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Sample records for adenosine receptor antagonism

  1. Continuous adenosine A2A receptor antagonism after focal cerebral ischemia in spontaneously hypertensive rats.

    PubMed

    Fronz, Ulrike; Deten, Alexander; Baumann, Frank; Kranz, Alexander; Weidlich, Sarah; Härtig, Wolfgang; Nieber, Karen; Boltze, Johannes; Wagner, Daniel-Christoph

    2014-02-01

    Antagonism of the adenosine A2A receptor (A2AR) has been shown to elicit substantial neuroprotective properties when given immediately after cerebral ischemia. We asked whether the continuous application of a selective A2AR antagonist within a clinically relevant time window will be a feasible and effective approach to treat focal cerebral ischemia. To answer this question, we subjected 20 male spontaneously hypertensive rats to permanent middle cerebral artery occlusion and randomized them equally to a verum and a control group. Two hours after stroke onset, the animals received a subcutaneous implantation of an osmotic minipump filled with 5 mg kg(-1) day(-1) 8-(3-chlorostyryl) caffeine (CSC) or vehicle solution. The serum level of CSC was measured twice a day for three consecutive days. The infarct volume was determined at days 1 and 3 using magnetic resonance imaging. We found the serum level of CSC showing a bell-shaped curve with its maximum at 36 h. The infarct volume was not affected by continuous CSC treatment. These results suggest that delayed and continuous CSC application was not sufficient to treat acute ischemic stroke, potentially due to unfavorable hepatic elimination and metabolization of the pharmaceutical. PMID:24170241

  2. 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

  3. 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

  4. 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.

  5. 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

  6. Fluorescent ligands for adenosine receptors.

    PubMed

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

    2013-01-01

    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.

  7. 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.

  8. Evidence for an antagonism between caffeine and adenosine in the human cardiovascular system.

    PubMed

    Smits, P; Boekema, P; De Abreu, R; Thien, T; van 't Laar, A

    1987-08-01

    A randomized, double-blind and placebo-controlled study was performed in 10 normotensive male subjects to analyze a possible antagonism between caffeine and adenosine with respect to their effects on the cardiovascular system in humans. Caffeine alone, 250 mg intravenously (i.v.), increased blood pressure by 9/12 mm Hg, and resulted in a fall of heart rate (HR) of 3 beats/min. Plasma epinephrine (E) rose by 114% after caffeine. Adenosine alone, in an increasing dose of 0.04-0.16 mg/kg/min, induced an increase in systolic blood pressure (SBP) (17 mm Hg), and HR (33 beats/min), a moderate fall in diastolic blood pressure (DBP) (-4 mm Hg), and no change of mean arterial pressure (MAP). At the highest adenosine infusion rate, forearm blood flow, skin temperature (ST), and transcutaneous oxygen tension were lowered, whereas plasma (nor)epinephrine was increased 227.2 and 215.9%, respectively. Adenosine infusion after caffeine induced comparable effects, but the fractional adenosine-induced changes of SBP, HR, plasma catecholamines, plasma renin activity (PRA), and aldosterone all were significantly reduced by previous administration of caffeine. Our results indicate an antagonism between caffeine and adenosine in humans, which may support the suggestion that some circulatory effects of caffeine are caused by an interaction with endogenous adenosine. PMID:2441163

  9. Mast Cell Adenosine Receptors Function: A Focus on the A3 Adenosine Receptor and Inflammation

    PubMed Central

    Rudich, Noam; Ravid, Katya; Sagi-Eisenberg, Ronit

    2012-01-01

    Adenosine is a metabolite, which has long been implicated in a variety of inflammatory processes. Inhaled adenosine provokes bronchoconstriction in asthmatics or chronic obstructive pulmonary disease patients, but not in non-asthmatics. This hyper responsiveness to adenosine appears to be mediated by mast cell activation. These observations have marked the receptor that mediates the bronchoconstrictor effect of adenosine on mast cells (MCs), as an attractive drug candidate. Four subtypes (A1, A2a, A2b, and A3) of adenosine receptors have been cloned and shown to display distinct tissue distributions and functions. Animal models have firmly established the ultimate role of the A3 adenosine receptor (A3R) in mediating hyper responsiveness to adenosine in MCs, although the influence of the A2b adenosine receptor was confirmed as well. In contrast, studies of the A3R in humans have been controversial. In this review, we summarize data on the role of different adenosine receptors in mast cell regulation of inflammation and pathology, with a focus on the common and distinct functions of the A3R in rodent and human MCs. The relevance of mouse studies to the human is discussed. PMID:22675325

  10. Adenosine Receptors: Expression, Function and Regulation

    PubMed Central

    Sheth, Sandeep; Brito, Rafael; Mukherjea, Debashree; Rybak, Leonard P.; Ramkumar, Vickram

    2014-01-01

    Adenosine receptors (ARs) comprise a group of G protein-coupled receptors (GPCR) which mediate the physiological actions of adenosine. To date, four AR subtypes have been cloned and identified in different tissues. These receptors have distinct localization, signal transduction pathways and different means of regulation upon exposure to agonists. This review will describe the biochemical characteristics and signaling cascade associated with each receptor and provide insight into how these receptors are regulated in response to agonists. A key property of some of these receptors is their ability to serve as sensors of cellular oxidative stress, which is transmitted by transcription factors, such as nuclear factor (NF)-κB, to regulate the expression of ARs. Recent observations of oligomerization of these receptors into homo- and heterodimers will be discussed. In addition, the importance of these receptors in the regulation of normal and pathological processes such as sleep, the development of cancers and in protection against hearing loss will be examined. PMID:24477263

  11. Adenosine receptors as drug targets — what are the challenges?

    PubMed Central

    Chen, Jiang-Fan; Eltzschig, Holger K.; Fredholm, Bertil B.

