1 receptor subtype expressed by neurons in the enteric nervous system. The influence of β-NAD on intestinal motility emerges from adenosine A1 receptor-mediated suppression of neurotransmitter release at inhibitory neuromuscular junctions. PMID:25813057

  20. β-Nicotinamide adenine dinucleotide acts at prejunctional adenosine A1 receptors to suppress inhibitory musculomotor neurotransmission in guinea pig colon and human jejunum

    PubMed Central

    Wang, Guo-Du; Wang, Xi-Yu; Liu, Sumei; Xia, Yun; Zou, Fei; Qu, Meihua; Needleman, Bradley J.; Mikami, Dean J.

    2015-01-01

    Intracellular microelectrodes were used to record neurogenic inhibitory junction potentials in the intestinal circular muscle coat. Electrical field stimulation was used to stimulate intramural neurons and evoke contraction of the smooth musculature. Exposure to β-nicotinamide adenine dinucleotide (β-NAD) did not alter smooth muscle membrane potential in guinea pig colon or human jejunum. ATP, ADP, β-NAD, and adenosine, as well as the purinergic P2Y1 receptor antagonists MRS 2179 and MRS 2500 and the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine, each suppressed inhibitory junction potentials in guinea pig and human preparations. β-NAD suppressed contractile force of twitch-like contractions evoked by electrical field stimulation in guinea pig and human preparations. P2Y1 receptor antagonists did not reverse this action. Stimulation of adenosine A1 receptors with 2-chloro-N6-cyclopentyladenosine suppressed the force of twitch contractions evoked by electrical field stimulation in like manner to the action of β-NAD. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine suppressed the inhibitory action of β-NAD on the force of electrically evoked contractions. The results do not support an inhibitory neurotransmitter role for β-NAD at intestinal neuromuscular junctions. The data suggest that β-NAD is a ligand for the adenosine A1 receptor subtype expressed by neurons in the enteric nervous system. The influence of β-NAD on intestinal motility emerges from adenosine A1 receptor-mediated suppression of neurotransmitter release at inhibitory neuromuscular junctions. PMID:25813057

  1. Alcohol Worsens Acute Lung Injury by Inhibiting Alveolar Sodium Transport through the Adenosine A1 Receptor

    PubMed Central

    Urich, Daniela; Soberanes, Saul; Manghi, Tomas S.; Chiarella, Sergio E.; Chandel, Navdeep S.; Budinger, G. R. Scott; Mutlu, Gökhan M.

    2012-01-01

    Objective Alcohol intake increases the risk of acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) and is associated with poor outcomes in patients who develop these syndromes. No specific therapies are currently available to treat or decrease the risk of ARDS in patients with alcoholism. We have recently shown increased levels of lung adenosine inhibit alveolar fluid clearance, an important predictor of outcome in patients with ARDS. We hypothesized that alcohol might worsen lung injury by increasing lung adenosine levels, resulting in impaired active Na+ transport in the lung. Methods We treated wild-type mice with alcohol administered i.p. to achieve blood alcohol levels associated with moderate to severe intoxication and measured the rate of alveolar fluid clearance and Na,K-ATPase expression in peripheral lung tissue and assessed the effect of alcohol on survival during exposure to hyperoxia. We used primary rat alveolar type II cells to investigate the mechanisms by which alcohol regulates alveolar Na+ transport. Results Exposure to alcohol reduced alveolar fluid clearance, downregulated Na,K-ATPase in the lung tissue and worsened hyperoxia-induced lung injury. Alcohol caused an increase in BAL fluid adenosine levels. A similar increase in lung adenosine levels was observed after exposure to hyperoxia. In primary rat alveolar type II cells alcohol and adenosine decreased the abundance of the Na,K-ATPase at the basolateral membrane via a mechanism that required activation of the AMPK. Conclusions Alcohol decreases alveolar fluid clearance and impairs survival from acute lung injury. Alcohol induced increases in lung adenosine levels may be responsible for reduction in alveolar fluid clearance and associated worsening of lung injury. PMID:22272351

  2. The G-protein-coupled bile acid receptor Gpbar1 (TGR5) suppresses gastric cancer cell proliferation and migration through antagonizing STAT3 signaling pathway

    PubMed Central

    Guo, Cong; Su, Jia; Li, Zhijun; Xiao, Rui; Wen, Jianxun; Li, Yanyan; Zhang, Meng; Zhang, Xueting; Yu, Donna; Huang, Wendong

    2015-01-01

    Gpbar1 (TGR5), a membrane-bound bile acid receptor, is well known for its roles in regulation of energy homeostasis and glucose metabolism. Here we show that TGR5 is a suppressor of gastric cancer cell proliferation and migration through antagonizing STAT3 signaling pathway. We firstly show that TGR5 activation greatly inhibited proliferation and migration of human gastric cancer cells and strongly induced gastric cancer cell apoptosis. We then found that TGR5 activation antagonized STAT3 signaling pathway through suppressing the phosphorylation of STAT3 and its transcription activity induced by lipopolysaccharide (LPS) or interleukin-6. TGR5 overexpression with ligand treatment inhibited gene expression mediated by STAT3. It suggests that TGR5 antagonizes gastric cancer proliferation and migration at least in part by inhibiting STAT3 signaling. These findings identify TGR5 as a suppressor of gastric cancer cell proliferation and migration that may serve as an attractive therapeutic tool for human gastric cancer. PMID:26417930

  3. Chronic hypoxia reduces adenosine A2A receptor-mediated inhibition of calcium current in rat PC12 cells via downregulation of protein kinase A

    PubMed Central

    Kobayashi, Shuichi; Beitner-Johnson, Dana; Conforti, Laura; Millhorn, David E

    1998-01-01

    Adenosine has been shown to decrease Ca2+ current (ICa) and attenuate the hypoxia-induced enhancement of intracellular free Ca2+ ([Ca2+]i) in oxygen-sensitive rat phaeochromocytoma (PC12) cells. These effects are mediated via the adenosine A2A receptor and protein kinase A (PKA). The current study was undertaken to determine the effects of adenosine on Ca2+ current and hypoxia-induced change in [Ca2+]i during chronic hypoxia.Whole cell patch-clamp studies revealed that the effect of adenosine on ICa was significantly reduced when PC12 cells were exposed to hypoxia (10 % O2) for 24 and 48 h.Ca2+ imaging studies using fura-2 revealed that the anoxia-induced increase in [Ca2+]i was significantly enhanced when PC12 cells were exposed to 10 % O2 for up to 48 h. In contrast, the inhibitory effects of adenosine on anoxia-induced elevation of [Ca2+]i was significantly blunted in PC12 cells exposed to hypoxia for 48 h.Northern blot analysis revealed that mRNA for the A2A receptor, which is the only adenosine receptor subtype expressed in PC12 cells, was significantly upregulated by hypoxia. Radioligand binding analysis with [3H]CGS21680, a selective A2A receptor ligand, showed that the number of adenosine A2A receptor binding sites was similarly increased during exposure to 10 % O2 for 48 h.PKA enzyme activity was significantly inhibited when PC12 cells were exposed to 10 % O2 for 24 and 48 h. However, we found that hypoxia failed to induce change in adenosine- and forskolin-stimulated adenylate cyclase enzyme activity. Chronic hypoxia also did not alter the immunoreactivity level of the G protein Gsα, an effector of the A2 signalling pathway.Whole cell patch-clamp analysis showed that the effect of 8-bromo-cAMP, an activator of PKA, on ICa was significantly attenuated during 48 h exposure to 10 % O2.We conclude therefore that the reduced effect of adenosine on ICa and [Ca2+]i in PC12 cells exposed to chronic hypoxia is due to hypoxia-induced downregulation of PKA. This

  4. Chronic hypoxia reduces adenosine A2A receptor-mediated inhibition of calcium current in rat PC12 cells via downregulation of protein kinase A.

    PubMed

    Kobayashi, S; Beitner-Johnson, D; Conforti, L; Millhorn, D E

    1998-10-15

    1. Adenosine has been shown to decrease Ca2+ current (ICa) and attenuate the hypoxia-induced enhancement of intracellular free Ca2+ ([Ca2+]i) in oxygen-sensitive rat phaeochromocytoma (PC12) cells. These effects are mediated via the adenosine A2A receptor and protein kinase A (PKA). The current study was undertaken to determine the effects of adenosine on Ca2+ current and hypoxia-induced change in [Ca2+]i during chronic hypoxia. 2. Whole cell patch-clamp studies revealed that the effect of adenosine on ICa was significantly reduced when PC12 cells were exposed to hypoxia (10 % O2) for 24 and 48 h. 3. Ca2+ imaging studies using fura-2 revealed that the anoxia-induced increase in [Ca2+]i was significantly enhanced when PC12 cells were exposed to 10 % O2 for up to 48 h. In contrast, the inhibitory effects of adenosine on anoxia-induced elevation of [Ca2+]i was significantly blunted in PC12 cells exposed to hypoxia for 48 h. 4. Northern blot analysis revealed that mRNA for the A2A receptor, which is the only adenosine receptor subtype expressed in PC12 cells, was significantly upregulated by hypoxia. Radioligand binding analysis with [3H]CGS21680, a selective A2A receptor ligand, showed that the number of adenosine A2A receptor binding sites was similarly increased during exposure to 10% O2 for 48 h. 5. PKA enzyme activity was significantly inhibited when PC12 cells were exposed to 10% O2 for 24 and 48 h. However, we found that hypoxia failed to induce change in adenosine- and forskolin-stimulated adenylate cyclase enzyme activity. Chronic hypoxia also did not alter the immunoreactivity level of the G protein Gsalpha, an effector of the A2 signalling pathway. 6. Whole cell patch-clamp analysis showed that the effect of 8-bromo-cAMP, an activator of PKA, on ICa was significantly attenuated during 48 h exposure to 10% O2.7. We conclude therefore that the reduced effect of adenosine on ICa and [Ca2+]i in PC12 cells exposed to chronic hypoxia is due to hypoxia

  5. Blockade of striatal neurone responses to morphine by aminophylline: evidence for adenosine mediation of opiate action.

    PubMed Central

    Perkins, M. N.; Stone, T. W.

    1980-01-01

    1 The responses of cortical and striatal neurones to morphine and adenosine applied iontophoretically have been studied in the male rat. 2 The majority of cells (57%) within the corpus striatum were profoundly inhibited, and a smaller proportion (18%) excited by morphine. Adenosine depressed the firing rate of 30/44 cells in the striatum, excitation never being observed. In contrast, the responses of cortical cells to morphine were typically weak and required longer ejection pulses to effect comparable changes in firing rate. 3 Aminophylline applied iontophoretically, as an anion, proved able to antagonize reversibly both morphine and adenosine in parallel. 4 On a number of cells, gamma-aminobutyric acid (GABA) was used as a control depressant but aminophylline did not appear to reduce these responses. 5 The responses to morphine (both inhibitory and excitatory) were not easily antagonized by naloxone. Typically, excitatory reponses were easier to antagonize than the inhibitory ones. 6 It is concluded that a consequence of the interaction of morphine with its receptors may be the release of adenosine which subsequently produces the inhibition observed with morphine. PMID:7378652

  6. [Adenosine and its role in physiology].

    PubMed

    Novotný, J

    2015-01-01

    Adenosine is not just a major component of adenine nucleotides and ribonucleic acids, but also has its own signaling functions. ExtraceIlular level of adenosine in an organism is strictly maintained through the balance between its formation, degradation and transport. Adenosine is formed by enzymatic degradation of adenosine triphosphate and eliminated by phosphorylation to adenosine monophosphate or by deamination to inosine. Transport of adenosine across the cell membrane is ensured by equilibrative and concentrative nucleoside transporters. All these processes participate in maintenance of adenosine level under normal conditions, but a balanced equilibrium can be disrupted in some pathophysiological situations. Extracellular adenosine as a signaling molecule binds to adenosine receptors, which may trigger via their cognate trimeric G proteins different signaling pathways. In this way, adenosine regulates energy homeostasis and affects the function of various organs. Targeted pharmacological manipulations of specific adenosine receptor subtypes or enzymes involved in its metabolism can potentially be used for therapeutic purposes. PMID:26738245

  7. Histamine H2 receptor antagonism by T-593: studies on cAMP generation in Hepa cells expressing histamine H2 receptor.

    PubMed

    Tashiro, T; Ono, K; Watanabe, T; Inoie, M; Arai, H; Kimura, S; Kurokawa, K

    1999-07-01

    Histamine H2 receptor antagonism by T-593 was investigated in Hepa cells expressing canine histamine H2 receptors. T-593 inhibited generation of cAMP in Hepa cells stimulated by 10(-5) mol/l histamine with an IC50 value of 2.3 x 10(-6) mol/l, (S)-(-)-T-593, one of the enantiomers comprising racemic T-593, inhibited cAMP generation with an IC50 value of 6.1 x 10(-7) mol/l. On the other hand, the other enantiomer (R)-(+)-T-593 exhibited only a negligible effect. Incubation of the cell with (S)-(-)-T-593 for 60 min depressed the maximal response of the concentration-response curve of histamine with a nonparallel rightward shift. The slope of a Schild plot was 1.27. In contrast, (S)-(-)-T-593 caused a parallel rightward shift of the curve, with a Schild plot slope that did not significantly differ from unity, by treating the cells for 15 min. The H2 receptor-blocking action of (S)-(-)-T-593 remained almost unaffected after washing out the drug, whereas the effect of ranitidine was reversible after washing. These results suggest that T-593 possesses a time-dependent insurmountable antagonistic action against histamine H2 receptor. T-593 may interact with the histamine H2 receptor molecule in a slowly associable and dissociable manner. PMID:10352421

  8. NK1 receptor antagonism lowers occupancy requirement for antidepressant-like effects of SSRIs in the gerbil forced swim test.

    PubMed

    Lelas, Snjezana; Li, Yu-Wen; Wallace-Boone, Tanya L; Taber, Matthew T; Newton, Amy E; Pieschl, Rick L; Davis, Carl D; Molski, Thaddeus F; Newberry, Kimberly S; Parker, Michael F; Gillman, Kevin W; Bronson, Joanne J; Macor, John E; Lodge, Nicholas J

    2013-10-01

    The known interactions between the serotonergic and neurokinin systems suggest that serotonin reuptake inhibitor (SSRIs) efficacy may be improved by neurokinin-1 receptor (NK1R) antagonism. In the current studies combination of a subeffective dose of an SSRI (0.3 mg/kg fluoxetine or 0.03 mg/kg citalopram) with a subeffective dose of an NK1R antagonist (0.3 mg/kg aprepitant or 1 mg/kg CP-122,721) produced efficacy in the gerbil forced swim test (FST). Serotonin transporter (SERT) occupancy produced by 1 mg/kg fluoxetine (lowest efficacious dose) was 52 ± 5% and was reduced to 29 ± 4% at 0.3 mg/kg, a dose that was efficacious in combination with 0.3 mg/kg aprepitant or 1 mg/kg CP-122,721; the corresponding NK1R occupancies were 79 ± 4% and 61 ± 4% for aprepitant and CP-122,721, respectively. For citalopram, SERT occupancy at the lowest efficacious dose (0.1 mg/kg) was 50 ± 4% and was reduced to 20 ± 5% at 0.03 mg/kg, a dose that was efficacious when combined with aprepitant (0.3 mg/kg). Aprepitant (10 mg/kg) augmented the serotonin elevation produced by fluoxetine (1 or 10 mg/kg) in the gerbil prefrontal cortex; i.e. NK1R antagonism can modulate serotonin responses. A novel orally-available dual-acting NK1R antagonist/SERT inhibitor BMS-795176 is described; gerbil Ki = 1.4 and 1 nM at NK1R and SERT, respectively. BMS-795176 was efficacious in the gerbil FST; efficacy was observed with 35 ± 3% SERT occupancy and 73 ± 3% NK1R occupancy. The interaction between NK1R antagonism and SERT inhibition to lower the SERT occupancy required for antidepressant-like efficacy suggests that BMS-795176 has the potential to improve efficacy with a reduction in SSRI-associated side effects. PMID:23770339

  9. Excess adenosine A2B receptor signaling contributes to priapism through HIF-1α mediated reduction of PDE5 gene expression

    PubMed Central

    Ning, Chen; Wen, Jiaming; Zhang, Yujin; Dai, Yingbo; Wang, Wei; Zhang, Weiru; Qi, Lin; Grenz, Almut; Eltzschig, Holger K.; Blackburn, Michael R.; Kellems, Rodney E.; Xia, Yang

    2014-01-01

    Priapism is featured with prolonged and painful penile erection and is prevalent among males with sickle cell disease (SCD). The disorder is a dangerous urological and hematological emergency since it is associated with ischemic tissue damage and erectile disability. Here we report that phosphodiesterase-5 (PDE5) gene expression and PDE activity is significantly reduced in penile tissues of two independent priapic models: SCD mice and adenosine deaminase (ADA)-deficient mice. Moreover, using ADA enzyme therapy to reduce adenosine or a specific antagonist to block A2B adenosine receptor (ADORA2B) signaling, we successfully attenuated priapism in both ADA−/− and SCD mice by restoring penile PDE5 gene expression to normal levels. This finding led us to further discover that excess adenosine signaling via ADORA2B activation directly reduces PDE5 gene expression in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. Overall, we reveal that excess adenosine-mediated ADORA2B signaling underlies reduced penile PDE activity by decreasing PDE5 gene expression in a HIF-1α-dependent manner and provide new insight for the pathogenesis of priapism and novel therapies for the disease.—Ning, C., Wen, J., Zhang, Y., Dai, Y., Wang, W., Zhang, W., Qi, L., Grenz, A., Eltzschig, H. K., Blackburn, M. R., Kellems, R. E., Xia, Y. Excess adenosine A2B receptor signaling contributes to priapism through HIF-1α mediated reduction of PDE5 gene expression. PMID:24614760