    2014-01-01

    Adenosine signalling has long been a target for drug development, with adenosine itself or its derivatives being used clinically since the 1940s. In addition, methylxanthines such as caffeine have profound biological effects as antagonists at adenosine receptors. Moreover, drugs such as dipyridamole and methotrexate act by enhancing the activation of adenosine receptors. There is strong evidence that adenosine has a functional role in many diseases, and several pharmacological compounds specifically targeting individual adenosine receptors — either directly or indirectly — have now entered the clinic. However, only one adenosine receptor-specific agent — the adenosine A2A receptor agonist regadenoson (Lexiscan; Astellas Pharma) — has so far gained approval from the US Food and Drug Administration (FDA). Here, we focus on the biology of adenosine signalling to identify hurdles in the development of additional pharmacological compounds targeting adenosine receptors and discuss strategies to overcome these challenges. PMID:23535933

  12. Endothelin ETA receptor antagonism in cardiovascular disease.

    PubMed

    Nasser, Suzanne A; El-Mas, Mahmoud M

    2014-08-15

    Since the discovery of the endothelin system in 1988, it has been implicated in numerous physiological and pathological phenomena. In the cardiovascular system, endothelin-1 (ET-1) acts through intracellular pathways of two endothelin receptors (ETA and ETB) located mainly on smooth muscle and endothelial cells to regulate vascular tone and provoke mitogenic and proinflammatory reactions. The endothelin ETA receptor is believed to play a pivotal role in the pathogenesis of several cardiovascular disease including systemic hypertension, pulmonary arterial hypertension (PAH), dilated cardiomyopathy, and diabetic microvascular dysfunction. Growing evidence from recent experimental and clinical studies indicates that the blockade of endothelin receptors, particularly the ETA subtype, grasps promise in the treatment of major cardiovascular pathologies. The simultaneous blockade of endothelin ETB receptors might not be advantageous, leading possibly to vasoconstriction and salt and water retentions. This review summarizes the role of ET-1 in cardiovascular modulation and the therapeutic potential of endothelin receptor antagonism.

  13. MOLECULAR PROBES FOR EXTRACELLULAR ADENOSINE RECEPTORS

    PubMed Central

    Jacobson, Kenneth A.; Ukena, Dieter; Padgett, William; Kirk, Kenneth L.; Daly, John W.

    2012-01-01

    Derivatives of adenosine receptor agonists (N6-phenyladenosines) and antagonists (1,3-dialkyl-8-phenylxanthines) bearing functionalized chains suitable for attachment to other molecules have been reported [Jacobson et al., J. med. Chem. 28, 1334 and 1341 (1985)]. The “functionalized congener” approach has been extended to the synthesis of spectroscopic and other probes for adenosine receptors that retain high affinity (Ki ~ 10−9 −10−8 M) in A1-receptor binding. The probes have been synthesized from an antagonist xanthine amine congener (XAC) and an adenosine amine congener (ADAC). [3H]ADAC has been synthesized and found to bind highly specifically to A1-adenosine receptors of rat and calf cerebral cortical membranes with KD values of 1.4 and 0.34 nM respectively. The higher affinity in the bovine brain, seen also with many of the probes derived from ADAC and XAC, is associated with phenyl substituents. The spectroscopic probes contain a reporter group attached at a distal site of the functionalized chain. These bifunctional ligands may contain a spin label (e.g. the nitroxyl radical TEMPO) for electron spin resonance spectroscopy, or a fluorescent dye, including fluorescein and 4-nitrobenz-2-oxa-1,3-diazole (NBD), or labels for 19F nuclear magnetic resonance spectroscopy. Potential applications of the spectroscopic probes in characterization of adenosine receptors are discussed. PMID:3036153

  14. 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.

  15. Pain-relieving prospects for adenosine receptors and ectonucleotidases

    PubMed Central

    Zylka, Mark J.

    2010-01-01

    Adenosine receptor agonists have potent antinociceptive effects in diverse preclinical models of chronic pain. In contrast, the efficacy of adenosine or adenosine receptor agonists at treating pain in humans is unclear. Two ectonucleotidases that generate adenosine in nociceptive neurons were recently identified. When injected spinally, these enzymes have long-lasting adenosine A1 receptor (A1R)-dependent antinociceptive effects in inflammatory and neuropathic pain models. Furthermore, recent findings indicate that spinal adenosine A2A receptor activation can enduringly inhibit neuropathic pain symptoms. Collectively, these studies suggest the possibility of treating chronic pain in humans by targeting specific adenosine receptor subtypes in anatomically defined regions with agonists or with ectonucleotidases that generate adenosine. PMID:21236731

  16. The adenosine receptor affinities and monoamine oxidase B inhibitory properties of sulfanylphthalimide analogues.

    PubMed

    Van der Walt, Mietha M; Terre'Blanche, Gisella; Petzer, Anél; Petzer, Jacobus P

    2015-04-01

    Based on a report that sulfanylphthalimides are highly potent monoamine oxidase (MAO) B selective inhibitors, the present study examines the adenosine receptor affinities and MAO-B inhibitory properties of a series of 4- and 5-sulfanylphthalimide analogues. Since adenosine antagonists (A1 and A2A subtypes) and MAO-B inhibitors are considered agents for the therapy of neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease, dual-target-directed drugs that antagonize adenosine receptors and inhibit MAO-B may have enhanced therapeutic value. The results document that the sulfanylphthalimide analogues are selective for the adenosine A1 receptor over the A2A receptor subtype, with a number of compounds also possessing MAO-B inhibitory properties. Among the compounds evaluated, 5-[(4-methoxybenzyl)sulfanyl]phthalimide was found to possess the highest binding affinity to adenosine A1 receptors with a Ki value of 0.369 μM. This compound is reported to also inhibit MAO-B with an IC50 value of 0.020 μM. Such dual-target-directed compounds may act synergistic in the treatment of Parkinson's disease: antagonism of the A1 receptor may facilitate dopamine release, while MAO-B inhibition may reduce dopamine metabolism. Additionally, dual-target-directed compounds may find therapeutic value in Alzheimer's disease: antagonism of the A1 receptor may be beneficial in the treatment of cognitive dysfunction, while MAO-B inhibition may exhibit neuroprotective properties. In neurological diseases, such as Parkinson's disease and Alzheimer's disease, dual-target-directed drugs are expected to be advantageous over single-target treatments.

  17. Glucagon receptor antagonism induces increased cholesterol absorption.

    PubMed

    Guan, Hong-Ping; Yang, Xiaodong; Lu, Ku; Wang, Sheng-Ping; Castro-Perez, Jose M; Previs, Stephen; Wright, Michael; Shah, Vinit; Herath, Kithsiri; Xie, Dan; Szeto, Daphne; Forrest, Gail; Xiao, Jing Chen; Palyha, Oksana; Sun, Li-Ping; Andryuk, Paula J; Engel, Samuel S; Xiong, Yusheng; Lin, Songnian; Kelley, David E; Erion, Mark D; Davis, Harry R; Wang, Liangsu

    2015-11-01

    Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report findings that link MK-0893 with increased glucagon-like peptide 2 and cholesterol absorption. There was not, however, a GRA-related modulation of cholesterol synthesis. These findings were replicated using structurally diverse GRAs. To examine potential pharmacologic mitigation, coadministration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice abrogated the GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results provide a novel finding by which glucagon and, hence, GCGR antagonism govern cholesterol metabolism.

  18. 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

  19. Role of adenosine receptor subtypes in methamphetamine reward and reinforcement.

    PubMed

    Kavanagh, Kevin A; Schreiner, Drew C; Levis, Sophia C; O'Neill, Casey E; Bachtell, Ryan K

    2015-02-01

    The neurobiology of methamphetamine (MA) remains largely unknown despite its high abuse liability. The present series of studies explored the role of adenosine receptors on MA reward and reinforcement and identified alterations in the expression of adenosine receptors in dopamine terminal areas following MA administration in rats. We tested whether stimulating adenosine A1 or A2A receptor subtypes would influence MA-induced place preference or MA self-administration on fixed and progressive ratio schedules in male Sprague-Dawley rats. Stimulation of either adenosine A1 or A2A receptors significantly reduced the development of MA-induced place preference. Stimulating adenosine A1, but not A2A, receptors reduced MA self-administration responding. We next tested whether repeated experimenter-delivered MA administration would alter the expression of adenosine receptors in the striatal areas using immunoblotting. We observed no change in the expression of adenosine receptors. Lastly, rats were trained to self-administer MA or saline for 14 days and we detected changes in adenosine A1 and A2A receptor expression using immunoblotting. MA self-administration significantly increased adenosine A1 in the nucleus accumbens shell, caudate-putamen and prefrontal cortex. MA self-administration significantly decreased adenosine A2A receptor expression in the nucleus accumbens shell, but increased A2A receptor expression in the amygdala. These findings demonstrate that MA self-administration produces selective alterations in adenosine receptor expression in the nucleus accumbens shell and that stimulation of adenosine receptors reduces several behavioral indices of MA addiction. Together, these studies shed light onto the neurobiological alterations incurred through chronic MA use that may aid in the development of treatments for MA addiction.

  20. Activation of nicotinic ACh receptors with α4 subunits induces adenosine release at the rat carotid body

    PubMed Central

    Conde, Sílvia V; Monteiro, Emília C

    2006-01-01

    The effect of ACh on the release of adenosine was studied in rat whole carotid bodies, and the nicotinic ACh receptors involved in the stimulation of this release were characterized. ACh and nicotinic ACh receptor agonists, cytisine, DMPP and nicotine, caused a concentration-dependent increase in adenosine production during normoxia, with nicotine being more potent and efficient in stimulating adenosine release from rat CB than cytisine and DMPP. D-Tubocurarine, mecamylamine, DHβE and α-bungarotoxin, nicotinic ACh receptor antagonists, caused a concentration-dependent reduction in the release of adenosine evoked by hypoxia. The rank order of potency for nicotinic ACh receptor antagonists that inhibit adenosine release was DHβE>mecamylamine>D-tubocurarine>α-bungarotoxin. The effect of the endogenous agonist, ACh, which was mimicked by nicotine, was antagonized by DHβE, a selective nicotinic receptor antagonist. The ecto-5′-nucleotidase inhibitor AOPCP produces a 72% inhibition in the release of adenosine from CB evoked by nicotine. Taken together, these data indicate that ACh induced the production of adenosine, mainly from extracellular ATP catabolism at the CB through a mechanism that involves the activation of nicotinic receptors with α4 and β2 receptor subunits. PMID:16444287

  1. Caffeine intensifies taste of certain sweeteners: role of adenosine receptor.

    PubMed

    Schiffman, S S; Diaz, C; Beeker, T G

    1986-03-01

    Caffeine, a potent antagonist of adenosine receptors, potentiates the taste of some but not all sweeteners. It significantly enhances the taste of acesulfam-K, neohesperidin dihydrochalcone, d-tryptophan, thaumatin, stevioside, and sodium saccharin. Adenosine reverses the enhancement. Caffeine has no effect on aspartame, sucrose, fructose, and calcium cyclamate. These results suggest that the inhibitory A1 adenosine receptor plays an important local role in modulating the taste intensity of certain sweeteners and that several transduction mechanisms mediate sweet taste.