  10. A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice.

    PubMed

    Eckle, Tobias; Grenz, Almut; Laucher, Stefanie; Eltzschig, Holger K

    2008-10-01

    Although acute lung injury contributes significantly to critical illness, resolution often occurs spontaneously via activation of incompletely understood pathways. We recently found that mechanical ventilation of mice increases the level of pulmonary adenosine, and that mice deficient for extracellular adenosine generation show increased pulmonary edema and inflammation after ventilator-induced lung injury (VILI). Here, we profiled the response to VILI in mice with genetic deletions of each of the 4 adenosine receptors (ARs) and found that deletion of the A2BAR gene was specifically associated with reduced survival time and increased pulmonary albumin leakage after injury. In WT mice, treatment with an A2BAR-selective antagonist resulted in enhanced pulmonary inflammation, edema, and attenuated gas exchange, while an A2BAR agonist attenuated VILI. In bone marrow-chimeric A2BAR mice, although the pulmonary inflammatory response involved A2BAR signaling from bone marrow-derived cells, A2BARs located on the lung tissue attenuated VILI-induced albumin leakage and pulmonary edema. Furthermore, measurement of alveolar fluid clearance (AFC) demonstrated that A2BAR signaling enhanced amiloride-sensitive fluid transport and elevation of pulmonary cAMP levels following VILI, suggesting that A2BAR agonist treatment protects by drying out the lungs. Similar enhancement of pulmonary cAMP and AFC were also observed after beta-adrenergic stimulation, a pathway known to promote AFC. Taken together, these studies reveal a role for A2BAR signaling in attenuating VILI and implicate this receptor as a potential therapeutic target during acute lung injury. PMID:18787641

  11. A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival.

    PubMed

    Vyas, Falguni S; Hargreaves, Alan J; Bonner, Philip L R; Boocock, David J; Coveney, Clare; Dickenson, John M

    2016-05-01

    The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells. H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection. PMID:27005940

  12. Membrane omega-3 fatty acids modulate the oligomerisation kinetics of adenosine A2A and dopamine D2 receptors

    PubMed Central

    Guixà-González, Ramon; Javanainen, Matti; Gómez-Soler, Maricel; Cordobilla, Begoña; Domingo, Joan Carles; Sanz, Ferran; Pastor, Manuel; Ciruela, Francisco; Martinez-Seara, Hector; Selent, Jana

    2016-01-01

    Membrane levels of docosahexaenoic acid (DHA), an essential omega-3 polyunsaturated fatty acid (ω-3 PUFA), are decreased in common neuropsychiatric disorders. DHA modulates key cell membrane properties like fluidity, thereby affecting the behaviour of transmembrane proteins like G protein-coupled receptors (GPCRs). These receptors, which have special relevance for major neuropsychiatric disorders have recently been shown to form dimers or higher order oligomers, and evidence suggests that DHA levels affect GPCR function by modulating oligomerisation. In this study, we assessed the effect of membrane DHA content on the formation of a class of protein complexes with particular relevance for brain disease: adenosine A2A and dopamine D2 receptor oligomers. Using extensive multiscale computer modelling, we find a marked propensity of DHA for interaction with both A2A and D2 receptors, which leads to an increased rate of receptor oligomerisation. Bioluminescence resonance energy transfer (BRET) experiments performed on living cells suggest that this DHA effect on the oligomerisation of A2A and D2 receptors is purely kinetic. This work reveals for the first time that membrane ω-3 PUFAs play a key role in GPCR oligomerisation kinetics, which may have important implications for neuropsychiatric conditions like schizophrenia or Parkinson’s disease. PMID:26796668

  13. Membrane omega-3 fatty acids modulate the oligomerisation kinetics of adenosine A2A and dopamine D2 receptors

    NASA Astrophysics Data System (ADS)

    Guixà-González, Ramon; Javanainen, Matti; Gómez-Soler, Maricel; Cordobilla, Begoña; Domingo, Joan Carles; Sanz, Ferran; Pastor, Manuel; Ciruela, Francisco; Martinez-Seara, Hector; Selent, Jana

    2016-01-01

    Membrane levels of docosahexaenoic acid (DHA), an essential omega-3 polyunsaturated fatty acid (ω-3 PUFA), are decreased in common neuropsychiatric disorders. DHA modulates key cell membrane properties like fluidity, thereby affecting the behaviour of transmembrane proteins like G protein-coupled receptors (GPCRs). These receptors, which have special relevance for major neuropsychiatric disorders have recently been shown to form dimers or higher order oligomers, and evidence suggests that DHA levels affect GPCR function by modulating oligomerisation. In this study, we assessed the effect of membrane DHA content on the formation of a class of protein complexes with particular relevance for brain disease: adenosine A2A and dopamine D2 receptor oligomers. Using extensive multiscale computer modelling, we find a marked propensity of DHA for interaction with both A2A and D2 receptors, which leads to an increased rate of receptor oligomerisation. Bioluminescence resonance energy transfer (BRET) experiments performed on living cells suggest that this DHA effect on the oligomerisation of A2A and D2 receptors is purely kinetic. This work reveals for the first time that membrane ω-3 PUFAs play a key role in GPCR oligomerisation kinetics, which may have important implications for neuropsychiatric conditions like schizophrenia or Parkinson’s disease.

  14. Type 2 Interleukin-4 Receptor Signaling in Neutrophils Antagonizes Their Expansion and Migration during Infection and Inflammation.

    PubMed

    Woytschak, Janine; Keller, Nadia; Krieg, Carsten; Impellizzieri, Daniela; Thompson, Robert W; Wynn, Thomas A; Zinkernagel, Annelies S; Boyman, Onur

    2016-07-19

    Neutrophils are the first immune cells recruited to sites of inflammation and infection. However, patients with allergic disorders such as atopic dermatitis show a paucity of skin neutrophils and are prone to bacterial skin infections, suggesting that allergic inflammation curtails neutrophil responses. Here we have shown that the type 2 cell signature cytokine interleukin-4 (IL-4) hampers neutrophil expansion and migration by antagonizing granulocyte colony-stimulating factor (G-CSF) and chemokine receptor-mediated signals. Cutaneous bacterial infection in mice was exacerbated by IL-4 signaling and improved with IL-4 inhibition, each outcome inversely correlating with neutrophil migration to skin. Likewise, systemic bacterial infection was worsened by heightened IL-4 activity, with IL-4 restricting G-CSF-induced neutrophil expansion and migration to tissues by affecting CXCR2-CXCR4 chemokine signaling in neutrophils. These effects were dependent on IL-4 acting through type 2 IL-4 receptors on neutrophils. Thus, targeting IL-4 might be beneficial in neutropenic conditions with increased susceptibility to bacterial infections. PMID:27438770

  15. AT1 receptor antagonism before ischemia prevents the transition of acute kidney injury to chronic kidney disease.

    PubMed

    Rodríguez-Romo, Roxana; Benítez, Kenia; Barrera-Chimal, Jonatan; Pérez-Villalva, Rosalba; Gómez, Arturo; Aguilar-León, Diana; Rangel-Santiago, Jesús F; Huerta, Sara; Gamba, Gerardo; Uribe, Norma; Bobadilla, Norma A

    2016-02-01

    Despite clinical recovery of patients from an episode of acute kidney injury (AKI), progression to chronic kidney disease (CKD) is possible on long-term follow-up. However, mechanisms of this are poorly understood. Here, we determine whether activation of angiotensin-II type 1 receptors during AKI triggers maladaptive mechanisms that lead to CKD. Nine months after AKI, male Wistar rats develop CKD characterized by renal dysfunction, proteinuria, renal hypertrophy, glomerulosclerosis, tubular atrophy, and tubulointerstitial fibrosis. Renal injury was associated with increased oxidative stress, inflammation, α-smooth muscle actin expression, and activation of transforming growth factor β; the latter mainly found in epithelial cells. Although administration of losartan prior to the initial ischemic insult did not prevent or reduce AKI severity, it effectively prevented eventual CKD. Three days after AKI, renal dysfunction, tubular structural injury, and elevation of urinary biomarkers were present. While the losartan group had similar early renal injury, renal perfusion was completely restored as early as day 3 postischemia. Further, there was increased vascular endothelial growth factor expression and an early activation of hypoxia-inducible factor 1 α, a transcription factor that regulates expression of many genes that help reduce renal injury. Thus, AT1 receptor antagonism prior to ischemia prevented AKI to CKD transition by improving early renal blood flow recovery, lesser inflammation, and increased hypoxia-inducible factor 1 α activity. PMID:26509589

  16. Potential Role of A2B Adenosine Receptors on Proliferation/Migration of Fetal Endothelium Derived from Preeclamptic Pregnancies

    PubMed Central

    Acurio, Jesenia; Troncoso, Felipe; Salomon, Carlos; Aguayo, Claudio; Sobrevia, Luis

    2014-01-01

    To investigate the functionality of A2B adenosine receptor (A2BAR) and the nitric oxide (NO) and vascular endothelial growth factor (VEGF) signaling pathway in the endothelial cell proliferation/migration during preeclampsia, we used human umbilical vein endothelial cells (HUVECs) isolated from normal pregnancies (n = 15) or pregnancies with preeclampsia (n = 15). Experiments were performed in presence or absence of the nonselective adenosine receptor agonist NECA, the A2BAR selective antagonist MRS-1754, and the nitric oxide synthase (NOS) inhibitor L-NAME. Results indicated that cells from preeclampsia exhibited a significant higher protein level of A2BAR and logEC50 for NECA-mediated proliferation than normotensive pregnancies. The stimulatory effect of NECA (10 μM, 24 h) on cell proliferation was prevented by MRS-1754 (5 nM) coincubation only in cells from normotensive pregnancies. Nevertheless, L-NAME (100 μM, 24 h) reduced the NECA-induced cell proliferation/migration in HUVEC from normal pregnancy; however in preeclampsia only NECA-induced cell proliferation was reduced by L-NAME. Moreover, NECA increased protein nitration and abundance of VEGF in cells from normal pregnancy and effect prevented by MRS-1754 coincubation. Nevertheless, in preeclampsia NECA did not affect the protein level of VEGF. In conclusion HUVECs from preeclampsia exhibit elevated protein level of A2BAR and impairment of A2BAR-mediated NO/VEGF signaling pathway. PMID:24877077

  17. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor.

    PubMed

    Watson, Michael J; Lee, Shernita L; Marklew, Abigail J; Gilmore, Rodney C; Gentzsch, Martina; Sassano, Maria F; Gray, Michael A; Tarran, Robert

    2016-01-01

    CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR's function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR's PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs. PMID:27278076

  18. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor

    PubMed Central

    Watson, Michael J.; Lee, Shernita L.; Marklew, Abigail J.; Gilmore, Rodney C.; Gentzsch, Martina; Sassano, Maria F.; Gray, Michael A.; Tarran, Robert

    2016-01-01

    CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR’s function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR’s PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs. PMID:27278076

  19. Neuroprotection by caffeine in the MPTP model of parkinson's disease and its dependence on adenosine A2A receptors.

    PubMed

    Xu, K; Di Luca, D G; Orrú, M; Xu, Y; Chen, J-F; Schwarzschild, M A

    2016-05-13

    Considerable epidemiological and laboratory data have suggested that caffeine, a nonselective adenosine receptor antagonist, may protect against the underlying neurodegeneration of parkinson's disease (PD). Although both caffeine and more specific antagonists of the A2A subtype of adenosine receptor (A2AR) have been found to confer protection in animal models of PD, the dependence of caffeine's neuroprotective effects on the A2AR is not known. To definitively determine its A2AR dependence, the effect of caffeine on 1-methyl-4-phenyl-1,2,3,6 tetra-hydropyridine (MPTP) neurotoxicity was compared in wild-type (WT) and A2AR gene global knockout (A2A KO) mice, as well as in central nervous system (CNS) cell type-specific (conditional) A2AR knockout (cKO) mice that lack the receptor either in postnatal forebrain neurons or in astrocytes. In WT and in heterozygous A2AR KO mice caffeine pretreatment (25mg/kgip) significantly attenuated MPTP-induced depletion of striatal dopamine. By contrast in homozygous A2AR global KO mice caffeine had no effect on MPTP toxicity. In forebrain neuron A2AR cKO mice, caffeine lost its locomotor stimulant effect, whereas its neuroprotective effect was mostly preserved. In astrocytic A2AR cKO mice, both caffeine's locomotor stimulant and protective properties were undiminished. Taken together, these results indicate that neuroprotection by caffeine in the MPTP model of PD relies on the A2AR, although the specific cellular localization of these receptors remains to be determined. PMID:26905951

  20. The role of CB1 receptors in sweet versus fat reinforcement: effect of CB1 receptor deletion, CB1 receptor antagonism (SR141716A) and CB1 receptor agonism (CP-55940).

    PubMed

    Ward, S J; Dykstra, L A

    2005-09-01

    It is well established that Cannabis sativa can increase appetite, particularly for sweet and palatable foods. In laboratory animals, cannabinoid CB1 receptor antagonism decreases motivation for palatable foods, and most recently, the CB1 receptor antagonist SR141716A, or rimonabant (Acomplia), was reported to produce weight loss in obese human subjects. Indeed, the endocannabinoid system plays a select role in the rewarding properties of palatable foods, and this is well characterized in laboratory animals with sweet sucrose solutions. In the present study, CB1 knockout mice (CB1 KO) and wild-type littermate mice (WT) were trained to respond for a complex sweet as well as a pure fat reinforcer under a progressive ratio (PR) schedule, to determine whether motivation to consume different palatable foods is tonically regulated by CB1 receptors. To assess sweet reinforcement, several concentrations of the liquid nutritional drink, Ensure, were presented under the PR schedule. For fat reinforcement, several concentrations of corn oil (emulsified in 3% xanthan gum) were made available. Additionally, to compare the result of genetic invalidation of the CB1 receptor to antagonism of the CB1 receptor system, the effect of SR141716A (3.0 mg/kg) on responding for Ensure and corn oil were also assessed using the PR schedule. We also assessed the effect of the CB1 agonist CP-55940 (30 microg/kg) on responding for Ensure and corn oil. CB1 KOs took significantly longer to acquire operant responding maintained by Ensure, and responding for Ensure under the PR schedule was significantly reduced in CB1 KOs as well as in WTs pretreated with SR141716A, as compared to WT controls. Additionally, pretreatment with the CB1 agonist CP-55940 increased responding for Ensure. In contrast, responding for corn oil during acquisition and under the PR schedule was not significantly different in CB1 KOs versus wild-type mice. However, SR141716A did reduce responding for corn oil in WTs, and CP

  1. Differential Expression of Adenosine P1 Receptor ADORA1 and ADORA2A Associated with Glioma Development and Tumor-Associated Epilepsy.