  2. Adenosine modulation of [Ca2+]i in cerebellar granular cells: multiple adenosine receptors involved.

    PubMed

    Vacas, Javier; Fernández, Mercedes; Ros, Manuel; Blanco, Pablo

    2003-12-01

    Elimination of adenosine by addition of adenosine deaminase (ADA) to the media leads to alterations in intracellular free calcium concentration ([Ca(2+)](i)) in cerebellar granular cells. Adenosine deaminase brings about increases or decreases in [Ca(2+)](i) depending on the previous activation state of the cell. These effects are dependent on the catalytic activity of adenosine deaminase, since its previous catalytic inactivation with Hg(2+) prevents the above-mentioned changes in intracellular calcium. Extracellular calcium is required for the increase in [Ca(2+)](i) promoted by ADA. This rise is insensitive to thapsigargin, but sensitive to micromolar concentrations of Ni(2+). Toxins specific for L, N and P/Q calcium channels do not overtly reduce this effect. N(6)-Cyclopentyl adenosine (CPA), an A(1) receptor agonist, produces a partial reversion of ADA effects, while CGS21680, A(2A)/A(2B) receptor agonist, slightly enhances them. Expression of A(1), A(2A), A(2B) and A(3) adenosine receptor mRNAs was detected in cerebellar granular cell cultures. These results suggest that adenosine modulate [Ca(2+)](i) in cerebellar granule cells through different adenosine receptor subtypes which, at least in part, seem to act through R-type calcium channels.

  3. Introduction to Adenosine Receptors as Therapeutic Targets

    PubMed Central

    Jacobson, Kenneth A.

    2012-01-01

    Adenosine acts as a cytoprotective modulator in response to stress to an organ or tissue. Although short-lived in the circulation, it can activate four sub-types of G protein-coupled adenosine receptors (ARs): A1, A2A, A2B, and A3. The alkylxanthines caffeine and theophylline are the prototypical antagonists of ARs, and their stimulant actions occur primarily through this mechanism. For each of the four AR subtypes, selective agonists and antagonists have been introduced and used to develop new therapeutic drug concepts. ARs are notable among the GPCR family in the number and variety of agonist therapeutic candidates that have been proposed. The selective and potent synthetic AR agonists, which are typically much longer lasting in the body than adenosine, have potential therapeutic applications based on their anti-inflammatory (A2A and A3), cardioprotective (preconditioning by A1 and A3 and postconditioning by A2B), cerebroprotective (A1 and A3), and antinociceptive (A1) properties. Potent and selective AR antagonists display therapeutic potential as kidney protective (A1), antifibrotic (A2A), neuroprotective (A2A), and antiglaucoma (A3) agents. AR agonists for cardiac imaging and positron-emitting AR antagonists are in development for diagnostic applications. Allosteric modulators of A1 and A3 ARs have been described. In addition to the use of selective agonists/antagonists as pharmacological tools, mouse strains in which an AR has been genetically deleted have aided in developing novel drug concepts based on the modulation of ARs. PMID:19639277

  4. KW-3902, a selective high affinity antagonist for adenosine A1 receptors.

    PubMed Central

    Nonaka, H.; Ichimura, M.; Takeda, M.; Kanda, T.; Shimada, J.; Suzuki, F.; Kase, H.

    1996-01-01

    1. We demonstrate that 8-(noradamantan-3-yl)-1,3-dipropylxanthine (KW-3902) is a very potent and selective adenosine A1 receptor antagonist, assessed by radioligand binding and cyclic AMP response in cells. 2. In rat forebrain adenosine A1 receptors labelled with [3H]-cyclohexyladenosine (CHA), KW-3902 had a Ki value of 0.19 nM, whereas it showed a Ki value of 170 nM in rat striatal A2A receptors labelled with [3H]-2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoad enosine (CGS21680), indicating 890 fold A1 receptor selectivity versus the A2A receptor. KW-3902 at 10 microM showed no effect on recombinant rat A3 receptors expressed on CHO cells. 3. Saturation studies with [3H]-KW-3902 revealed that it bound with high affinity (Kd = 77 pM) and limited capacity (Bmax = 470 fmol mg-1 of protein) to a single class of recognition sites. A high positive correlation was observed between the pharmacological profile of adenosine ligands inhibiting the binding of [3H]-KW-3902 and that of [3H]-CHA. 4. KW-3902 showed potent A1 antagonism against the inhibition of forskolin-induced cyclic AMP accumulation in DDT1 MF-2 cells by the A1-selective agonist, cyclopentyladenosine with a dissociation constant (KB value) of 0.34 nM. KW-3902 antagonized 5'-N-ethylcarboxamidoadenosine-elicited cyclic AMP accumulation via A2B receptors with a KB value of 52 nM. 5. KW-3902 exhibited marked species-dependent differences in the binding affinities. The highest affinity was for the rat A1 receptor (ki = 0.19 nM) and these values for guinea-pig and dog A1 receptors were 1.3 and 10 nM, respectively. PMID:8732272

  5. Determination of adenosine effects and adenosine receptors in murine corpus cavernosum.

    PubMed

    Tostes, Rita C; Giachini, Fernanda R C; Carneiro, Fernando S; Leite, Romulo; Inscho, Edward W; Webb, R Clinton

    2007-08-01

    This study tested the hypothesis that adenosine, in murine corpora cavernosa, produces direct relaxation of smooth muscle cells and inhibition of contractile responses mediated by sympathetic nerve stimulation. Penes were excised from anesthetized male C57BL/6 mice, dissected, and cavernosal strips were mounted to record isometric force. Adenosine, 2-chloroadenosine (stable analog of adenosine), and 2-phenylaminoadenosine (CV1808) (A2(A)/A2(B) agonist) produced concentration-dependent relaxations of phenylephrine-contracted tissues. Relaxation to 2-chloroadenosine was inhibited, in a concentration-dependent manner, by 2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH58261; A2(A) antagonist; 10(-9)-10(-6) M) and N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamida (MRS1706; A2(B) antagonist; 10(-8)-10(-6) M). The combination of both antagonists abrogated 2-chloroadenosine-induced relaxation. Electrical field stimulation (EFS; 1-32 Hz) of adrenergic nerves produced frequency-dependent contractions that were inhibited by compounds that increase adenosine levels, such as 5'-iodotubercidin (adenosine kinase inhibitor), erythro-9-(2-hydroxy-3-nonyl)adenine (adenosine deaminase inhibitor), and dipyridamole (inhibitor of adenosine transport). The adenosine A1 receptor agonist N(6)-cyclopentyladenosine (C8031) right-shifted contractile responses to EFS, with a significant inhibitory effect at 10(-6) M. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine (C101) (10(-7) M) enhanced contractile responses to EFS and eliminated the inhibitory effects of 5'-iodotubercidin. Dipyridamole and 5'-iodotubercidin had no effect on adenosine-mediated relaxation. In summary, adenosine directly relaxes cavernosal smooth muscle cells, by the activation of A2(A)/A2(B) receptor subtypes. In addition, adenosine negatively modulates sympathetic neurotransmission, by A1 receptor