    PubMed

    Huang, Jun; Chen, Ming-Na; Du, Juan; Liu, Hao; He, Yu-Jiao; Li, Guo-Liang; Li, Shu-Yu; Liu, Wei-Ping; Long, Xiao-Yan

    2016-07-01

    Level of adenosine, an endogenous astrocyte-based neuromodulator, is primarily regulated by adenosine P1 receptors. This study assessed expression of adenosine P1 receptors, ADORA1 (adenosine A1 receptor) and ADORA2A (adenosine A2a receptor) and their association with glioma development and epilepsy in glioma patients. Expression of ADORA1/ADORA2A was assessed immunohistochemically in 65 surgically removed glioma tissue and 21 peri-tumor tissues and 8 cases of normal brain tissues obtained from hematoma patients with cerebral trauma. Immunofluorescence, Western blot, and qRT-PCR were also used to verify immunohistochemical data. Adenosine P1 receptor ADORA1 and ADORA2A proteins were localized in the cell membrane and cytoplasm and ADORA1/ADORA2A immunoreactivity was significantly stronger in glioma and peri-tumor tissues that contained infiltrating tumor cells than in normal brain tissues (p < 0.05). The World Health Organization (WHO) grade III gliomas expressed even higher level of ADORA1 and ADORA2A. Western blot and qRT-PCR confirmed immunohistochemical data. Moreover, higher levels of ADORA1 and ADORA2A expression occurred in high-grade gliomas, in which incidence of epilepsy were lower (p < 0.05). In contrast, a lower level of ADORA1/ADORA2A expression was found in peri-tumor tissues with tumor cell presence from patients with epilepsy compared to patients without epilepsy (p < 0.05). The data from the current study indicates that dysregulation in ADORA1/ADORA2A expression was associated with glioma development, whereas low level of ADORA1/ADORA2A expression could increase susceptibility of tumor-associated epilepsy. PMID:27038930

  2. NAAG fails to antagonize synaptic and extrasynaptic NMDA receptors in cerebellar granule neurons.

    PubMed

    Losi, G; Vicini, S; Neale, J

    2004-03-01

    The peptide transmitter N-acetylaspartylglutamate (NAAG) selectively activates the group II metabotropic glutamate receptors. Several reports also suggest that this peptide acts as a partial agonist at N-methyl-D-aspartate (NMDA) receptors but its putative antagonist effects have not been directly tested. To do this, we used whole cell recordings from cerebellar granule cells (CGC) in culture that allow the highest possible resolution of NMDA channel activation. When CGC were activated with equimolar concentrations of NMDA and NAAG, the peptide failed to alter the peak current elicited by NMDA. Very high concentrations of NAAG (100-200 microM) did not significantly reduce the current elicited by 10 microM NMDA or 0.1 microM glutamate, while 400 microM NAAG produced only a very small (less than 15%) reduction in these whole cell currents. Similarly, NAAG (400 microM) failed to significantly alter the average decay time constant or the peak amplitude of NMDA receptor-mediated miniature excitatory post-synaptic currents (mEPSCs). We conclude that high concentrations of the peptide do not exert physiologically relevant antagonist actions on synaptic NMDA receptor activation following vesicular release of glutamate. As an agonist, purified NAAG was found to be at least 10,000-fold less potent than glutamate in increasing "background" current via NMDA receptors on CGC. Inasmuch as it is difficult to confirm that NAAG preparations are completely free from contamination with glutamate at the 0.01% level, the peptide itself appears unlikely to have a direct agonist activity at the NMDA receptor subtypes found in CGC. Recent reports indicate that enhancing the activity of endogenous NAAG may be an important therapeutic approach to excitotoxicity and chronic pain perception. These effects are likely mediated by group II mGluRs, not NMDA receptors. PMID:14975672

  3. Adenosine-to-Inosine RNA Editing Affects Trafficking of the γ-Aminobutyric Acid Type A (GABAA) Receptor*

    PubMed Central

    Daniel, Chammiran; Wahlstedt, Helene; Ohlson, Johan; Björk, Petra; Öhman, Marie

    2011-01-01

    Recoding by adenosine-to-inosine RNA editing plays an important role in diversifying proteins involved in neurotransmission. We have previously shown that the Gabra-3 transcript, coding for the α3 subunit of the GABAA receptor is edited in mouse, causing an isoleucine to methionine (I/M) change. Here we show that this editing event is evolutionarily conserved from human to chicken. Analyzing recombinant GABAA receptor subunits expressed in HEK293 cells, our results suggest that editing at the I/M site in α3 has functional consequences on receptor expression. We demonstrate that I/M editing reduces the cell surface and the total number of α3 subunits. The reduction in cell surface levels is independent of the subunit combination as it is observed for α3 in combination with either the β2 or the β3 subunit. Further, an amino acid substitution at the corresponding I/M site in the α1 subunit has a similar effect on cell surface presentation, indicating the importance of this site for receptor trafficking. We show that the I/M editing during brain development is inversely related to the α3 protein abundance. Our results suggest that editing controls trafficking of α3-containing receptors and may therefore facilitate the switch of subunit compositions during development as well as the subcellular distribution of α subunits in the adult brain. PMID:21030585

  4. Adenosine-to-inosine RNA editing affects trafficking of the gamma-aminobutyric acid type A (GABA(A)) receptor.

    PubMed

    Daniel, Chammiran; Wahlstedt, Helene; Ohlson, Johan; Björk, Petra; Ohman, Marie

    2011-01-21

    Recoding by adenosine-to-inosine RNA editing plays an important role in diversifying proteins involved in neurotransmission. We have previously shown that the Gabra-3 transcript, coding for the α3 subunit of the GABA(A) receptor is edited in mouse, causing an isoleucine to methionine (I/M) change. Here we show that this editing event is evolutionarily conserved from human to chicken. Analyzing recombinant GABA(A) receptor subunits expressed in HEK293 cells, our results suggest that editing at the I/M site in α3 has functional consequences on receptor expression. We demonstrate that I/M editing reduces the cell surface and the total number of α3 subunits. The reduction in cell surface levels is independent of the subunit combination as it is observed for α3 in combination with either the β2 or the β3 subunit. Further, an amino acid substitution at the corresponding I/M site in the α1 subunit has a similar effect on cell surface presentation, indicating the importance of this site for receptor trafficking. We show that the I/M editing during brain development is inversely related to the α3 protein abundance. Our results suggest that editing controls trafficking of α3-containing receptors and may therefore facilitate the switch of subunit compositions during development as well as the subcellular distribution of α subunits in the adult brain. PMID:21030585

  5. NMDA receptor subunits and associated signaling molecules mediating antidepressant-related effects of NMDA-GluN2B antagonism

    PubMed Central

    Kiselycznyk, Carly; Jury, Nicholas; Halladay, Lindsay; Nakazawa, Kazu; Mishina, Masayoshi; Sprengel, Rolf; Grant, Seth G.N.; Svenningsson, Per; Holmes, Andrew

    2015-01-01

    Drugs targeting the glutamate N-methyl-D-aspartate receptor (NMDAR) may be efficacious for treating mood disorders, as exemplified by the rapid antidepressant effects produced by single administration of the NMDAR antagonist ketamine. Though the precise mechanisms underlying the antidepressant-related effects of NMDAR antagonism remain unclear, recent studies implicate specific NMDAR subunits, including GluN2A and GluN2B, as well as the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) subunit glutamate receptor interacting molecule, PSD-95. Here, integrating mutant and pharmacological in mice, we investigated the contribution of these subunits and molecules to antidepressant-related behaviors and the antidepressant-related effects of the GluN2B blocker, Ro 25-6981. We found that global deletion of GluA1 or PSD-95 reduced forced swim test (FST) immobility, mimicking the antidepressant-related effect produced by systemically administered Ro 25-6981 in C57BL/6J mice. Moreover, the FST antidepressant-like effects of systemic Ro 25-6981 were intact in mutants with global GluA1 deletion or GluN1 deletion in forebrain interneurons, but were absent in mutants constitutively lacking GluN2A or PSD-95. Next, we found that microinfusing Ro 25-6981 into the medial prefrontal cortex (mPFC), but not basolateral amygdala, of C57BL/6J mice was sufficient to produce an antidepressant-like effect. Together, these findings extend and refine current understanding of the mechanisms mediating antidepressant-like effects produced by NMDAR-GluN2B antagonists, and may inform the development of a novel class of medications for treating depression that target the GluN2B subtype of NMDAR. PMID:25800971

  6. Using prepulse inhibition to detect functional D3 receptor antagonism: effects of WC10 and WC44.

    PubMed

    Weber, Martin; Chang, Wei-Li; Durbin, John P; Park, Paula E; Luedtke, Robert R; Mach, Robert H; Swerdlow, Neal R

    2009-08-01

    Prepulse inhibition of startle (PPI) is an operational measure of sensorimotor gating that is impaired in schizophrenia. Treatment with mixed dopamine D2/D3 antagonists diminishes schizophrenia symptoms, and opposes dopamine agonist-induced PPI deficits in rats. There are reasons to believe that functional D3 receptor antagonists might offer more favorable therapeutic profiles compared to current antipsychotics. However, D3-related drug discovery is hampered by the absence of assays sensitive to D3-mediated (antipsychotic) properties in vivo. Here, we characterized two putative D3-active compounds - WC10 and WC44 - in a PPI-based screening assay, comparing the sensitivity of test compounds to oppose PPI deficits induced by the mixed D1/D2-like agonist apomorphine vs. the preferential D3 agonist pramipexole in rats. WC10, WC44 (0, 1, 3, 10 mg/kg, each), and the preferential D2 antagonist L741,626 (0, 1 mg/kg) were studied, in combination with apomorphine (0, 0.5 mg/kg), or pramipexole (0, 1 mg/kg). L741,626 prevented apomorphine-, but not pramipexole-induced PPI deficits. WC10, but not WC44, prevented apomorphine-induced PPI deficits; both compounds opposed pramipexole-induced PPI deficits, suggesting functional D3 and D1/D2 antagonist profiles for WC10, and functional D3 receptor antagonism for WC44. This assay may be valuable for detecting predominantly D3 vs. D2 receptor-linked mechanisms of action in vivo. PMID:19426754

  7. Polymerization of actin in RBL-2H3 cells can be triggered through either the IgE receptor or the adenosine receptor but different signaling pathways are used.

    PubMed Central

    Apgar, J R

    1994-01-01

    Crosslinking of the IgE receptor on rat basophilic leukemia (RBL) cells using the multivalent antigen DNP-BSA leads to a rapid and sustained increase in the filamentous actin content of the cells. Stimulation of RBL cells through the adenosine receptor also induces a very rapid polymerization of actin, which peaks in 45-60 s and is equivalent in magnitude to the F-actin response elicited through stimulation of the IgE receptor. However, in contrast to the IgE mediated response, which remains elevated for over 30 min, the F-actin increase induced by the adenosine analogue 5'-(N-ethylcarboxamido)-adenosine (NECA) is relatively transient and returns to baseline values within 5-10 min. While previous work has shown that the polymerization of actin in RBL cells stimulated through the IgE receptor is mediated by protein kinase C (PKC), protein kinase inhibitors have no effect on the F-actin response activated through the adenosine receptor. In contrast, pretreatment of the cells with pertussis toxin completely inhibits the F-actin response to NECA but has relatively little effect on the response induced through the IgE receptor. Stimulation of RBL cells through either receptor causes increased production of phosphatidylinositol mono-phosphate (PIP) and phosphatidylinositol bis-phosphate (PIP2), which correlates with the F-actin response. Production of PIP and PIP2 may be important downstream signals since these polyphosphoinositides are able to regulate the interaction of gelsolin and profilin with actin. Thus the polymerization of actin can be triggered through either the adenosine receptor or the IgE receptor, but different upstream signaling pathways are being used. The IgE mediated response requires the activation of PKC while stimulation through the adenosine receptor is PKC independent but involves a G protein. PMID:8049523

  8. Differential Modulation of Reinforcement Learning by D2 Dopamine and NMDA Glutamate Receptor Antagonism

    PubMed Central

    Klein, Tilmann A.; Ullsperger, Markus

    2014-01-01

    The firing pattern of midbrain dopamine (DA) neurons is well known to reflect reward prediction errors (PEs), the difference between obtained and expected rewards. The PE is thought to be a crucial signal for instrumental learning, and interference with DA transmission impairs learning. Phasic increases of DA neuron firing during positive PEs are driven by activation of NMDA receptors, whereas phasic suppression of firing during negative PEs is likely mediated by inputs from the lateral habenula. We aimed to determine the contribution of DA D2-class and NMDA receptors to appetitively and aversively motivated reinforcement learning. Healthy human volunteers were scanned with functional magnetic resonance imaging while they performed an instrumental learning task under the influence of either the DA D2 receptor antagonist amisulpride (400 mg), the NMDA receptor antagonist memantine (20 mg), or placebo. Participants quickly learned to select (“approach”) rewarding and to reject (“avoid”) punishing options. Amisulpride impaired both approach and avoidance learning, while memantine mildly attenuated approach learning but had no effect on avoidance learning. These behavioral effects of the antagonists were paralleled by their modulation of striatal PEs. Amisulpride reduced both appetitive and aversive PEs, while memantine diminished appetitive, but not aversive PEs. These data suggest that striatal D2-class receptors contribute to both approach and avoidance learning by detecting both the phasic DA increases and decreases during appetitive and aversive PEs. NMDA receptors on the contrary appear to be required only for approach learning because phasic DA increases during positive PEs are NMDA dependent, whereas phasic decreases during negative PEs are not. PMID:25253860

  9. Differential modulation of reinforcement learning by D2 dopamine and NMDA glutamate receptor antagonism.

    PubMed

    Jocham, Gerhard; Klein, Tilmann A; Ullsperger, Markus

    2014-09-24

    The firing pattern of midbrain dopamine (DA) neurons is well known to reflect reward prediction errors (PEs), the difference between obtained and expected rewards. The PE is thought to be a crucial signal for instrumental learning, and interference with DA transmission impairs learning. Phasic increases of DA neuron firing during positive PEs are driven by activation of NMDA receptors, whereas phasic suppression of firing during negative PEs is likely mediated by inputs from the lateral habenula. We aimed to determine the contribution of DA D2-class and NMDA receptors to appetitively and aversively motivated reinforcement learning. Healthy human volunteers were scanned with functional magnetic resonance imaging while they performed an instrumental learning task under the influence of either the DA D2 receptor antagonist amisulpride (400 mg), the NMDA receptor antagonist memantine (20 mg), or placebo. Participants quickly learned to select ("approach") rewarding and to reject ("avoid") punishing options. Amisulpride impaired both approach and avoidance learning, while memantine mildly attenuated approach learning but had no effect on avoidance learning. These behavioral effects of the antagonists were paralleled by their modulation of striatal PEs. Amisulpride reduced both appetitive and aversive PEs, while memantine diminished appetitive, but not aversive PEs. These data suggest that striatal D2-class receptors contribute to both approach and avoidance learning by detecting both the phasic DA increases and decreases during appetitive and aversive PEs. NMDA receptors on the contrary appear to be required only for approach learning because phasic DA increases during positive PEs are NMDA dependent, whereas phasic decreases during negative PEs are not. PMID:25253860

  10. Nucleoside-derived antagonists to A3 adenosine receptors lower mouse intraocular pressure and act across species.

    PubMed

    Wang, Zhao; Do, Chi Wai; Avila, Marcel Y; Peterson-Yantorno, Kim; Stone, Richard A; Gao, Zhan-Guo; Joshi, Bhalchandra; Besada, Pedro; Jeong, Lak Shin; Jacobson, Kenneth A; Civan, Mortimer M

    2010-01-01

    The purpose of the study was to determine whether novel, selective antagonists of human A3 adenosine receptors (ARs) derived from the A3-selective agonist Cl-IB-MECA lower intraocular pressure (IOP) and act across species. IOP was measured invasively with a micropipette by the Servo-Null Micropipette System (SNMS) and by non-invasive pneumotonometry during topical drug application. Antagonist efficacy was also assayed by measuring inhibition of adenosine-triggered shrinkage of native bovine nonpigmented ciliary epithelial (NPE) cells. Five agonist-based A3AR antagonists lowered mouse IOP measured with SNMS tonometry by 3-5 mm Hg within minutes of topical application. Of the five agonist derivatives, LJ 1251 was the only antagonist to lower IOP measured by pneumotonometry. No effect was detected pneumotonometrically over 30 min following application of the other four compounds, consonant with slower, smaller responses previously measured non-invasively following topical application of A3AR agonists and the dihydropyridine A3AR antagonist MRS 1191. Latanoprost similarly lowered SNMS-measured IOP, but not IOP measured non-invasively over 30 min. Like MRS 1191, agonist-based A3AR antagonists applied to native bovine NPE cells inhibited adenosine-triggered shrinkage. In summary, the results indicate that antagonists of human A3ARs derived from the potent, selective A3 agonist Cl-IB-MECA display efficacy in mouse and bovine cells, as well. When intraocular delivery was enhanced by measuring mouse IOP invasively, five derivatives of the A3AR agonist Cl-IB-MECA lowered IOP but only one rapidly reduced IOP measured non-invasively after topical application. We conclude that derivatives of the highly-selective A3AR agonist Cl-IB-MECA can reduce IOP upon reaching their intraocular target, and that nucleoside-based derivatives are promising A3 antagonists for study in multiple animal models. PMID:19878673

  11. Nucleoside-Derived Antagonists to A3 Adenosine Receptors Lower Mouse Intraocular Pressure and Act across Species

    PubMed Central

    Wang, Zhao; Do, Chi Wai; Avila, Marcel Y.; Peterson-Yantorno, Kim; Stone, Richard A.; Gao, Zhan-Guo; Joshi, Bhalchandra; Besada, Pedro; Jeong, Lak Shin; Jacobson, Kenneth A.; Civan, Mortimer M.

    2009-01-01

    The purpose of the study was to determine whether novel, selective antagonists of human A3 adenosine receptors (ARs) derived from the A3-selective agonist Cl-IB-MECA lower intraocular pressure (IOP) and act across species. IOP was measured invasively with a micropipette by the Servo-Null Micropipette System (SNMS) and by non-invasive pneumotonometry during topical drug application. Antagonist efficacy was also assayed by measuring inhibition of adenosine-triggered shrinkage of native bovine nonpigmented ciliary epithelial (NPE) cells. Five agonist-based A3AR antagonists lowered mouse IOP measured with SNMS tonometry by 3–5 mm Hg within minutes of topical application. Of the five agonist derivatives, LJ 1251 was the only antagonist to lower IOP measured by pneumotonometry. No effect was detected pneumotonometrically over 30 min following application of the other four compounds, consonant with slower, smaller responses previously measured non-invasively following topical application of A3AR agonists and the dihydropyridine A3AR antagonist MRS 1191. Latanoprost similarly lowered SNMS-measured IOP, but not IOP measured non-invasively over 30 minutes. Like MRS 1191, agonist-based A3AR antagonists applied to native bovine NPE cells inhibited adenosine-triggered shrinkage. In summary, the results indicate that antagonists of human A3ARs derived from the potent, selective A3 agonist Cl-IB-MECA display efficacy in mouse and bovine cells, as well. When intraocular delivery was enhanced by measuring mouse IOP invasively, five derivatives of the A3AR agonist Cl-IB-MECA lowered IOP but only one rapidly reduced IOP measured non-invasively after topical application. We conclude that derivatives of the highly selective A3AR agonist Cl-IB-MECA can reduce IOP upon reaching their intraocular target, and that nucleoside-based derivatives are promising A3 antagonists for study in multiple animal models. PMID:19878673

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

    PubMed

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

    2012-08-01

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

  13. CF102 an A3 Adenosine Receptor Agonist Mediates Anti-Tumor and Anti-Inflammatory Effects in the Liver

    PubMed Central

    COHEN, S.; STEMMER, S.M.; ZOZULYA, G.; OCHAION, A.; PATOKA, R.; BARER, F.; BAR-YEHUDA, S.; RATH-WOLFSON, L.; JACOBSON, K.A.; FISHMAN, P.

    2012-01-01

    The Gi protein-associated A3 adenosine receptor (A3AR) is a member of the adenosine receptor family. Selective agonists at the A3AR, such as CF101 and CF102 were found to induce anti-inflammatory and anti-cancer effects. In this study, we examined the differential effect of CF102 in pathological conditions of the liver. The anti-inflammatory protective effect of CF101 was tested in a model of liver inflammation induced by Concanavalin A (Con. A) and the anti-cancer effect of CF102 was examined in vitro and in a xenograft animal model utilizing Hep-3B hepatocellular carcinoma (HCC) cells. The mechanism of action was explored by following the expression levels of key signaling proteins in the inflamed and tumor liver tissues, utilizing Western blot (WB) analysis. In the liver inflammation model, CF102 (100 μg/kg) markedly reduced the secretion of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase in comparison to the vehicle-treated group. Mechanistically, CF102 treatment decreased the expression level of phosphorylated glycogen synthase kinase-3β, NF-κB, and TNF-α and prevented apoptosis in the liver. This was demonstrated by decreased expression levels of Fas receptor (FasR) and of the pro-apoptotic proteins Bax and Bad in liver tissues. In addition, CF102-induced apoptosis of Hep-3B cells both in vitro and in vivo via de-regulation of the PI3K-NF-κB signaling pathway, resulting in up-regulation of pro-apoptotic proteins. Taken together, CF102 acts as a protective agent in liver inflammation and inhibits HCC tumor growth. These results suggest that CF102 through its differential effect is a potential drug candidate to treat various pathological liver conditions. PMID:21660967

  14. CF102 an A3 adenosine receptor agonist mediates anti-tumor and anti-inflammatory effects in the liver.

    PubMed

    Cohen, S; Stemmer, S M; Zozulya, G; Ochaion, A; Patoka, R; Barer, F; Bar-Yehuda, S; Rath-Wolfson, L; Jacobson, K A; Fishman, P

    2011-09-01

    The Gi protein-associated A(3) adenosine receptor (A(3) AR) is a member of the adenosine receptor family. Selective agonists at the A(3) AR, such as CF101 and CF102 were found to induce anti-inflammatory and anti-cancer effects. In this study, we examined the differential effect of CF102 in pathological conditions of the liver. The anti-inflammatory protective effect of CF101 was tested in a model of liver inflammation induced by Concanavalin A (Con. A) and the anti-cancer effect of CF102 was examined in vitro and in a xenograft animal model utilizing Hep-3B hepatocellular carcinoma (HCC) cells. The mechanism of action was explored by following the expression levels of key signaling proteins in the inflamed and tumor liver tissues, utilizing Western blot (WB) analysis. In the liver inflammation model, CF102 (100 µg/kg) markedly reduced the secretion of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase in comparison to the vehicle-treated group. Mechanistically, CF102 treatment decreased the expression level of phosphorylated glycogen synthase kinase-3β, NF-κB, and TNF-α and prevented apoptosis in the liver. This was demonstrated by decreased expression levels of Fas receptor (FasR) and of the pro-apoptotic proteins Bax and Bad in liver tissues. In addition, CF102-induced apoptosis of Hep-3B cells both in vitro and in vivo via de-regulation of the PI3K-NF-κB signaling pathway, resulting in up-regulation of pro-apoptotic proteins. Taken together, CF102 acts as a protective agent in liver inflammation and inhibits HCC tumor growth. These results suggest that CF102 through its differential effect is a potential drug candidate to treat various pathological liver conditions. PMID:21660967

  15. Angiotensin and mineralocorticoid receptor antagonism attenuates cardiac oxidative stress in angiotensin II-infused rats.

    PubMed

    Minas, Jacqueline N; Thorwald, Max A; Conte, Debra; Vázquez-Medina, Jose-Pablo; Nishiyama, Akira; Ortiz, Rudy M

    2015-11-01

    Angiotensin II (Ang II) and aldosterone contribute to hypertension, oxidative stress and cardiovascular damage, but the contributions of aldosterone during Ang II-dependent hypertension are not well defined because of the difficulty to assess each independently. To test the hypothesis that during Ang II infusion, oxidative and nitrosative damage is mediated through both the mineralocorticoid receptor (MR) and angiotensin type 1 receptor (AT1), five groups of Sprague-Dawley rats were studied: (i) control; (ii) Ang II infused (80 ng/min × 28 days); (iii) Ang II + AT1 receptor blocker (ARB; 10 mg losartan/kg per day × 21 days); (iv) Ang II + mineralocorticoid receptor (MR) antagonist (Epl; 100 mg eplerenone/day × 21 days); and (v) Ang II + ARB + Epl (Combo; × 21 days). Both ARB and combination treatments completely alleviated the Ang II-induced hypertension, whereas eplerenone treatment only prolonged the onset of the hypertension. Eplerenone treatment exacerbated the Ang II-mediated increase in plasma and heart aldosterone 2.3- and 1.8-fold, respectively, while ARB treatment reduced both. Chronic MR blockade was sufficient to ameliorate the AT1-mediated increase in oxidative damage. All treatments normalized protein oxidation (nitrotyrosine) levels; however, only ARB and Combo treatments completely reduced lipid peroxidation (4-hydroxynonenal) to control levels. Collectively, these data suggest that receptor signalling, and not the elevated arterial blood pressure, is the principal culprit in the oxidative stress-associated cardiovascular damage in Ang II-dependent hypertension. PMID:26234762

  16. Interaction of the adenosine A1 receptor agonist N6-cyclopentyladenosine and κ-opioid receptors in rat spinal cord nociceptive reflexes.

    PubMed

    Ramos-Zepeda, Guillermo A; Herrero-Zorita, Carlos; Herrero, Juan F

    2014-12-01

    Antinociception induced by the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) is linked to opioid receptors. We studied the subtype of receptors to which CPA action is related, as well as a possible enhancement of antinociception when CPA is coadministered with opioid receptor agonists. Spinal cord neuronal nociceptive responses of male Wistar rats with inflammation were recorded using the single motor unit technique. CPA antinociception was challenged with naloxone or norbinaltorphimine. The antinociceptive activity of fentanyl and U-50488H was studied alone and combined with CPA. Reversal of CPA antinociception was observed with norbinaltorphimine (82.9±13% of control) but not with low doses of naloxone (27±8% of control), indicating an involvement of κ-opioid but not µ-opioid receptors. Low doses of CPA did not modify fentanyl antinociception. However, a significant enhancement of the duration of antinociception was seen when U-50488H was coadministered with CPA. We conclude that antinociception mediated by CPA in the spinal cord is associated with activation of κ-opioid but not µ-opioid receptors in inflammation. In addition, coadministration of CPA and κ-opioid receptor agonists is followed by significantly longer antinociception, opening new perspectives in the treatment of chronic inflammatory pain. PMID:25325292

  17. Prevention of adenosine A2A receptor activation diminishes beat-to-beat alternation in human atrial myocytes.

    PubMed

    Molina, Cristina E; Llach, Anna; Herraiz-Martínez, Adela; Tarifa, Carmen; Barriga, Montserrat; Wiegerinck, Rob F; Fernandes, Jacqueline; Cabello, Nuria; Vallmitjana, Alex; Benitéz, Raúl; Montiel, José; Cinca, Juan; Hove-Madsen, Leif

    2016-01-01

    Atrial fibrillation (AF) has been associated with increased spontaneous calcium release from the sarcoplasmic reticulum and linked to increased adenosine A2A receptor (A2AR) expression and activation. Here we tested whether this may favor atrial arrhythmogenesis by promoting beat-to-beat alternation and irregularity. Patch-clamp and confocal calcium imaging was used to measure the beat-to-beat response of the calcium current and transient in human atrial myocytes. Responses were classified as uniform, alternating or irregular and stimulation of Gs-protein coupled receptors decreased the frequency where a uniform response could be maintained from 1.0 ± 0.1 to 0.6 ± 0.1 Hz; p < 0.01 for beta-adrenergic receptors and from 1.4 ± 0.1 to 0.5 ± 0.1 Hz; p < 0.05 for A2ARs. The latter was linked to increased spontaneous calcium release and after-depolarizations. Moreover, A2AR activation increased the fraction of non-uniformly responding cells in HL-1 myocyte cultures from 19 ± 3 to 51 ± 9 %; p < 0.02, and electrical mapping in perfused porcine atria revealed that adenosine induced electrical alternans at longer cycle lengths, doubled the fraction of electrodes showing alternation, and increased the amplitude of alternations. Importantly, protein kinase A inhibition increased the highest frequency where uniform responses could be maintained from 0.84 ± 0.12 to 1.86 ± 0.11 Hz; p < 0.001 and prevention of A2AR-activation with exogenous adenosine deaminase selectively increased the threshold from 0.8 ± 0.1 to 1.2 ± 0.1 Hz; p = 0.001 in myocytes from patients with AF. In conclusion, A2AR-activation promotes beat-to-beat irregularities in the calcium transient in human atrial myocytes, and prevention of A2AR activation may be a novel means to maintain uniform beat-to-beat responses at higher beating frequencies in patients with atrial fibrillation. PMID:26611209

  18. Fibroblast and endothelial outgrowth from human Tenon's explants: inhibition of fibroblast growth by 5HT receptor antagonism.

    PubMed

    Mullaney, P; Curren, B; Collum, L; Kilfeather, S

    1991-11-01

    Tenon's layer capsule is a major source of fibroplasia associated with post-traumatic wound healing in the external eye and failure of filtration surgery for glaucoma (trabeculectomy). We have investigated the cell types involved in outgrowth from human Tenon's layer explants in culture. Outgrowth from explants maintained in fetal bovine serum (5%) and fibroblast conditioned medium (25%) consisted of endothelial cells exhibiting factor VIII antibody staining and were located to sectioned capillary ends. In media supplemented with human serum (10%), fibroblast outgrowth was observed from the entire perimeter of explants. The mitogenic stimulus of human serum on fibroblasts, monitored by 3H-thymidine uptake, was dose-dependent and accompanied by a 200% increase in inositol phosphate production. 5HT induced a significant increase in fibroblast proliferation and 5HT receptor antagonism with methysergide (20 microM) reduced serum-induced mitogenesis by 30%. These findings support the role of fibroblasts in failure of filtration surgery for glaucoma and suggest a role for 5HT in serum-derived Tenon's fibroplasia. PMID:1811281

  19. Antagonism of histamine H4 receptors exacerbates clinical and pathological signs of experimental autoimmune encephalomyelitis

    PubMed Central

    Ballerini, C; Aldinucci, A; Luccarini, I; Galante, A; Manuelli, C; Blandina, P; Katebe, M; Chazot, P L; Masini, E; Passani, M B

    2013-01-01

    Background and Purpose The histamine H4 receptor has a primary role in inflammatory functions, making it an attractive target for the treatment of asthma and refractory inflammation. These observations suggested a facilitating action on autoimmune diseases. Here we have assessed the role of H4 receptors in experimental autoimmune encephalomyelitis (EAE) a model of multiple sclerosis (MS). Experimental Approach We induced EAE with myelin oligodendrocyte glycoprotein (MOG35–55) in C57BL/6 female mice as a model of MS. The histamine H4 receptor antagonist 5-chloro-2-[(4-methylpiperazin-1-yl)carbonyl]-1H-indole (JNJ7777120) was injected i.p. daily starting at day 10 post-immunization (D10 p.i.). Disease severity was monitored by clinical and histopathological evaluation of inflammatory cells infiltrating into the spinal cord, anti-MOG35–55 antibody production, assay of T-cell proliferation by [3H]-thymidine incorporation, mononucleate cell phenotype by flow cytometry, cytokine production by elisa assay and transcription factor quantification of mRNA expression. Key Results Treatment with JNJ7777120 exacerbated EAE, increased inflammation and demyelination in the spinal cord of EAE mice and increased IFN-γ expression in lymph nodes, whereas it suppressed IL-4 and IL-10, and augmented expression of the transcription factors Tbet, FOXP3 and IL-17 mRNA in lymphocytes. JNJ7777120 did not affect proliferation of anti-MOG35–55 T-cells, anti-MOG35–55 antibody production or mononucleate cell phenotype. Conclusions and Implications H4 receptor blockade was detrimental in EAE. Given the interest in the development of H4 receptor antagonists as anti-inflammatory compounds, it is important to understand the role of H4 receptors in immune diseases to anticipate clinical benefits and also predict possible detrimental effects. Linked Articles This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http

  20. 5'-Substituted Amiloride Derivatives as Allosteric Modulators Binding in the Sodium Ion Pocket of the Adenosine A2A Receptor.

    PubMed

    Massink, Arnault; Louvel, Julien; Adlere, Ilze; van Veen, Corine; Huisman, Berend J H; Dijksteel, Gabrielle S; Guo, Dong; Lenselink, Eelke B; Buckley, Benjamin J; Matthews, Hayden; Ranson, Marie; Kelso, Michael; IJzerman, Adriaan P

    2016-05-26

    The sodium ion site is an allosteric site conserved among many G protein-coupled receptors (GPCRs). Amiloride 1 and 5-(N,N-hexamethylene)amiloride 2 (HMA) supposedly bind in this sodium ion site and can influence orthosteric ligand binding. The availability of a high-resolution X-ray crystal structure of the human adenosine A2A receptor (hA2AAR), in which the allosteric sodium ion site was elucidated, makes it an appropriate model receptor for investigating the allosteric site. In this study, we report the synthesis and evaluation of novel 5'-substituted amiloride derivatives as hA2AAR allosteric antagonists. The potency of the amiloride derivatives was assessed by their ability to displace orthosteric radioligand [(3)H]4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol ([(3)H]ZM-241,385) from both the wild-type and sodium ion site W246A mutant hA2AAR. 4-Ethoxyphenethyl-substituted amiloride 12l was found to be more potent than both amiloride and HMA, and the shift in potency between the wild-type and mutated receptor confirmed its likely binding to the sodium ion site. PMID:27124340

  1. Grape powder attenuates the negative effects of GLP-1 receptor antagonism by exendin-3 (9-39) in a normoglycemic mouse model.

    PubMed

    Haufe, T C; Gilley, A D; Goodrich, K M; Ryan, C M; Smithson, A T; Hulver, M W; Liu, D; Neilson, A P

    2016-06-15

    Prediabetes is a condition affecting 35% of US adults and about 50% of US adults age 65+. Foods rich in polyphenols, including flavanols and other flavonoids, have been studied for their putative beneficial effects on many different health conditions including type 2 diabetes mellitus and prediabetes. Studies have shown that some flavanols increase glucagon-like peptide 1 (GLP-1) secretion. GLP-1 is a feeding hormone that increases insulin secretion after carbohydrate consumption, and increased GLP-1 secretion may be responsible for some of the beneficial effects on glycemic control after flavanol consumption. The present study explored the effects of grape powder consumption on metrics of glycemic health in normoglycemic and prediabetic C57BL/6J mice; additionally, the mechanism of action of grape powder polyphenols was investigated. Grape powder significantly reduced (p < 0.01) blood glucose levels following oral glucose gavage after GLP-1 receptor antagonism by exendin-3 (9-39) compared to sugar-matched control, indicating that it was able to attenuate the hyperglycemic effects of GLP-1 receptor antagonism. Grape powder was employed in acute (1.6 g grape powder per kg bodyweight) and long-term high fat diet (grape powder incorporated into treatment diets at 5% w/w) feeding studies in normoglycemic and prediabetic (diet-induced obesity) mice; grape powder did not impove glycemic control in these studies versus sugar-matched control. The mechanisms by which grape powder ameliorates the deleterious effects of GLP-1 receptor antagonism warrant further study. PMID:27189193

  2. A2B adenosine receptor contributes to penile erection via PI3K/AKT signaling cascade-mediated eNOS activation

    PubMed Central

    Wen, Jiaming; Grenz, Almut; Zhang, Yujin; Dai, Yingbo; Kellems, Rodney E.; Blackburn, Michael R.; Eltzschig, Holger K.; Xia, Yang

    2011-01-01

    Normal penile erection is under the control of multiple factors and signaling pathways. Although adenosine signaling is implicated in normal and abnormal penile erection, the exact role and the underlying mechanism for adenosine signaling in penile physiology remain elusive. Here we report that shear stress leads to increased adenosine release from endothelial cells. Subsequently, we determined that ecto-5′-nucleotidase (CD73) is a key enzyme required for the production of elevated adenosine from ATP released by shear-stressed endothelial cells. Mechanistically, we demonstrate that shear stress-mediated elevated adenosine functions through the adenosine A2B receptor (A2BR) to activate the PI3K/AKT signaling cascade and subsequent increased endothelial nitric oxide synthase (eNOS) phosphorylation. These in vitro studies led us to discover further that adenosine was induced during sustained penile erection and contributes to PI3K/AKT activation and subsequent eNOS phosphorylation via A2BR signaling in intact animal. Finally, we demonstrate that lowering adenosine in wild-type mice or genetic deletion of A2BR in mutant mice significantly attenuated PI3K/AKT activation, eNOS phosphorylation, and subsequent impaired penile erection featured with the reduction of ratio of maximal intracavernosal pressure to systemic arterial pressure from 0.49 ± 0.03 to 0.41 ± 0.05 and 0.38 ± 0.04, respectively (both P<0.05). Overall, using biochemical, cellular, genetic, and physiological approaches, our findings reveal that adenosine is a novel molecule signaling via A2BR activation, contributing to penile erection via PI3K/AKT-dependent eNOS activation. These studies suggest that this signaling pathway may be a novel therapeutic target for erectile disorders.—Wen, J., Grenz, A., Zhang, Y., Dai, Y., Kellems, R. E., Blackburn, M. R., Eltzschig, H. K., Xia, Y. A2B adenosine receptor contributes to penile erection via PI3K/AKT signaling cascade-mediated eNOS activation. PMID

  3. Structure–Activity Relationships and Molecular Modeling of 3,5-Diacyl-2,4-dialkylpyridine Derivatives as Selective A3 Adenosine Receptor Antagonists

    PubMed Central

    Li, An-Hu; Moro, Stefano; Melman, Neli; Ji, Xiao-duo; Jacobson, Kenneth A.