  6. GIRK channel activation via adenosine or muscarinic receptors has similar effects on rat atrial electrophysiology.

    PubMed

    Wang, Xiaodong; Liang, Bo; Skibsbye, Lasse; Olesen, Søren-Peter; Grunnet, Morten; Jespersen, Thomas

    2013-08-01

    G protein-coupled inwardly rectifying K⁺ channels (GIRK) are important in the regulation of heart rate and atrial electrophysiology. GIRK channels are activated by G protein-coupled receptors, including muscarinic M₂ receptors and adenosine A₁ receptors. The aim of this study was to characterize and compare the electrophysiological effects of acetylcholine (ACh) and adenosine on GIRK channels in rat atria. Action potential duration at 90% repolarization (APD₉₀), effective refractory period (ERP), and resting membrane potential (RMP) were investigated in isolated rat atria by intracellular recordings. Both the adenosine analog N6-cyclopentyladenosine (CPA) and ACh profoundly shortened APD₉₀ and ERP and hyperpolarized the RMP. No additive or synergistic effect of CPA and ACh coapplication was observed. To antagonize GIRK channel activation, the specific inhibitor rTertiapin Q (TTQ) was applied. The coapplication of TTQ reversed the CPA and ACh-induced effects. When TTQ was applied without exogenous receptor activator, both APD₉₀ and ERP were prolonged and RMP was depolarized, confirming a basal activity of the GIRK current. The results reveal that activation of A₁ and M₂ receptors has a profound and equal effect on the electrophysiology in rat atrium. This effect is to a major extent mediated through GIRK channels. Furthermore, these results support the notion that atrial GIRK currents from healthy hearts have a basal component and additional activation can be mediated via at least 2 different receptor mechanisms. PMID:23609329

  7. Adenosine receptor ligands: differences with acute versus chronic treatment

    PubMed Central

    Jacobson, Kenneth A.; von Lubitz, Dag K. J. E.; Daly, John W.; Fredholm, Bertil B.

    2012-01-01

    Adenosine receptors have been the target of intense research with respect to potential use of selective ligands in a variety of therapeutic areas. Caffeine and theophylline are adenosine receptor antagonists, and over the past three decades a wide range of selective agonists and antagonists for adenosine receptor subtypes have been developed. A complication to the therapeutic use of adenosine receptor ligands is the observation that the effects of acute administration of a particular ligand can be diametrically opposite to the chronic effects of the same ligand. This ‘effect inversion’ is discussed here by Ken Jecobson and colleagues, and has been observed for effects on cognitive processes, seizures and ischaemic damage. PMID:8936347

  8. 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...

  9. 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.

  10. 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

  11. Evidence for an atypical receptor mediating the augmented bronchoconstrictor response to adenosine induced by allergen challenge in actively sensitized Brown Norway rats.

    PubMed

    Hannon, J P; Tigani, B; Wolber, C; Williams, I; Mazzoni, L; Howes, C; Fozard, J R

    2002-02-01

    The bronchoconstrictor response to adenosine is markedly and selectively increased following ovalbumin (OA) challenge in actively sensitized, Brown Norway rats. We present a pharmacological analysis of the receptor mediating this response. Like adenosine, the broad-spectrum adenosine receptor agonist, NECA, induced dose-related bronchoconstriction in actively sensitized, OA-challenged animals. In contrast, CPA, CGS 21680 and 2-Cl-IB-MECA, agonists selective for A(1) A(2A) and A(3) receptors, respectively, induced no, or minimal, bronchoconstriction. Neither the selective A(1) receptor antagonist, DPCPX, nor the selective A(2A) receptor antagonist, ZM 241385, blocked the bronchoconstrictor response to adenosine. MRS 1754, which has similar affinity for rat A(2B) and A(1) receptors, failed to block the bronchoconstrictor response to adenosine despite blockade of the A(1) receptor-mediated bradycardia induced by NECA. 8-SPT and CGS 15943, antagonists at A(1), A(2A), and A(2B) but not A(3) receptors, inhibited the bronchoconstrictor response to adenosine. However, the degree of blockade (approximately 3 fold) did not reflect the plasma concentrations, which were 139 and 21 times greater than the K(B) value at the rat A(2B) receptor, respectively. Adenosine and NECA, but not CPA, CGS 21680 or 2-Cl-IB-MECA, induced contraction of parenchymal strip preparations from actively sensitized OA-challenged animals. Responses to adenosine could not be antagonized by 8-SPT or MRS 1754 at concentrations >50 times their affinities at the rat A(2B) receptor. The receptor mediating the bronchoconstrictor response to adenosine augmented following allergen challenge in actively sensitized BN rats cannot be categorized as one of the four recognized adenosine receptor subtypes.

  12. CGRP receptor antagonism and migraine therapy.

    PubMed

    Edvinsson, Lars; Warfvinge, Karin

    2013-08-01

    Migraine is the most prevalent of the neurological disorders and can affect the patient throughout the lifetime. Calcitonin gene-related peptide (CGRP) is a neuropeptide that is expressed in the central and peripheral nervous systems. It is now 2 decades since it was proposed to be involved in migraine pathophysiology. The cranial sensory system contains C-fibers storing CGRP and trigeminal nerve activation and acute migraine attacks result in release of CGRP. The CGRP receptor consists of a complex of calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1) and receptor component protein (RCP). At the central synapses in the trigeminal nucleus caudalis, CGRP acts postjunctionally on second-order neurons to transmit pain signals centrally via brainstem and midbrain to thalamus and higher cortical pain regions. CLR and RAMPs are widely expressed throughout the brain, in the trigeminal ganglion and in intracranial arteries. CGRP does not induce neurogenic inflammation or sensitization at peripheral meningeal sites but relays nociceptive information from trigeminal primary afferent neurons to the second-order neurons in the spinal trigeminal nucleus neurons. CGRP receptor antagonists have been developed as novel antimigraine drugs and found to be effective in the treatment of acute migraine attacks. Other ways to stop CGRP activity has been introduced recently through antibodies against CGRP and the CGRP receptor. While the CGRP receptors are expressed both in the CNS and at various places related to the trigeminal system the exact site of action for their therapy effect is still unresolved but the new approaches may resolve this. PMID:23745702

  13. 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.

  14. N6-(2-Hydroxyethyl)-Adenosine Exhibits Insecticidal Activity against Plutella xylostella via Adenosine Receptors

    PubMed Central

    Fang, Ming; Chai, Yiqiu; Chen, Guanjv; Wang, Huidong; Huang, Bo

    2016-01-01

    The diamondback moth, Plutella xylostella, is one of the most important pests of cruciferous crops. We have earlier shown that N6-(2-hydroxyethyl)-adenosine (HEA) exhibits insecticidal activity against P. xylostella. In the present study we investigated the possible mechanism of insecticidal action of HEA on P. xylostella. HEA is a derivative of adenosine, therefore, we speculated whether it acts via P. xylostella adenosine receptor (PxAdoR). We used RNAi approach to silence PxAdoR gene and used antagonist of denosine receptor (AdoR) to study the insecticidal effect of HEA. We cloned the whole sequence of PxAdoR gene. A BLAST search using NCBI protein database showed a 61% identity with the Drosophila adenosine receptor (DmAdoR) and a 32–35% identity with human AdoR. Though the amino acids sequence of PxAdoR was different compared to other adenosine receptors, most of the amino acids that are known to be important for adenosine receptor ligand binding and signaling were present. However, only 30% binding sites key residues was similar between PxAdoR and A1R. HEA, at a dose of 1 mg/mL, was found to be lethal to the second-instar larvae of P. xylostella, and a significant reduction of mortality and growth inhibition ratio were obtained when HEA was administered to the larvae along with PxAdoR-dsRNA or antagonist of AdoR (SCH58261) for 36, 48, or 60 h. Especially at 48 h, the rate of growth inhibition of the PxAdoR knockdown group was 3.5-fold less than that of the HEA group, and the corrected mortality of SCH58261 group was reduced almost 2-fold compared with the HEA group. Our findings show that HEA may exert its insecticidal activity against P. xylostella larvae via acting on PxAdoR. PMID:27668428

  15. Induction of oral tremor in mice by the acetylcholinesterase inhibitor galantamine: Reversal with adenosine A2A antagonism.

    PubMed

    Podurgiel, Samantha J; Spencer, Tiahna; Kovner, Rotem; Baqi, Younis; Müller, Christa E; Correa, Merce; Salamone, John D

    2016-01-01

    Tremulous jaw movements (TJMs) have become a commonly used rat model of Parkinsonian tremor. TJMs can be induced by a number of neurochemical conditions that parallel those seen in human Parkinsonism, including DA depletion, DA antagonism, and cholinomimetic administration, and can be reduced by various antiparkinsonian agents. TJMs typically occur in bursts with the peak frequency in the range of 3-7.5 Hz, which is similar to the Parkinsonian tremor frequency range. While the vast majority of this work has been done using rats, current efforts have focused on extending the TJM model to mice. The aim of the present studies was to establish a mouse model of Parkinsonian resting tremor using the anticholinesterase galantamine, and to investigate the effects of adenosine A2A antagonism on galantamine-induced TJMs. Galantamine significantly induced TJMs in a dose-dependent manner (0.5, 1.0, 1.5, 2.0, 2.5 mg/kg IP). The TJMs tended to occur in bursts in the 3-7.5 Hz frequency range, with a peak frequency of approximately 6 Hz. Systemic administration of the adenosine A2A antagonist MSX-3 (2.5, 5.0, 10.0 mg/kg) significantly attenuated galantamine-induced TJMs. Co-administration of MSX-3 also altered the local frequency of galantamine-induced TJMs, decreasing the peak frequency from approximately 6 Hz to 5 Hz, though the vast majority of TJMs remained in the frequency range characteristic of Parkinsonian resting tremor. These results indicate that adenosine A2A antagonism is capable of reducing anticholinesterase-induced TJMs in mice. Extending the TJM model to mice gives researchers an additional avenue for investigating drug-induced Parkinsonism and tremorogenesis, and could be a useful addition to the study of motor abnormalities observed in mouse genetic models of Parkinsonism. PMID:26459156

  16. Mechanism of A2 adenosine receptor activation. I. Blockade of A2 adenosine receptors by photoaffinity labeling

    SciTech Connect

    Lohse, M.J.; Klotz, K.N.; Schwabe, U.

    1991-04-01

    It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxy-phenylisopropyladenosine ((R)-AHPIA) into the A1 adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of cellular cAMP levels. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenylate cyclase stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies indicate that up to 90% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20% of A2 receptors are sufficient to mediate an adenylate cyclase stimulation of up to 50% of the control value. Similarly, the activation via these 10-20% of receptors occurs with a half-life that is only 2 times longer than that in control membranes. This indicates the presence of a receptor reserve, with respect to both the extent and the rate of adenylate cyclase stimulation. These observations require a modification of the models of receptor-adenylate cyclase coupling.

  17. 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

  18. 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.

  19. 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.