    2012-01-01

    The structure-activity relationships of 6-phenyl-1,4-dihydropyridine derivatives as selective antagonists at human A3 adenosine receptors have been explored (Jiang et al. J. Med. Chem. 1997, 39, 4667-4675). In the present study, related pyridine derivatives have been synthesized and tested for affinity at adenosine receptors in radioligand binding assays. Ki values in the nanomolar range were observed for certain 3,5-diacyl-2,4-dialkyl-6-phenylpyridine derivatives in displacement of [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5′-N-methylcarbamoyladenosine) at recombinant human A3 adenosine receptors. Selectivity for A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors. Structure–activity relationships at various positions of the pyridine ring (the 3- and 5-acyl substituents and the 2- and 4-alkyl substituents) were probed. A 4-phenylethynyl group did not enhance A3 selectivity of pyridine derivatives, as it did for the 4-substituted dihydropyridines. At the 2-and 4-positions ethyl was favored over methyl. Also, unlike the dihydropyridines, a thioester group at the 3-position was favored over an ester for affinity at A3 adenosine receptors, and a 5-position benzyl ester decreased affinity. Small cycloalkyl groups at the 6-position of 4-phenylethynyl-1,4-dihydropyridines were favorable for high affinity at human A3 adenosine receptors, while in the pyridine series a 6-cyclopentyl group decreased affinity. 5-Ethyl 2,4-diethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate, 38, was highly potent at human A3 receptors, with a Ki value of 20 nM. A 4-propyl derivative, 39b, was selective and highly potent at both human and rat A3 receptors, with Ki values of 18.9 and 113 nM, respectively. A 6-(3-chlorophenyl) derivative, 44, displayed a Ki value of 7.94 nM at human A3 receptors and selectivity of 5200-fold. Molecular modeling, based on the steric and electrostatic alignment (SEAL) method, defined common pharmacophore

  4. Pharmacologically targeted NMDA receptor antagonism by NitroMemantine for cerebrovascular disease

    PubMed Central

    Takahashi, Hiroto; Xia, Peng; Cui, Jiankun; Talantova, Maria; Bodhinathan, Karthik; Li, Wenjun; Holland, Emily A.; Tong, Gary; Piña-Crespo, Juan; Zhang, Dongxian; Nakanishi, Nobuki; Larrick, James W.; McKercher, Scott R.; Nakamura, Tomohiro; Wang, Yuqiang; Lipton, Stuart A.

    2015-01-01

    Stroke and vascular dementia are leading causes of morbidity and mortality. Neuroprotective therapies have been proposed but none have proven clinically tolerated and effective. While overstimulation of N-methyl-d-aspartate-type glutamate receptors (NMDARs) is thought to contribute to cerebrovascular insults, the importance of NMDARs in physiological function has made this target, at least in the view of many in ‘Big Pharma,’ ‘undruggable’ for this indication. Here, we describe novel NitroMemantine drugs, comprising an adamantane moiety that binds in the NMDAR-associated ion channel that is used to target a nitro group to redox-mediated regulatory sites on the receptor. The NitroMemantines are both well tolerated and effective against cerebral infarction in rodent models via a dual allosteric mechanism of open-channel block and NO/redox modulation of the receptor. Targeted S-nitrosylation of NMDARs by NitroMemantine is potentiated by hypoxia and thereby directed at ischemic neurons. Allosteric approaches to tune NMDAR activity may hold therapeutic potential for cerebrovascular disorders. PMID:26477507

  5. Antagonism of Neuronal Prostaglandin E2 Receptor Subtype 1 Mitigates Amyloid β Neurotoxicity In Vitro

    PubMed Central

    Li, Xianwu; Rose, Shannon; Montine, Kathleen; Keene, C. Dirk; Montine, Thomas J.

    2013-01-01

    Multiple lines of evidence indicate that regional brain eicosanoid signaling is important in initiation and progression of neurodegenerative conditions that have a neuroinflammatory pathologic component, such as AD. We hypothesized that PGE2 receptor subtype 1 (EP1) signaling (linked to intracellular Ca2+ release) regulates Aβ peptide neurotoxicity and tested this in two complementary in vitro models: a human neuroblastoma cell line (MC65) producing Aβ1-40 through conditional expression of the APP C-terminal portion, and murine primary cortical neuron cultures exposed to Aβ1-42. In MC65 cells, EP1 receptor antagonist SC-51089 reduced Aβ neurotoxicity ~50% without altering high molecular weight Aβ immunoreactive species formation. Inositol-3-phosphate receptor antagonist 2-aminoethoxy-diphenyl borate offered similar protection. SC-51089 largely protected the neuron cultures from synthetic Aβ1-42 neurotoxicity. Nimodipine, a Ca2+ channel blocker, was completely neuroprotective in both models. Based on these data, we conclude that suppressing neuronal EP1 signaling may represent a promising therapeutic approach to ameliorate Aβ peptide neurotoxicity. PMID:22718277

  6. Central V1b receptor antagonism in lactating rats: impairment of maternal care but not of maternal aggression.

    PubMed

    Bayerl, D S; Klampfl, S M; Bosch, O J

    2014-12-01

    Maternal behaviour in rodents is mediated by the central oxytocin and vasopressin systems, amongst others. The role of vasopressin, acting via the V1a receptor (V1aR), on maternal care and maternal aggression has recently been described. However, a potential involvement of the V1b receptor (V1bR) in maternal behaviour has only been demonstrated in knockout mice. The present study aimed to examine the effects of central pharmacological manipulation of the V1bR on maternal behaviour in lactating Wistar rats. On pregnancy day 18, female rats were implanted with a guide cannula targeting the lateral ventricle. After parturition, dams received an acute central infusion of a specific V1bR agonist (d[Leu4,Lys8]VP) or V1bR antagonist (SSR149415) once daily, followed by observations of maternal care [lactation day (LD) 1], maternal motivation in the pup retrieval test (LD 2), anxiety-related behaviour on the elevated plus-maze (LD 3) and maternal aggression in the maternal defence test followed by maternal care monitoring (LD 4). Our data demonstrate that, under nonstress conditions, the V1bR antagonist decreased the occurrence of both nursing and mother-pup interaction, whereas the V1bR agonist did not affect either parameter. Under stress conditions (i.e. after the maternal defence test), mother-pup interaction was decreased by infusion of the V1bR antagonist. During the maternal defence test, neither treatment affected aggressive or non-aggressive behaviour. Finally, neither treatment altered maternal motivation or anxiety. In conclusion, central V1bR antagonism modulates aspects of maternal care but not of maternal aggression or maternal motivation in lactating rats. These findings further extend our knowledge on the vasopressin system as a vital mediator of maternal behaviour. PMID:25283607

  7. Subgingival Plaque in Periodontal Health Antagonizes at Toll-Like Receptor 4 and Inhibits E-Selectin Expression on Endothelial Cells

    PubMed Central

    Gümüş, Pinar; Nizam, Nejat; Buduneli, Nurcan

    2015-01-01

    The ability of the subgingival microbial community to induce an inappropriate inflammatory response ultimately results in the destruction of bone and gingival tissue. In this study, subgingival plaque samples from both healthy and diseased sites in the same individual were obtained from adults with chronic periodontitis and screened for their ability to either activate Toll-like receptor 2 (TLR2) or TLR4 and to antagonize TLR4-specific activation by agonist, Fusobacterium nucleatum LPS. Subgingival plaque from diseased sites strongly activated TLR4, whereas matched plaque samples obtained from healthy sites were significantly more variable, with some samples displaying strong TLR4 antagonism, while others were strong TLR4 agonists when combined with F. nucleatum LPS. Similar results were observed when TLR4 dependent E-selectin expression by endothelial cells was determined. These results are the first to demonstrate TLR4 antagonism from human plaque samples and demonstrate that healthy but not diseased sites display a wide variation in TLR4 agonist and antagonist behavior. The results have identified a novel characteristic of clinically healthy sites and warrant further study on the contribution of TLR4 antagonism in the progression of a healthy periodontal site to a diseased one. PMID:26483407

  8. Selective antagonism of the GABAA receptor by ciprofloxacin and biphenylacetic acid

    PubMed Central

    Green, M A; Halliwell, R F

    1997-01-01

    Previous studies have shown that ciprofloxacin and biphenylacetic acid (BPAA) synergistically inhibit γ-aminobutyric acid (GABA)A receptors. In the present study, we have investigated the actions of these two drugs on other neuronal ligand-gated ion channels. Agonist-evoked depolarizations were recorded from rat vagus and optic nerves in vitro by use of an extracellular recording technique. GABA (50 μM)-evoked responses, in the vagus nerve in vitro, were inhibited by bicuculline (0.3–10 μM) and picrotoxin (0.3–10 μM), with IC50 values and 95% confidence intervals (CI) of 1.2 μM (1.1–1.4) and 3.6 μM (3.0–4.3), respectively, and were potentiated by sodium pentobarbitone (30 μM) and diazepam (1 μM) to (mean±s.e.mean) 168±18% and 117±4% of control, respectively. 5-Hydroxytryptamine (5-HT; 0.5 μM)-evoked responses were inhibited by MDL 72222 (1 μM) to 10±4% of control; DMPP (10 μM)-evoked responses were inhibited by hexamethonium (100 μM) to 12±5% of control, and αbMeATP (30 μM)-evoked responses were inhibited by PPADS (10 μM) to 21±5% of control. Together, these data are consistent with activation of GABAA, 5-HT3, nicotinic ACh and P2X receptors, respectively. Ciprofloxacin (10–3000 μM) inhibited GABAA-mediated responses in the vagus nerve with an IC50 (and 95% CI) of 202 μM (148–275). BPAA (1–1000 μM) had little or no effect on the GABAA-mediated response but concentration-dependently potentiated the effects of ciprofloxacin by up to 33,000 times. Responses mediated by 5-HT3, nicotinic ACh and P2X receptors in the vagus nerve and strychnine-sensitive glycine receptors in the optic nerve were little or unaffected by ciprofloxacin (100 μM), BPAA (100 μM) or the combination of these drugs (both at 100 μM). GABA (1 mM)-evoked responses in the optic nerve were inhibited by bicuculline with an IC50 of 3.6 μM (2.8–4.5), a value not significantly different from that determined in the vagus

  9. Selective antagonism of the GABA(A) receptor by ciprofloxacin and biphenylacetic acid.

    PubMed

    Green, M A; Halliwell, R F

    1997-10-01

    1. Previous studies have shown that ciprofloxacin and biphenylacetic acid (BPAA) synergistically inhibit y-aminobutyric acid (GABA)A receptors. In the present study, we have investigated the actions of these two drugs on other neuronal ligand-gated ion channels. 2. Agonist-evoked depolarizations were recorded from rat vagus and optic nerves in vitro by use of an extracellular recording technique. 3. GABA (50 microM)-evoked responses, in the vagus nerve in vitro, were inhibited by bicuculline (0.3-10 microM) and picrotoxin (0.3-10 microM), with IC50 values and 95% confidence intervals (CI) of 1.2 microM (1.1-1.4) and 3.6 microM (3.0-4.3), respectively, and were potentiated by sodium pentobarbitone (30 microM) and diazepam (1 microM) to (mean+/-s.e.mean) 168+/-18% and 117+/-4% of control, respectively. 5-Hydroxytryptamine (5-HT; 0.5 microM)-evoked responses were inhibited by MDL 72222 (1 microM) to 10+/-4% of control; DMPP (10 microM)-evoked responses were inhibited by hexamethonium (100 microM) to 12+/-5% of control, and alphabetaMeATP (30 microM)-evoked responses were inhibited by PPADS (10 microM) to 21+/-5% of control. Together, these data are consistent with activation of GABA(A), 5-HT3, nicotinic ACh and P2X receptors, respectively. 4 Ciprofloxacin (10-3000 microM) inhibited GABA(A)-mediated responses in the vagus nerve with an IC50 (and 95% CI) of 202 microM (148-275). BPAA (1-1000 microM) had little or no effect on the GABA(A)-mediated response but concentration-dependently potentiated the effects of ciprofloxacin by up to 33,000 times. 5. Responses mediated by 5-HT3, nicotinic ACh and P2X receptors in the vagus nerve and strychnine-sensitive glycine receptors in the optic nerve were little or unaffected by ciprofloxacin (100 microM), BPAA (100 microM) or the combination of these drugs (both at 100 microM). 6. GABA (1 mM)-evoked responses in the optic nerve were inhibited by bicuculline with an IC50 of 3.6 microM (2.8-4.5), a value not significantly different

  10. Corticotropin releasing factor-1 receptor antagonism alters the biochemical, but not behavioral effects of repeated interleukin-1β administration

    PubMed Central

    Wilhelm, Clare J.; Murphy-Crews, Aaron; Menasco, Daniel J.; Huckans, Marilyn S.; Loftis, Jennifer M.

    2011-01-01

    Activation of the immune system via administration of cytokines is used for the treatment of chronic viral infections such as hepatitis C and for cancers resistant to radiotherapy. Cytokine-based treatments induce a range of “sickness” behaviors (e.g. depression, anxiety, pain, anorexia, and fatigue). Activation of the hypothalamic pituitary-adrenal axis via the induction of corticotropin releasing factor (CRF) may underlie these unwanted side effects. This study used repeated systemic injections of the pro-inflammatory cytokine interleukin-1β (IL-1β) to model the sickness behaviors and biochemical effects of immune system activation. We assessed the ability of CRF type I receptor (CRF1) antagonism to reduce biochemical and behavioral signs of sickness induced by IL-1β treatment. Forty Wistar rats were assigned to one of four groups: 1) saline + vehicle; 2) saline + DMP904 (CRF1 antagonist); 3) IL-1β + vehicle; 4) IL-1β + DMP904. Rats received intraperitoneal injections of either DMP904 or vehicle and of IL-1β or saline for six days. Sickness behavior was evaluated using body weight assessments and forced swim testing (FST). Blood and brain samples were collected to measure cytokine, p38 mitogen activated protein kinase (MAPK), and phospho-p38 MAPK levels using multiplex techniques. There were significant reductions in body weights and FST immobility times associated with IL-1β administration. Rats administered IL-1β had significantly higher serum levels of IL-10, but not interferon-γ. Within the hippocampus, IL-1β reduced levels of p38 MAPK, but had no impact on levels of phospho-p38 MAPK except in the presence of DMP904. When administered alone, DMP904 had no significant effect on p38 MAPK or phospho-p38 MAPK in the hippocampus, but when given with IL-1β led to increased phosphorylation of p38 MAPK. IL-1β and DMP904 reduced levels of p38 MAPK within the hypothalamus, while co-administration of IL-1β and DMP904 abolished the effects of either drug

  11. Corticotropin releasing factor-1 receptor antagonism alters the biochemical, but not behavioral effects of repeated interleukin-1β administration.

    PubMed

    Wilhelm, Clare J; Murphy-Crews, Aaron; Menasco, Daniel J; Huckans, Marilyn S; Loftis, Jennifer M

    2012-01-01

    Activation of the immune system via administration of cytokines is used for the treatment of chronic viral infections such as hepatitis C and for cancers resistant to radiotherapy. Cytokine-based treatments induce a range of "sickness" behaviors (e.g. depression, anxiety, pain, anorexia, and fatigue). Activation of the hypothalamic pituitary-adrenal axis via the induction of corticotropin releasing factor (CRF) may underlie these unwanted side effects. This study used repeated systemic injections of the pro-inflammatory cytokine interleukin-1β (IL-1β) to model the sickness behaviors and biochemical effects of immune system activation. We assessed the ability of CRF type I receptor (CRF(1)) antagonism to reduce biochemical and behavioral signs of sickness induced by IL-1β treatment. Forty Wistar rats were assigned to one of four groups: 1) saline+vehicle; 2) saline+DMP904 (CRF(1) antagonist); 3) IL-1β+vehicle; 4) IL-1β+DMP904. Rats received intraperitoneal injections of either DMP904 or vehicle and of IL-1β or saline for six days. Sickness behavior was evaluated using body weight assessments and forced swim testing (FST). Blood and brain samples were collected to measure cytokine, p38 mitogen-activated protein kinase (MAPK), and phospho-p38 MAPK levels using multiplex techniques. There were significant reductions in body weights and FST immobility times associated with IL-1β administration. Rats administered IL-1β had significantly higher serum levels of IL-10, but not interferon-γ. Within the hippocampus, IL-1β reduced levels of p38 MAPK, but had no impact on levels of phospho-p38 MAPK except in the presence of DMP904. When administered alone, DMP904 had no significant effect on p38 MAPK or phospho-p38 MAPK in the hippocampus, but when given with IL-1β led to increased phosphorylation of p38 MAPK. IL-1β and DMP904 reduced levels of p38 MAPK within the hypothalamus, while co-administration of IL-1β and DMP904 abolished the effects of either drug alone

  12. Adenosine 2A receptor agonism: A single intrathecal administration attenuates motor paralysis in experimental autoimmune encephalopathy in rats.

    PubMed

    Loram, Lisa C; Strand, Keith A; Taylor, Frederick R; Sloane, Evan; Van Dam, Anne-Marie; Rieger, Jayson; Maier, Steven F; Watkins, Linda R

    2015-05-01

    A single intrathecal dose of adenosine 2A receptor (A2AR) agonist was previously reported to produce a multi-week reversal of allodynia in two different models of neuropathic pain in addition to downregulating glial activation markers in the spinal cord. We aimed to determine whether a single intrathecal administration of an A2AR agonist was able to attenuate motor symptoms induced by experimental autoimmune encephalopathy. Two A2AR agonists (CGS21680 and ATL313) significantly attenuated progression of motor symptoms following a single intrathecal administration at the onset of motor symptoms. OX-42, a marker of microglial activation, was significantly attenuated in the lumbar spinal cord following A2AR administration compared to vehicle. Therefore, A2AR agonists attenuate motor symptoms of EAE by acting on A2AR in the spinal cord. PMID:25653191

  13. Similarities and differences in affinity and binding modes of tricyclic pyrimido- and pyrazinoxanthines at human and rat adenosine receptors.

    PubMed

    Szymańska, Ewa; Drabczyńska, Anna; Karcz, Tadeusz; Müller, Christa E; Köse, Meryem; Karolak-Wojciechowska, Janina; Fruziński, Andrzej; Schabikowski, Jakub; Doroz-Płonka, Agata; Handzlik, Jadwiga; Kieć-Kononowicz, Katarzyna

    2016-09-15

    A new series of 32 pyrimido- and 5 tetrahydropyrazino[2,1-f]purinediones was obtained and evaluated for their adenosine receptors (ARs) affinities. The 1,3-dibutyl derivative of 9-(4-(2-(dimethylamino)ethoxy)phenyl)-6,7,8,9-tetrahydropyrimido[1,2-f]purine-2,4(1H,3H)-dione was found to be the most potent A1 AR antagonist of the present series, showing selectivity over the other AR subtypes. The structure-activity for the obtained purinediones was established. Docking experiments of the investigated library to homology models of the human and rat A1 and A2A ARs allowed to compare the expected binding modes for selected compounds. The detailed analysis of binding cavities within individual AR subtypes indicated small but significant structural variations that may underlie the observed differences in binding affinities of purinediones at particular subtypes and species. PMID:27485602

  14. The adenosine/neutrophil paradox resolved: human neutrophils possess both A1 and A2 receptors that promote chemotaxis and inhibit O2 generation, respectively.

    PubMed Central

    Cronstein, B N; Daguma, L; Nichols, D; Hutchison, A J; Williams, M

    1990-01-01

    Occupancy of specific receptors on neutrophils by adenosine or its analogues diminishes the stimulated release of toxic oxygen metabolites from neutrophils, while paradoxically promoting chemotaxis. We now report evidence that two distinct adenosine receptors are found on neutrophils (presumably the A1 and A2 receptors of other cell types). These adenosine receptors modulate chemotaxis and O2- generation, respectively. N6-Cyclopentyladenosine (CPA), a selective A1 agonist, promoted neutrophil chemotaxis to the chemoattractant FMLP as well as or better than 5'N-ethylcarboxamidoadenosine (NECA). In contrast, CPA did not inhibit O2- generation stimulated by FMLP. Pertussis toxin completely abolished promotion of chemotaxis by CPA but enhanced inhibition by NECA of O2- generation. Disruption of microtubules by colchicine or vinblastine also abrogated the enhancement by NECA of chemotaxis whereas these agents did not markedly interfere with inhibition by NECA of O2- generation. FMLP receptors, once they have bound ligand, shift to a high affinity state and become associated with the cytoskeleton. NECA significantly increased association of [3H]FMLP with cytoskeletal preparations as it inhibited O2-. Disruption of microtubules did not prevent NECA from increasing association of [3H]FMLP with cytoskeletal preparations. Additionally, CPA (A1 agonist) did not increase binding of [3H]FMLP to the cytoskeleton as well as NECA (A2 agonist). These studies indicate that occupancy of one class of adenosine receptors (A1) promotes chemotaxis by a mechanism requiring intact microtubules and G proteins whereas engagement of a second class of receptors (A2) inhibits O2- generation. Signalling via A2 receptors is independent of microtubules, insensitive to pertussis toxin and is associated with binding of [3H]FMLP to cytoskeletal preparations. PMID:2156895

  15. Inflammatory responses of airway smooth muscle cells and effects of endothelin receptor antagonism.

    PubMed

    Knobloch, Jürgen; Lin, Yingfeng; Konradi, Jürgen; Jungck, David; Behr, Juergen; Strauch, Justus; Stoelben, Erich; Koch, Andrea

    2013-07-01

    Endothelin receptor antagonists (ETRAs), authorized for pulmonary hypertension, have failed to prove their utility in chronic lung diseases with corticosteroid-resistant airway inflammation when applied at late disease stages with emphysema/fibrosis. Earlier administration might prove effective by targeting the interaction between airway inflammation and tissue remodeling. We hypothesized that human airway smooth muscle cells (HASMCs) participate in linking inflammation with remodeling and that associated genes become differentially suppressed by ambrisentan (A-receptor selective ETRA) and bosentan (nonselective/dual ETRA). Inflammatory responses of ex vivo-cultivated HASMCs to TNF-α were investigated by whole-genome microarray analyses. qRT-PCR and ELISA were used to test inflammatory and remodeling genes for sensitivity to bosentan and ambrisentan and to investigate differential sensitivities mechanistically. ETRA and corticosteroid effects were compared in HASMCs from patients with chronic obstructive pulmonary disease. TNF-α induced the expression of 18 cytokines/chemokines and five tissue remodeling genes involved in severe, corticosteroid-insensitive asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and/or pulmonary hypertension. Thirteen cytokines/chemokines, MMP13, and WISP1 were suppressed by ETRAs. Eight genes had differential sensitivity to bosentan and ambrisentan depending on the endothelin-B receptor impact on transcriptional regulation and mRNA stabilization. Chemokine (C-C motif) ligands 2 and 5, granulocyte macrophage colony-stimulating factor, and MMP13 had increased sensitivity to bosentan or bosentan/dexamethasone combination versus dexamethasone alone. Suppression of cytokine and remodeling gene expression by ETRAs was confirmed in TNF-α-activated human bronchial epithelial cells. HASMCs and human bronchial epithelial cells participate in the interaction of inflammation and tissue remodeling. This interaction is

  16. Growth hormone receptor antagonism suppresses tumour regrowth after radiotherapy in an endometrial cancer xenograft model.

    PubMed

    Evans, Angharad; Jamieson, Stephen M F; Liu, Dong-Xu; Wilson, William R; Perry, Jo K

    2016-08-28

    Human GH expression is associated with poor survival outcomes for endometrial cancer patients, enhanced oncogenicity of endometrial cancer cells and reduced sensitivity to ionising radiation in vitro, suggesting that GH is a potential target for anticancer therapy. However, whether GH receptor inhibition sensitises to radiotherapy in vivo has not been tested. In the current study, we evaluated whether the GH receptor antagonist, pegvisomant (Pfizer), sensitises to radiotherapy in vivo in an endometrial tumour xenograft model. Subcutaneous administration of pegvisomant (20 or 100 mg/kg/day, s.c.) reduced serum IGF1 levels by 23% and 68%, respectively, compared to vehicle treated controls. RL95-2 xenografts grown in immunodeficient NIH-III mice were treated with vehicle or pegvisomant (100 mg/kg/day), with or without fractionated gamma radiation (10 × 2.5 Gy over 5 days). When combined with radiation, pegvisomant significantly increased the median time tumours took to reach 3× the pre-radiation treatment volume (49 days versus 72 days; p = 0.001). Immunohistochemistry studies demonstrated that 100 mg/kg pegvisomant every second day was sufficient to abrogate MAP Kinase signalling throughout the tumour. In addition, treatment with pegvisomant increased hypoxic regions in irradiated tumours, as determined by immunohistochemical detection of pimonidazole adducts, and decreased the area of CD31 labelling in unirradiated tumours, suggesting an anti-vascular effect. Pegvisomant did not affect intratumoral staining for HIF1α, VEGF-A, CD11b, or phospho-EGFR. Our results suggest that blockade of the human GH receptor may improve the response of GH and/or IGF1-responsive endometrial tumours to radiation. PMID:27241667

  17. The adenosine 2A receptor agonist GW328267C improves lung function after acute lung injury in rats.

    PubMed

    Folkesson, Hans G; Kuzenko, Stephanie R; Lipson, David A; Matthay, Michael A; Simmons, Mark A

    2012-08-01

    There is a significant unmet need for treatments of patients with acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS). The primary mechanism that leads to resolution of alveolar and pulmonary edema is active vectorial Na(+) and Cl(-) transport across the alveolar epithelium. Several studies have suggested a role for adenosine receptors in regulating this fluid transport in the lung. Furthermore, these studies point to the A(2A) subtype of adenosine receptor (A(2A)R) as playing a role to enhance fluid transport, suggesting that activation of the A(2A)R may enhance alveolar fluid clearance (AFC). The current studies test the potential therapeutic value of the A(2A)R agonist GW328267C to accelerate resolution of alveolar edema and ALI/ARDS in rats. GW328267C, at concentrations of 10(-5) M to 10(-3) M, instilled into the airspaces, increased AFC in control animals. GW328267C did not increase AFC beyond that produced by maximal β-adrenergic stimulation. The effect of GW328267C was inhibited by amiloride but was not affected by cystic fibrosis transmembrane conductance regulator inhibition. The drug was tested in three models of ALI, HCl instillation 1 h, LPS instillation 16 h, and live Escherichia coli instillation 2 h before GW328267C instillation. After either type of injury, GW328267C (10(-4) M) decreased pulmonary edema formation and restored AFC, measured 1 h after GW328267C instillation. These findings show that GW328267C has beneficial effects in experimental models of ALI and may be a useful agent for treating patients with ALI or prophylactically to prevent ALI. PMID:22659881

  18. 3D-pharmacophore models for selective A2A and A2B adenosine receptor antagonists.

    PubMed

    Wei, Jing; Wang, Songqing; Gao, Shaofen; Dai, Xuedong; Gao, Qingzhi

    2007-01-01

    Three-dimensional pharmacophore models were generated for A2A and A2B adenosine receptors (ARs) based on highly selective A2A and A2B antagonists using the Catalyst program. The best pharmacophore model for selective A2A antagonists (Hypo-A2A) was obtained through a careful validation process. Four features contained in Hypo-A2A (one ring aromatic feature (R), one positively ionizable feature (P), one hydrogen bond acceptor lipid feature (L), and one hydrophobic feature (H)) seem to be essential for antagonists in terms of binding activity and A2A AR selectivity. The best pharmacophore model for selective A2B antagonists (Hypo-A2B) was elaborated by modifying the Catalyst common features (HipHop) hypotheses generated from the selective A2B antagonists training set. Hypo-A2B also consists of four features: one ring aromatic feature (R), one hydrophobic aliphatic feature (Z), and two hydrogen bond acceptor lipid features (L). All features play an important role in A2B AR binding affinity and are essential for A2B selectivity. Both A2A and A2B pharmacophore models have been validated toward a wide set of test molecules containing structurally diverse selective antagonists of all AR subtypes. They are capable of identifying correspondingly high potent antagonists and differentiating antagonists between subtypes. The results of our study will act as a valuable tool for retrieving structurally diverse compounds with desired biological activities and designing novel selective adenosine receptor ligands. PMID:17330954

  19. Season primes the brain in an arctic hibernator to facilitate entrance into torpor mediated by adenosine A1 receptors

    PubMed Central

    Jinka, Tulasi R.; Tøien, Øivind; Drew, Kelly L.

    2011-01-01

    Torpor in hibernating mammals defines the nadir in mammalian metabolic demand and body temperature that accommodates seasonal periods of reduced energy availability. The mechanism of metabolic suppression during torpor onset is unknown although the central nervous system (CNS) is a key regulator of torpor. Seasonal hibernators such as the arctic ground squirrel (AGS) display torpor only during the winter, hibernation season. The seasonal character of hibernation thus provides a clue to its regulation. In the present study we delivered adenosine receptor agonists and antagonists into the lateral ventricle of AGS at different times of the year while monitoring the rate of O2 consumption and core body temperature as indicators of torpor. The A1 antagonist, cyclopentyltheophylline (CPT) reversed spontaneous entrance into torpor. The adenosine A1 receptor agonist, N6-cyclohexyladenosine (CHA) induced torpor in 6 out of 6 AGS tested during the mid-hibernation season, 2 out of 6 AGS tested early in the hibernation season and none of the 6 AGS tested during the summer, off-season. CHA-induced torpor within the hibernation season was specific to A1AR activation; the A3AR agonist 2-Cl-IB MECA failed to induce torpor and the A2aR antagonist MSX-3, failed to reverse spontaneous onset of torpor. CHA-induced torpor was similar to spontaneous entrance into torpor. These results show that metabolic suppression during torpor onset is regulated within the CNS via A1AR activation and requires a seasonal switch in the sensitivity of purinergic signaling. PMID:21795527

  20. Induction of murine adenosine A(2A) receptor expression by LPS: analysis of the 5' upstream promoter.

    PubMed

    Elson, G; Eisenberg, M; Garg, C; Outram, S; Ferrante, C J; Hasko, G; Leibovich, S J

    2013-04-01

    Non-activated macrophages express low levels of A(2A)Rs and lipopolysaccharides (LPS) upregulates A(2A)R expression in an NF-κB-dependent manner. The murine A(2A)R gene is encoded by three exons, m1, m2 and m3. Exons m2 and m3 are conserved, while m1 encodes the 5' untranslated UTR. Three m1 variants have been defined, m1A, m1B and m1C, with m1C being farthest from the transcriptional start site. LPS upregulates A(2A)Rs in primary murine peritoneal and bone-marrow-derived macrophages and RAW264.7 cells by selectively splicing m1C to m2, through a promoter located upstream of m1C. We have cloned ∼1.6 kb upstream of m1C into pGL4.16(luc2CP/Hygro) promoterless vector. This construct in RAW 264.7 cells responds to LPS, and adenosine receptor agonists augmented LPS responsiveness. The NF-κB inhibitors BAY-11 and triptolide inhibited LPS-dependent induction. Deletion of a key proximal NF-κB site (402-417) abrogated LPS responsiveness, while deletion of distal NF-κB and C/EBPβ sites did not. Site-directed mutagenesis of CREB (309-320), STAT1 (526-531) and AP2 (566-569) sites had little effect on LPS and adenosine receptor agonist responsiveness; however, mutation of a second STAT1 site (582-588) abrogated this responsiveness. Further analysis of this promoter should provide valuable insights into regulation of A(2A)R expression in macrophages in response to inflammatory stimuli. PMID:23328845

  1. Caffeine acts through neuronal adenosine A2A receptors to prevent mood and memory dysfunction triggered by chronic stress.

    PubMed

    Kaster, Manuella P; Machado, Nuno J; Silva, Henrique B; Nunes, Ana; Ardais, Ana Paula; Santana, Magda; Baqi, Younis; Müller, Christa E; Rodrigues, Ana Lúcia S; Porciúncula, Lisiane O; Chen, Jiang Fan; Tomé, Ângelo R; Agostinho, Paula; Canas, Paula M; Cunha, Rodrigo A

    2015-06-23

    The consumption of caffeine (an adenosine receptor antagonist) correlates inversely with depression and memory deterioration, and adenosine A2A receptor (A2AR) antagonists emerge as candidate therapeutic targets because they control aberrant synaptic plasticity and afford neuroprotection. Therefore we tested the ability of A2AR to control the behavioral, electrophysiological, and neurochemical modifications caused by chronic unpredictable stress (CUS), which alters hippocampal circuits, dampens mood and memory performance, and enhances susceptibility to depression. CUS for 3 wk in adult mice induced anxiogenic and helpless-like behavior and decreased memory performance. These behavioral changes were accompanied by synaptic alterations, typified by a decrease in synaptic plasticity and a reduced density of synaptic proteins (synaptosomal-associated protein 25, syntaxin, and vesicular glutamate transporter type 1), together with an increased density of A2AR in glutamatergic terminals in the hippocampus. Except for anxiety, for which results were mixed, CUS-induced behavioral and synaptic alterations were prevented by (i) caffeine (1 g/L in the drinking water, starting 3 wk before and continued throughout CUS); (ii) the selective A2AR antagonist KW6002 (3 mg/kg, p.o.); (iii) global A2AR deletion; and (iv) selective A2AR deletion in forebrain neurons. Notably, A2AR blockade was not only prophylactic but also therapeutically efficacious, because a 3-wk treatment with the A2AR antagonist SCH58261 (0.1 mg/kg, i.p.) reversed the mood and synaptic dysfunction caused by CUS. These results herald a key role for synaptic A2AR in the control of chronic stress-induced modifications and suggest A2AR as candidate targets to alleviate the consequences of chronic stress on brain function. PMID:26056314

  2. Caffeine acts through neuronal adenosine A2A receptors to prevent mood and memory dysfunction triggered by chronic stress

    PubMed Central

    Kaster, Manuella P.; Machado, Nuno J.; Silva, Henrique B.; Nunes, Ana; Ardais, Ana Paula; Santana, Magda; Baqi, Younis; Müller, Christa E.; Rodrigues, Ana Lúcia S.; Porciúncula, Lisiane O.; Chen, Jiang Fan; Tomé, Ângelo R.; Agostinho, Paula; Canas, Paula M.; Cunha, Rodrigo A.