  20. Adenosine receptor agonists for promotion of dermal wound healing

    PubMed Central

    Valls, María D.; Cronstein, Bruce N.; Montesinos, M. Carmen

    2009-01-01

    Wound healing is a dynamic and complex process that involves a well coordinated, highly regulated series of events including inflammation, tissue formation, revascularization and tissue remodeling. However, this orderly sequence is impaired in certain pathophysiological conditions such as diabetes mellitus, venous insufficiency, chronic glucocorticoid use, aging and malnutrition. Together with proper wound care, promotion of the healing process is the primary objective in the management of chronic poorly healing wounds. Recent studies have demonstrated that A2A adenosine receptor agonists promote wound healing in normal and diabetic animals and one such agonist, Sonedenoson, is currently being evaluated as a prospective new therapy of diabetic foot ulcers. We will review the mechanisms by which adenosine receptor activation affects the function of the cells and tissues that participate in wound healing, emphasizing the potential beneficial impact of adenosine receptor agonists in diabetic impaired healing. PMID:19041853

  1. 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

  2. Presynaptic adenosine A1 receptors regulate retinohypothalamic neurotransmission in the hamster suprachiasmatic nucleus.

    PubMed

    Hallworth, Richard; Cato, Matthew; Colbert, Costa; Rea, Michael A

    2002-09-01

    Adenosine has been implicated as a modulator of retinohypothalamic neurotransmission in the suprachiasmatic nucleus (SCN), the seat of the light-entrainable circadian clock in mammals. Intracellular recordings were made from SCN neurons in slices of hamster hypothalamus using the in situ whole-cell patch clamp method. A monosynaptic, glutamatergic, excitatory postsynaptic current (EPSC) was evoked by stimulation of the optic nerve. The EPSC was blocked by bath application of the adenosine A(1) receptor agonist cyclohexyladenosine (CHA) in a dose-dependent manner with a half-maximal concentration of 1.7 microM. The block of EPSC amplitude by CHA was antagonized by concurrent application of the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The adenosine A(2A) receptor agonist CGS21680 was ineffective in attenuating the EPSC at concentrations up to 50 microM. Trains of four consecutive stimuli at 25 ms intervals usually depressed the EPSC amplitude. However, after application of CHA, consecutive responses displayed facilitation of EPSC amplitude. The induction of facilitation by CHA suggested a presynaptic mechanism of action. After application of CHA, the frequency of spontaneous EPSCs declined substantially, while their amplitude distribution was unchanged or slightly reduced, again suggesting a mainly presynaptic site of action for CHA. Application of glutamate by brief pressure ejection evoked a long-lasting inward current that was unaffected by CHA at concentrations sufficient to reduce the evoked EPSC amplitude substantially (1 to 5 microM), suggesting that postsynaptic glutamate receptor-gated currents were unaffected by the drug. Taken together, these observations indicate that CHA inhibits optic nerve-evoked EPSCs in SCN neurons by a predominantly presynaptic mechanism. PMID:12210106

  3. 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.

  4. 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

  5. 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

  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. Therapeutic potential of endothelin receptor antagonism in kidney disease.

    PubMed

    Czopek, Alicja; Moorhouse, Rebecca; Webb, David J; Dhaun, Neeraj

    2016-03-01

    Our growing understanding of the role of the endothelin (ET) system in renal physiology and pathophysiology is from emerging studies of renal disease in animal models and humans. ET receptor antagonists reduce blood pressure and proteinuria in chronic kidney disease and cause regression of renal injury in animals. However, the therapeutic potential of ET receptor antagonism has not been fully explored and clinical studies have been largely limited to patients with diabetic nephropathy. There remains a need for more work in nondiabetic chronic kidney disease, end-stage renal disease (patients requiring maintenance dialysis and those with a functioning kidney transplant), ischemia reperfusion injury, and sickle cell disease. The current review summarizes the most recent advances in both preclinical and clinical studies of ET receptor antagonists in the field of kidney disease.

  8. Agonist Derived Molecular Probes for A2A Adenosine Receptors

    PubMed Central

    Jacobson, Kenneth A.; Pannell, Lewis K.; Ji, Xiao-duo; Jarvis, Michael F.; Williams, Michael; Hutchison, Alan J.; Barrington, William W.; Stiles, Gary L.

    2011-01-01

    The adenosine agonist 2-(4-(2-carboxyethyl)phenylethylamino)-5′-N-ethylcarboxamidoadenosine (CGS21680) was recently reported to be selective for the A2A adenosine receptor subtype, which mediates its hypotensive action. To investigate structurelactivity relationships at a distal site, CGS21680 was derivatized using a functionalized congener approach. The carboxylic group of CGS21680 has been esterified to form a methyl ester, which was then treated with ethylenediamine to produce an amine congener. The amine congener was an intermediate for acylation reactions, in which the reactive acyl species contained a reported group, or the precursor for such. For radioiodination, derivatives of p-hydroxyphenylpropionic, 2-thiophenylacetic, and p-aminophenylacetic acids were prepared. The latter derivative (PAPA-APEC) was iodinated electrophilically using [125I]iodide resulting in a radioligand which was used for studies of competition of binding to striatal A, adenosine receptors in bovine brain. A biotin conjugate and an aryl sulfonate were at least 350-fold selective for A, receptors. For spectroscopic detection, a derivative of the stable free radical tetramethyl-1-piperidinyloxy (TEMPO) was prepared. For irreversible inhibition of receptors, meta- and para-phenylenediisothiocyanate groups were incorporated in the analogs. We have demonstrated that binding at A2A receptors is relatively insensitive to distal structural changes at the 2-position, and we report high affinity molecular probes for receptor characterization by radioactive, spectroscopic and affinity labelling methodology. PMID:2561548