    2015-01-01

    The consumption of caffeine (an adenosine receptor antagonist) correlates inversely with depression and memory deterioration, and adenosine A2A receptor (A2AR) antagonists emerge as candidate therapeutic targets because they control aberrant synaptic plasticity and afford neuroprotection. Therefore we tested the ability of A2AR to control the behavioral, electrophysiological, and neurochemical modifications caused by chronic unpredictable stress (CUS), which alters hippocampal circuits, dampens mood and memory performance, and enhances susceptibility to depression. CUS for 3 wk in adult mice induced anxiogenic and helpless-like behavior and decreased memory performance. These behavioral changes were accompanied by synaptic alterations, typified by a decrease in synaptic plasticity and a reduced density of synaptic proteins (synaptosomal-associated protein 25, syntaxin, and vesicular glutamate transporter type 1), together with an increased density of A2AR in glutamatergic terminals in the hippocampus. Except for anxiety, for which results were mixed, CUS-induced behavioral and synaptic alterations were prevented by (i) caffeine (1 g/L in the drinking water, starting 3 wk before and continued throughout CUS); (ii) the selective A2AR antagonist KW6002 (3 mg/kg, p.o.); (iii) global A2AR deletion; and (iv) selective A2AR deletion in forebrain neurons. Notably, A2AR blockade was not only prophylactic but also therapeutically efficacious, because a 3-wk treatment with the A2AR antagonist SCH58261 (0.1 mg/kg, i.p.) reversed the mood and synaptic dysfunction caused by CUS. These results herald a key role for synaptic A2AR in the control of chronic stress-induced modifications and suggest A2AR as candidate targets to alleviate the consequences of chronic stress on brain function. PMID:26056314

  3. Adenosine A1 Receptors Selectively Modulate Oxygen-Induced Retinopathy at the Hyperoxic and Hypoxic Phases by Distinct Cellular Mechanisms

    PubMed Central

    Zhang, Shuya; Li, Haiyan; Li, Bo; Zhong, Dingjuan; Gu, Xuejiao; Tang, Lingyun; Wang, Yanyan; Wang, Cun; Zhou, Rong; Li, Yan; He, Yan; Chen, Mozi; Huo, Yuqing; Liu, Xiao-Ling; Chen, Jiang-Fan

    2015-01-01

    Purpose We critically evaluated the role of the adenosine A1 receptor (A1R) in normal development of retinal vasculature and pathogenesis of retinopathy of prematurity (ROP) by using the A1R knockout (KO) mice and oxygen-induced retinopathy (OIR) model. Methods Mice deficient in A1Rs and their wild-type (WT) littermates were examined during normal postnatal development or after being subjected to 75% oxygen from postnatal day (P) 7 to P12 and to room air from P12 to P17 (OIR model of ROP). Retinal vascularization was examined by whole-mount fluorescence and cross-sectional hematoxylin-eosin staining. Cellular proliferation, astrocyte and microglial activation, and tip cell function were determined by isolectin staining and immunohistochemistry. Apoptosis was determined by TUNEL assay. Results Genetic deletion of the A1R did not affect normal retinal vascularization during postnatal development with indistinguishable three-layer vascularization patterns in retina between WT and A1R KO mice. In the OIR model, genetic deletion of the A1R resulted in stage-specific effects: reduced hyperoxia-induced retinal vaso-obliteration at P12, but reduced avascular area and attenuated hypoxia-induced intraretinal revascularization without affecting intravitreal neovascularization at P17 and reduced avascular areas in retina at P21. These distinct effects of A1Rs on OIR were associated with A1R control of apoptosis mainly in inner and outer nuclear layers at the vaso-obliterative phase (P12) and the growth of endothelium tip cells at the vasoproliferative phase (P17), without modification of cellular proliferation, astrocytic activation, and tissue inflammation. Conclusions Adenosine A1 receptor activity is not required for normal postnatal development of retinal vasculature but selectively controls hyperoxia-induced vaso-obliteration and hypoxia-driven revascularization by distinct cellular mechanisms. PMID:26720463

  4. Adenosine and Bone Metabolism

    PubMed Central

    Mediero, Aránzazu; Cronstein, Bruce N.

    2013-01-01

    Bone is a dynamic organ that undergoes continuous remodeling whilst maintaining a balance between bone formation and resorption. Osteoblasts, which synthesize and mineralize new bone, and osteoclasts, the cells that resorb bone, act in concert to maintain bone homeostasis. In recent years, there has been increasing appreciation of purinergic regulation of bone metabolism. Adenosine, released locally, mediates its physiologic and pharmacologic actions via interactions with G-protein coupled receptors and recent work has indicated that these receptors are involved in the regulation of osteoclast differentiation and function, as well as osteoblast differentiation and bone formation. Moreover, adenosine receptors also regulate chondrocyte and cartilage homeostasis. These recent findings underscore the potential therapeutic importance of adenosine receptors in regulating bone physiology and pathology. PMID:23499155

  5. i-bodies, Human Single Domain Antibodies That Antagonize Chemokine Receptor CXCR4.

    PubMed

    Griffiths, Katherine; Dolezal, Olan; Cao, Benjamin; Nilsson, Susan K; See, Heng B; Pfleger, Kevin D G; Roche, Michael; Gorry, Paul R; Pow, Andrew; Viduka, Katerina; Lim, Kevin; Lu, Bernadine G C; Chang, Denison H C; Murray-Rust, Thomas; Kvansakul, Marc; Perugini, Matthew A; Dogovski, Con; Doerflinger, Marcel; Zhang, Yuan; Parisi, Kathy; Casey, Joanne L; Nuttall, Stewart D; Foley, Michael

    2016-06-10

    CXCR4 is a G protein-coupled receptor with excellent potential as a therapeutic target for a range of clinical conditions, including stem cell mobilization, cancer prognosis and treatment, fibrosis therapy, and HIV infection. We report here the development of a fully human single-domain antibody-like scaffold termed an "i-body," the engineering of which produces an i-body library possessing a long complementarity determining region binding loop, and the isolation and characterization of a panel of i-bodies with activity against human CXCR4. The CXCR4-specific i-bodies show antagonistic activity in a range of in vitro and in vivo assays, including inhibition of HIV infection, cell migration, and leukocyte recruitment but, importantly, not the mobilization of hematopoietic stem cells. Epitope mapping of the three CXCR4 i-bodies AM3-114, AM4-272, and AM3-523 revealed binding deep in the binding pocket of the receptor. PMID:27036939

  6. Bradykinin B2, but not B1, receptor antagonism has a neuroprotective effect after brain injury.

    PubMed

    Görlach, C; Hortobágyi, T; Hortobágyi, S; Benyó, Z; Relton, J; Whalley, E T; Wahl, M

    2001-08-01

    The aim of the present study was to measure the therapeutic effects of bradykinin antagonists on lesion volume and brain swelling induced by cold injury in the parietal cortex of rat and mouse, respectively. Cold lesion was induced by application of a precooled (-78 degrees C) copper cylinder (3 mm diameter) to the intact dura of rat and mouse for 6 and 30 sec, respectively. At 24 h after the injury, the brains were removed and lesion volume was determined by the triphenyltetrazolium chloride method in rats. In the mouse, brain swelling was expressed as percentage increase in weight of the injured hemisphere which is compared to the contralateral side. After a subcutaneous priming dose of 18 microg/kg, a 1-h pretreatment and 24-h posttreatment using osmotic minipumps (300 ng/kg x min) was applied. Hoe140, a bradykinin receptor 2 antagonist, revealed a 19% reduction of lesion volume (p < 0.05) in the rat and a 14% diminution of brain swelling (p < 0.05) in the mouse. In contrast, the bradykinin receptor 1 antagonist, B 9858, had no effect on lesion volume compared to sham treated rats. When B 9858 was given in combination with Hoe140, a significant reduction in lesion volume was seen which was equivalent to and not different from that seen with Hoe140 alone in the rat. We conclude that brain injury after cold lesion is partially mediated by bradykinin and can be successfully treated with B2 antagonists. PMID:11526989

  7. The role of CD4-Lck in T-cell receptor antagonism: evidence for negative signaling.

    PubMed Central

    Racioppi, L; Matarese, G; D'Oro, U; De Pascale, M; Masci, A M; Fontana, S; Zappacosta, S

    1996-01-01

    Small changes in the complex between a peptide and a molecule of the major histocompatibility complex generate ligands able to partially activate (partial agonist) or even inhibit (antagonist) T-cell functions. T-cell receptor engagement of antagonist complex results in a partial zeta chain phosphorylation without activation of the associated ZAP-70 kinase. Herein we show that, despite a strong inhibition of both inositol phospholipid hydrolysis and extracellular increasing antagonist concentrations increased the activity of the CD4-Lck kinase. Addition of anti-CD4 antibody to culture medium prevented inhibitory effects induced by antagonist ligand. We propose that CD4-Lck activation triggered by antagonist complexes may act in a dominant negative mode, thus overriding stimulatory signals coming from agonist ligand. These findings identify a new T-cell signaling profile that may explain the ability of some T-cell receptor variant ligands to inhibit specific biological activities or trigger alternative activation programs. Images Fig. 3 Fig. 4 PMID:8816805

  8. alpha1-noradrenergic receptor antagonism blocks dependence-induced increases in responding for ethanol.

    PubMed

    Walker, Brendan M; Rasmussen, Dennis D; Raskind, Murray A; Koob, George F

    2008-03-01

    The purpose of this study was to test the hypothesis that blockade of alpha1-adrenergic receptors may suppress the excessive ethanol consumption associated with acute withdrawal in ethanol-dependent rats. Following the acquisition and stabilization of operant ethanol self-administration in male Wistar rats, dependence was induced in half the animals by subjecting them to a 4-week intermittent vapor exposure period in which animals were exposed to ethanol vapor for 14h/day. Subsequent to dependence induction, the effect of alpha1-noradrenergic receptor antagonist prazosin (0.0, 0.25, 0.5, 1, 1.5, and 2.0mg/kg IP) was tested on operant responding for ethanol in vapor-exposed and control rats during acute withdrawal. In ethanol-dependent animals, prazosin significantly suppressed responding at the 1.5 and 2.0mg/kg doses, whereas only the 2.0mg/kg dose was effective in nondependent animals, identifying an increase in the sensitivity to prazosin in dependent animals. Conversely, at the lowest dose tested (0.25mg/kg), prazosin increased responding in nondependent animals, which is consistent with the effect of anxiolytics on ethanol self-administration in nondependent animals. None of the doses tested reliably affected concurrent water self-administration. These results suggest the involvement of the noradrenergic system in the excessive alcohol drinking seen during acute withdrawal in ethanol-dependent rats. PMID:18358987

  9. Adenosine A2A Receptor Up-Regulates Retinal Wave Frequency via Starburst Amacrine Cells in the Developing Rat Retina

    PubMed Central

    Huang, Pin-Chien; Hsiao, Yu-Tien; Kao, Shao-Yen; Chen, Ching-Feng; Chen, Yu-Chieh; Chiang, Chung-Wei; Lee, Chien-fei; Lu, Juu-Chin; Chern, Yijuang; Wang, Chih-Tien

    2014-01-01

    Background Developing retinas display retinal waves, the patterned spontaneous activity essential for circuit refinement. During the first postnatal week in rodents, retinal waves are mediated by synaptic transmission between starburst amacrine cells (SACs) and retinal ganglion cells (RGCs). The neuromodulator adenosine is essential for the generation of retinal waves. However, the cellular basis underlying adenosine's regulation of retinal waves remains elusive. Here, we investigated whether and how the adenosine A2A receptor (A2AR) regulates retinal waves and whether A2AR regulation of retinal waves acts via presynaptic SACs. Methodology/Principal Findings We showed that A2AR was expressed in the inner plexiform layer and ganglion cell layer of the developing rat retina. Knockdown of A2AR decreased the frequency of spontaneous Ca2+ transients, suggesting that endogenous A2AR may up-regulate wave frequency. To investigate whether A2AR acts via presynaptic SACs, we targeted gene expression to SACs by the metabotropic glutamate receptor type II promoter. Ca2+ transient frequency was increased by expressing wild-type A2AR (A2AR-WT) in SACs, suggesting that A2AR may up-regulate retinal waves via presynaptic SACs. Subsequent patch-clamp recordings on RGCs revealed that presynaptic A2AR-WT increased the frequency of wave-associated postsynaptic currents (PSCs) or depolarizations compared to the control, without changing the RGC's excitability, membrane potentials, or PSC charge. These findings suggest that presynaptic A2AR may not affect the membrane properties of postsynaptic RGCs. In contrast, by expressing the C-terminal truncated A2AR mutant (A2AR-ΔC) in SACs, the wave frequency was reduced compared to the A2AR-WT, but was similar to the control, suggesting that the full-length A2AR in SACs is required for A2AR up-regulation of retinal waves. Conclusions/Significance A2AR up-regulates the frequency of retinal waves via presynaptic SACs, requiring its full

  10. Cloning and chromosomal localization of the human A{sub 2b} adenosine receptor gene (ADORA2B) and its pseudogene

    SciTech Connect

    Jacobson, M.A.; Johnson, R.G.; Luneau, C.J.

    1995-05-20

    To determine the chromosomal localization of the human A{sub 2b} adenosine receptor, the corresponding genomic clone was isolated and used as a probe for fluorescence in situ hybridization to metaphase chromosomes. Partial sequence analysis of the A{sub 2b} gene (ADORA2B) revealed an intron that interrupted the coding region corresponding to the second intracellular loop similar to that reported for A{sub 1} and A{sub 2a} adenosine receptor genes. A pseudogene for the A{sub 2b} receptor was also identified; it exhibited 79% identity to the A{sub 2b} adenosine receptor cDNA coding sequence and contained multiple deletions, point mutations, and frame shifts and two in-frame stops. These changes would result in the inability to encode a functional receptor. The genomic clones were utilized to localize the A{sub 2b} receptor to chromosome 17p12 and the A{sub 2b} pseudogene to chromosome 1q32. 15 refs., 1 fig.

  11. The anti-inflammatory target A(3) adenosine receptor is over-expressed in rheumatoid arthritis, psoriasis and Crohn's disease.

    PubMed

    Ochaion, A; Bar-Yehuda, S; Cohen, S; Barer, F; Patoka, R; Amital, H; Reitblat, T; Reitblat, A; Ophir, J; Konfino, I; Chowers, Y; Ben-Horin, S; Fishman, P

    2009-01-01

    The Gi protein associated A(3) adenosine receptor (A(3)AR) was recently defined as a novel anti-inflammatory target. The aim of this study was to look at A(3)AR expression levels in peripheral blood mononuclear cells (PBMCs) of patients with autoimmune inflammatory diseases and to explore transcription factors involved receptor expression. Over-expression of A(3)AR was found in PBMCs derived from patients with rheumatoid arthritis (RA), psoriasis and Crohn's disease compared with PBMCs from healthy subjects. Bioinformatics analysis demonstrated the presence of DNA binding sites for nuclear factor-kappaB (NF-kappaB) and cyclic AMP-responsive element binding protein (CREB) in the A(3)AR gene promoter. Up-regulation of NF-kappaB and CREB was found in the PBMCs from patients with RA, psoriasis and Crohn's disease. The PI3K-PKB/Akt signaling pathway, known to regulate both the NF-kappaB and CREB, was also up-regulated in the patients' PBMCs. Taken together, NF-kappaB and CREB are involved with the over-expression of A(3)AR in patients with autoimmune inflammatory diseases. The receptor may be considered as a specific target to combat inflammation. PMID:19426966

  12. Presynaptic Adenosine Receptor-Mediated Regulation of Diverse Thalamocortical Short-Term Plasticity in the Mouse Whisker Pathway

    PubMed Central

    Ferrati, Giovanni; Martini, Francisco J.; Maravall, Miguel

    2016-01-01

    Short-term synaptic plasticity (STP) sets the sensitivity of a synapse to incoming activity and determines the temporal patterns that it best transmits. In “driver” thalamocortical (TC) synaptic populations, STP is dominated by depression during stimulation from rest. However, during ongoing stimulation, lemniscal TC connections onto layer 4 neurons in mouse barrel cortex express variable STP. Each synapse responds to input trains with a distinct pattern of depression or facilitation around its mean steady-state response. As a result, in common with other synaptic populations, lemniscal TC synapses express diverse rather than uniform dynamics, allowing for a rich representation of temporally varying stimuli. Here, we show that this STP diversity is regulated presynaptically. Presynaptic adenosine receptors of the A1R type, but not kainate receptors (KARs), modulate STP behavior. Blocking the receptors does not eliminate diversity, indicating that diversity is related to heterogeneous expression of multiple mechanisms in the pathway from presynaptic calcium influx to neurotransmitter release. PMID:26941610

  13. Neoceptor Concept Based on Molecular Complementarity in GPCRs: A Mutant Adenosine A3 Receptor with Selectively Enhanced Affinity for Amine-Modified Nucleosides

    PubMed Central

    Jacobson, Kenneth A.; Gao, Zhan-Guo; Chen, Aishe; Barak, Dov; Kim, Soon-Ai; Lee, Kyeong; Link, Andreas; Van Rompaey, Philippe; van Calenbergh, Serge; Liang, Bruce T.

    2012-01-01

    Adenosine A3 receptors are of interest in the treatment of cardiac ischemia, inflammation, and neurodegenerative diseases. In an effort to create a unique receptor mutant that would be activated by tailor-made synthetic ligands, we mutated the human A3 receptor at the site of a critical His residue in TM7, previously proposed to be involved in ligand recognition through interaction with the ribose moiety. The H272E mutant receptor displayed reduced affinity for most of the uncharged A3 receptor agonists and antagonists examined. For example, the nonselective agonist 1a was 19-fold less potent at the mutant receptor than at the wild-type receptor. The introduction of an amino group on the ribose moiety of adenosine resulted in either equipotency or enhanced binding affinity at the H272E mutant relative to wild-type A3 receptors, depending on the position of the amino group. 3′-Amino-3′-deoxyadenosine proved to be 7-fold more potent at the H272E mutant receptor than at the wild-type receptor, while the corresponding 2′- and 5′-amino analogues did not display significantly enhanced affinities. An 3′-amino-N6-iodobenzyl analogue showed only a small enhancement at the mutant (Ki = 320 nM) vs wild-type receptors. The 3′-amino group was intended for a direct electrostatic interaction with the negatively charged ribose-binding region of the mutant receptor, yet molecular modeling did not support this notion. This design approach is an example of engineering the structure of mutant receptors to recognize synthetic ligands for which they are selectively matched on the basis of molecular complementarity between the mutant receptor and the ligand. We have termed such engineered receptors “neoceptors”, since the ligand recognition profile of such mutant receptors need not correspond to the profile of the parent, native receptor. PMID:11708915

  14. The Androgen Receptor Antagonizes Wnt/β-Catenin Signaling in Epidermal Stem Cells

    PubMed Central

    Kretzschmar, Kai; Cottle, Denny L; Schweiger, Pawel J; Watt, Fiona M

    2015-01-01

    Activation of Wnt/β-catenin signaling in adult mouse epidermis leads to expansion of the stem cell compartment and redirects keratinocytes in the interfollicular epidermis and sebaceous glands (SGs) to differentiate along the hair follicle (HF) lineages. Here we demonstrate that during epidermal development and homeostasis there is reciprocal activation of the androgen receptor (AR) and β-catenin in cells of the HF bulb. AR activation reduced β-catenin-dependent transcription, blocked β-catenin-induced induction of HF growth, and prevented β-catenin-mediated conversion of SGs into HFs. Conversely, AR inhibition enhanced the effects of β-catenin activation, promoting HF proliferation and differentiation, culminating in the formation of benign HF tumors and a complete loss of SG identity. We conclude that AR signaling has a key role in epidermal stem cell fate selection by modulating responses to β-catenin in adult mouse skin. PMID:26121213

  15. Acute NMDA receptor antagonism disrupts synchronization of action potential firing in rat prefrontal cortex.

    PubMed

    Molina, Leonardo A; Skelin, Ivan; Gruber, Aaron J

    2014-01-01

    Antagonists of N-methyl-D-aspartate receptors (NMDAR) have psychotomimetic effects in humans and are used to model schizophrenia in animals. We used high-density electrophysiological recordings to assess the effects of acute systemic injection of an NMDAR antagonist (MK-801) on ensemble neural processing in the medial prefrontal cortex of freely moving rats. Although MK-801 increased neuron firing rates and the amplitude of gamma-frequency oscillations in field potentials, the synchronization of action potential firing decreased and spike trains became more Poisson-like. This disorganization of action potential firing following MK-801 administration is consistent with changes in simulated cortical networks as the functional connections among pyramidal neurons become less clustered. Such loss of functional heterogeneity of the cortical microcircuit may disrupt information processing dependent on spike timing or the activation of discrete cortical neural ensembles, and thereby contribute to hallucinations and other features of psychosis induced by NMDAR antagonists. PMID:24465743

  16. Differential antagonism of tetramethylenedisulfotetramine-induced seizures by agents acting at NMDA and GABAA receptors

    PubMed Central

    Shakarjian, Michael P.; Velíšková, Jana; Stanton, Patric K.; Velíšek, Libor

    2012-01-01

    Tetramethylenedisulfotetramine (TMDT) is a highly lethal neuroactive rodenticide responsible for many accidental and intentional poisonings in mainland China. Ease of synthesis, water solubility, potency, and difficulty to treat make TMDT a potential weapon for terrorist activity. We characterized TMDT-induced convulsions and mortality in male C57BL/6 mice. TMDT (ip) produced a continuum of twitches, clonic, and tonic-clonic seizures decreasing in onset latency and increasing in severity with increasing dose; 0.4 mg/kg was 100% lethal. The NMDA antagonist, ketamine (35 mg/kg) injected ip immediately after the first TMDT-induced seizure, did not change number of tonic-clonic seizures or lethality, but increased the number of clonic seizures. Doubling the ketamine dose decreased tonic-clonic seizures and eliminated lethality through a 60 min observation period. Treating mice with another NMDA antagonist, MK-801, 0.5 or 1 mg/kg ip, showed similar effects as low and high doses of ketamine, respectively, and prevented lethality, converting status epilepticus EEG activity to isolated interictal discharges. Treatment with these agents 15 min prior to TMDT administration did not increase their effectiveness. Post-treatment with the GABAA receptor allosteric enhancer diazepam (5 mg/kg) greatly reduced seizure manifestations and prevented lethality 60 min post-TMDT, but ictal events were evident in EEG recordings and, hours post-treatment, mice experienced status epilepticus and died. Thus, TMDT is a highly potent and lethal convulsant for which single-dose benzodiazepine treatment is inadequate in managing electrographic seizures or lethality. Repeated benzodiazepine dosing or combined application of benzodiazepines and NMDA receptor antagonists are more likely to be effective in treating TMDT poisoning. PMID:23022509

  17. Antagonism of mGlu receptors and potentiation of EPSCs at rat spinal motoneurones in vitro.

    PubMed

    Cao, C Q; Tse, H W; Jane, D E; Evans, R H; Headley, P M

    1997-03-01

    The patch-clamp technique has been used to record synaptic responses, elicited by electrical stimulation of dorsal roots, in 28 single motoneurones of in vitro spinal cord preparations from neonate (P5 to P8) rats. The effects of (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG) (200 microM), a potent antagonist at L-2-amino-4-phosphonobutanoate (AP4)-sensitive receptors, and (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) (500 microM), which is a less selective antagonist of mGluRs, were tested on EPSCs alone and as antagonists of AP4-induced depression of EPSCs. The EC50 for depression of EPSCs by AP4 (1.16 +/- 0.12 microM, n = 8) was increased to 18.9 +/- 0.7 microM (n = 6) by MPPG. MCPG (500 microM) had no significant effect on the depressant potency of AP4. Under control conditions, EPSCs had mean peak amplitudes of 983 pA +/- 64 SEM and mean charge transferred of 306 +/- 37 pC (n = 28). These values were increased significantly (p < 0.05) to 1168 +/- 68 pA and 363 +/- 39 pC by MPPG (n = 6), and 1150 +/- 54 pA and 358 +/- 33 pC (n = 6) by MCPG. There was no significant difference between the enhancement of the initial peak of the EPSCs (mean latency from stimulus artifact 5.9 +/- 0.3 ms) and later components, suggesting mGluRs to be present on primary afferent terminals presynaptic to motoneurones as well as in pathways via interneurones. These results are consistent with the presence of at least two types of presynaptic mGluR that modulate release of glutamate in segmental pathways convergent onto motoneurones. These receptors appear to be activated by interstitial glutamate tonically present in the present preparations. PMID:9175609

  18. Pharmacologic antagonism of thromboxane A2 receptors by trimetoquinol analogs in vitro and in vivo

    SciTech Connect

    Shin, Y.; Romstedt, K.J.; Doyle, K.; Harrold, M.W.; Gerhardt, M.A.; Miller, D.D.; Patil, P.N.; Feller, D.R. )

    1991-01-01

    Although (-)-(S)-trimetoquinol (1-(3,4,5-trimethoxy-benzyl)- 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; TMQ) is recognized as a potent bronchodilator, (+)-(R)-TMQ is a selective antagonist of human platelet aggregation and serotonin secretion induced by thromboxane A2 (TXA2) agonists. To confirm the pharmacological actions of TMQ analogs, the interaction of the drugs with TXA2 receptors was examined in human platelets and in a mouse sudden death model. The inhibitory potencies of TMQ analogs (pIC50 values) for displacement of (3H)SQ 29,548 binding to platelets showed excellent correlation with the respective pIC50 (-log IC50) values for U46619-induced aggregation (r = 0.99, P less than 0.01) and serotonin secretion (r = 0.99, P less than 0.01) in human platelet-rich plasma and for whole blood aggregation (r = 0.99, P less than 0.01). In each system, the rank order of inhibitory potencies was rac-iodoTMQ greater than or equal to (+)-(R)-TMQ greater than rac-TMQ much greater than (-)-(S)-TMQ. Antithrombotic effects of TMQ analogs were evaluated in a mouse sudden death model. In vivo antithrombotic potencies of these compounds were consistent with the in vitro potencies as TXA2 receptor antagonists in platelet systems. Administration of rac-iodoTMQ, (+)-(R)-TMQ and rac-TMQ 15 min before the injection of U46619 (800 micrograms/kg, iv) protected mice against U46619-induced sudden death. On the other hand, (-)-(S)-TMQ did not protect animals against death. Protection of U46619-induced cardiopulmonary thrombosis by TMQ analogs was seen at doses of 3-100 mg/kg.

  19. Differential inhibition of noradrenaline release mediated by inhibitory A₁-adenosine receptors in the mesenteric vein and artery from normotensive and hypertensive rats.

    PubMed

    Rocha-Pereira, C; Sousa, J B; Vieira-Rocha, M S; Fresco, P; Gonçalves, J; Diniz, C

    2013-03-01

    Mesenteric arteries and veins are densely innervated by sympathetic nerves and are crucial in the regulation of peripheral resistance and capacitance, respectively, thus, in the control of blood pressure. Presynaptic adenosine receptors are involved in vascular tonus regulation, by modulating noradrenaline release from vascular postganglionic sympathetic nerve endings. Some studies also suggest that adenosine receptors (AR) may have a role in hypertension. We aim at investigating the role of presynaptic adenosine receptors in mesenteric vessels and establish a relationship between their effects (in mesenteric vessels) and hypertension, using the spontaneously hypertensive rats (SHR) as a model of hypertension. Adenosine receptor-mediated modulation of noradrenaline release was investigated through the effects of selective agonists and antagonists on electrically-evoked [(3)H]-noradrenaline overflow. CPA (A1AR selective agonist: 1-100 nM) inhibited tritium overflow, but the inhibition was lower in SHR mesenteric vessels. IB-MECA (A3AR selective agonist: 1-100 nM) also inhibited tritium overflow but only in WKY mesenteric veins. CGS 21680 (A2AAR selective agonist: up to 100 nM) failed to facilitate noradrenaline release in mesenteric veins, from both strains, but induced a similar facilitation in the mesenteric arteries. NECA (non-selective AR agonist: 1, 3 and 10μM), in the presence of A1 (DPCPX, 20 nM) and A3 (MRS 1523, 1 μM) AR selective antagonists, failed to change tritium overflow. In summary, the modulatory effects mediated by presynaptic adenosine receptors were characterized, for the first time, in mesenteric vessels: a major inhibition exerted by the A1 subtype in both vessels; a slight inhibition mediated by A3 receptors in mesenteric vein; a facilitation mediated by A2A receptors only in mesenteric artery (from both strains). The less efficient prejunctional adenosine receptor mediated inhibitory effects can contribute to an increase of noradrenaline in

  20. CB1 receptor antagonism prevents long-term hyperexcitability after head injury by regulation of dynorphin-KOR system and mGluR5 in rat hippocampus.

    PubMed

    Wang, Xiu; Wang, Yao; Zhang, Chao; Liu, Chang; Zhao, Baotian; Wei, Naili; Zhang, Jian-Guo; Zhang, Kai

    2016-09-01

    Both endocannabinoids and dynorphin are feedback messengers in nervous system that act at the presynaptic nerve terminal to inhibit transmitter release. Many studies showed the cannabinoid-opioid cross-modulation in antinociception, hypothermia, sedation and reward. The aim of this study was to assess the influence of early application of cannabinoid type 1 (CB1) receptor antagonism SR141716A after brain injury on dynorphin-κ opioid receptor (KOR) system and the expression of metabotropic glutamate receptors (mGluRs) in a rat model of fluid percussion injury (FPI). Firstly, seizure latency induced by pentylenetetrazole was significantly prolonged 6 weeks after brain injury in group of SR141716A treatment. Then, PCR and western blot showed that SR141716A inhibited the long-term up-regulation of CB1 receptors in hippocampus. However, SR141716A resulted in long-term potentiation of dynorphin release and did not influence the up-regulation of KOR in hippocampus after brain injury. Furthermore, SR141716A reverse the overexpression of mGluR5 in the late stage of brain injury. We propose that during the induction of epileptogenesis after brain injury, early application of CB1 receptor antagonism could prevent long-term hyperexcitability by up-regulation of dynorphin-KOR system and prevention of mGluR5 induced epileptogenesis in hippocampus. PMID:27262683

  1. Beneficial effects of a novel agonist of the adenosine A2A receptor on monocrotaline-induced pulmonary hypertension in rats

    PubMed Central

    Alencar, Allan K N; Pereira, Sharlene L; Montagnoli, Tadeu L; Maia, Rodolfo C; Kümmerle, Arthur E; Landgraf, Sharon S; Caruso-Neves, Celso; Ferraz, Emanuelle B; Tesch, Roberta; Nascimento, José H M; de Sant'Anna, Carlos M R; Fraga, Carlos A M; Barreiro, Eliezer J; Sudo, Roberto T; Zapata-Sudo, Gisele

    2013-01-01

    Background and Purpose Pulmonary arterial hypertension (PAH) is characterized by enhanced pulmonary vascular resistance, right ventricular hypertrophy and increased right ventricular systolic pressure. Here, we investigated the effects of a N-acylhydrazone derivative, 3,4-dimethoxyphenyl-N-methyl-benzoylhydrazide (LASSBio-1359), on monocrotaline (MCT)-induced pulmonary hypertension in rats. Experimental Approach PAH was induced in male Wistar rats by a single i.p. injection of MCT (60 mg·kg−1) and 2 weeks later, oral LASSBio-1359 (50 mg·kg−1) or vehicle was given once daily for 14 days. Echocardiography was used to measure cardiac function and pulmonary artery dimensions, with histological assay of vascular collagen. Studies of binding to human recombinant adenosine receptors (A1, A2A, A3) and of docking with A2A receptors were also performed. Key Results MCT administration induced changes in vascular and ventricular structure and function, characteristic of PAH. These changes were reversed by treatment with LASSBio-1359. MCT also induced endothelial dysfunction in pulmonary artery, as measured by diminished relaxation of pre-contracted arterial rings, and this dysfunction was reversed by LASSBio-1359. In pulmonary artery rings from normal Wistar rats, LASSBio-1359 induced relaxation, which was decreased by the adenosine A2A receptor antagonist, ZM 241385. In adenosine receptor binding studies, LASSBio-1359 showed most affinity for the A2A receptor and in the docking analyses, binding modes of LASSBio-1359 and the A2A receptor agonist, CGS21680, were very similar. Conclusion and Implications In rats with MCT-induced PAH, structural and functional changes in heart and pulmonary artery were reversed by treatment with oral LASSBio-1359, most probably through the activation of adenosine A2A receptors. PMID:23530610

  2. Topological sub-structural molecular design (TOPS-MODE): a useful tool to explore key fragments of human A3 adenosine receptor ligands.

    PubMed

    Saíz-Urra, Liane; Teijeira, Marta; Rivero-Buceta, Virginia; Helguera, Aliuska Morales; Celeiro, Maria; Terán, Ma Carmen; Besada, Pedro; Borges, Fernanda

    2016-02-01

    Adenosine regulates tissue function by activating four G-protein-coupled adenosine receptors (ARs). Selective agonists and antagonists for A3 ARs have been investigated for the treatment of a variety of immune disorders, cancer, brain, and heart ischemic conditions. We herein present a QSAR study based on a Topological sub-structural molecular design (TOPS-MODE) approach, intended to predict the A3 ARs of a diverse dataset of 124 (94 training set/ 30 prediction set) adenosine derivatives. The final model showed good fit and predictive capability, displaying 85.1 % of the experimental variance. The TOPS-MODE approach afforded a better understanding and interpretation of the developed model based on the useful information extracted from the analysis of the contribution of different molecular fragments to the affinity. PMID:26205409

  3. Impact of purification conditions and history on A2A adenosine receptor activity: The role of CHAPS and lipids

    DOE PAGESBeta

    Naranjo, Andrea N.; McNeely, Patrick M.; Katsaras, John; Skaja Robinson, Anne

    2016-05-27

    The adenosine A2A receptor (A2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilization in DDM, DDM/CHAPS, ormore » DHPC micelles, although A2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. Furthermore, the studies presented in this paper also underline the importance of the protein’s purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.« less

  4. Adenosine (A)(2A)receptor modulation of nicotine-induced locomotor sensitization. A pharmacological and transgenic approach.

    PubMed

    Jastrzębska, Joanna; Nowak, Ewa; Smaga, Irena; Bystrowska, Beata; Frankowska, Małgorzata; Bader, Michael; Filip, Małgorzata; Fuxe, Kjell

    2014-06-01

    Preclinical evidence indicates an important role of adenosine (A)(2A) receptors in drug addiction while their therapeutic relevance is still a matter of debate. We examined the influence of the A(2A) receptor agonist CGS 21680 and the antagonist KW 6002 on nicotine sensitization and conditioned locomotor activity in adult (8-week old) male Sprague-Dawley rats (WT). Moreover, behavioral responses to nicotine were studied in rats overexpressing A(2A) receptors<