  9. Parasympathetic depression of vas deferens contraction in the guinea-pig involves adenosine receptors.

    PubMed Central

    Kurokawa, M; Tsunoo, A

    1988-01-01

    1. In the guinea-pig pelvic plexus-vas deferens preparation, stimulation of the parasympathetic pelvic nerves contracted the vas deferens then depressed the contractile responses to stimulation of the sympathetic hypogastric nerves. 2. The contraction caused by stimulation of the pelvic nerves was initially phasic then tonic. The contractions were almost abolished by application of hexamethonium to the plexus. The phasic contraction was abolished by alpha,beta-methylene adenosine triphosphate applied to the vas deferens. 3. Conditioning stimulation of the pelvic nerves preferentially depressed the phasic component of test contractions evoked by hypogastric nerve stimulation but did not affect the compound action potentials in postganglionic nerves evoked by test stimulation. 4. When the pelvic plexus was divided into two parts, one with the pelvic nerves and the other with the hypogastric nerves, conditioning stimulation of the pelvic nerves still depressed test contractions evoked by hypogastric nerve stimulation. 5. In the de-ganglionated vas deferens preparation, conditioning stimulation of some postganglionic nerves also depressed contractions evoked by test stimulation of the other postganglionic nerves. 6. 8-Phenyltheophylline (5-20 microM) applied to the vas deferens antagonized the conditioning stimulation-induced depression in both the pelvic plexus-vas deferens and the de-ganglionated preparations. 7. N6-Cyclohexyladenosine (CHA) and N6-(L-2-phenylisopropyl)-adenosine at 0.5 microM preferentially inhibited phasic contractions evoked by the postganglionic nerve stimulation. The effect of CHA was antagonized by 8-phenyltheophylline (10 microM). 8. The results indicate that the mechanism underlying the conditioning stimulation-induced depression of phasic contractions operates not in the ganglia, but through activation of adenosine receptors in the vas deferens. PMID:3256614

  10. Chemodetection in fluctuating environments: receptor coupling, buffering, and antagonism.

    PubMed

    Lalanne, Jean-Benoît; François, Paul

    2015-02-10

    Variability in the chemical composition of the extracellular environment can significantly degrade the ability of cells to detect rare cognate ligands. Using concepts from statistical detection theory, we formalize the generic problem of detection of small concentrations of ligands in a fluctuating background of biochemically similar ligands binding to the same receptors. We discover that in contrast with expectations arising from considerations of signal amplification, inhibitory interactions between receptors can improve detection performance in the presence of substantial environmental variability, providing an adaptive interpretation to the phenomenon of ligand antagonism. Our results suggest that the structure of signaling pathways responsible for chemodetection in fluctuating and heterogeneous environments might be optimized with respect to the statistics and dynamics of environmental composition. The developed formalism stresses the importance of characterizing nonspecific interactions to understand function in signaling pathways. PMID:25624502

  11. Chemodetection in fluctuating environments: Receptor coupling, buffering, and antagonism

    PubMed Central

    Lalanne, Jean-Benoît; François, Paul

    2015-01-01

    Variability in the chemical composition of the extracellular environment can significantly degrade the ability of cells to detect rare cognate ligands. Using concepts from statistical detection theory, we formalize the generic problem of detection of small concentrations of ligands in a fluctuating background of biochemically similar ligands binding to the same receptors. We discover that in contrast with expectations arising from considerations of signal amplification, inhibitory interactions between receptors can improve detection performance in the presence of substantial environmental variability, providing an adaptive interpretation to the phenomenon of ligand antagonism. Our results suggest that the structure of signaling pathways responsible for chemodetection in fluctuating and heterogeneous environments might be optimized with respect to the statistics and dynamics of environmental composition. The developed formalism stresses the importance of characterizing nonspecific interactions to understand function in signaling pathways. PMID:25624502

  12. 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

  13. 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

  14. Adenosine receptor modulation of seizure susceptibility in rats

    SciTech Connect

    Szot, P.

    1987-01-01

    Adenosine is considered to be a neuromodulator or cotransmitter in the periphery and CNS. This neuromodulatory action of adenosine may be observed as an anticonvulsant effect. Dose-response curves for R-phenylisopropyladenosine (PIA), cycohexyladenosine (CHA), 2-chloroadenosine (2-ClAdo), N-ethylcarboxamidoadenosine (NECA) and S-PIA were generated against PTZ seizure thresholds in the rat. The rank order of potency for adenosine agonists to elevate PTZ seizure threshold was R-PIA > 2-ClAdo > NECA > CHA > S-PIA. R-PIA was approximately 80-fold more potent than S-PIA. This 80-fold difference in potency between the diasteriomers of PIA was consistent with an A{sub 1} adenoise receptor-mediated response. The anticonvulsant action of 2-ClAdo was reversed by pretreatment with theoplylline. Chronic administration of theophylline significantly increased the specific binding of {sup 3}H-cyclohexyladenosine in membranes of the cerebral cortex and cerebellum of the rat. Chronic exposure to theophylline produced a significant increase in the densities of both the high- and low-affinity forms of A{sub 1} adenosine receptors in the cerebral cortex.

  15. 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.

  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. Binding kinetics differentiates functional antagonism of orexin-2 receptor ligands

    PubMed Central

    Mould, R; Brown, J; Marshall, FH; Langmead, CJ

    2014-01-01

    Orexin receptor antagonism represents a novel approach for the treatment of insomnia that directly targets sleep/wake regulation. Several such compounds have entered into clinical development, including the dual orexin receptor antagonists, suvorexant and almorexant. In this study, we have used equilibrium and kinetic binding studies with the orexin-2 (OX2) selective antagonist radioligand, [3H]-EMPA, to profile several orexin receptor antagonists. Furthermore, selected compounds were studied in cell-based assays of inositol phosphate accumulation and ERK-1/2 phosphorylation in CHO cells stably expressing the OX2 receptor that employ different agonist incubation times (30 and 5 min, respectively). EMPA, suvorexant, almorexant and TCS-OX-29 all bind to the OX2 receptor with moderate to high affinity (pKI values ≥ 7.5), whereas the primarily OX1 selective antagonists SB-334867 and SB-408124 displayed low affinity (pKI values ca. 6). Competition kinetic analysis showed that the compounds displayed a range of dissociation rates from very fast (TCS-OX2-29, koff = 0.22 min−1) to very slow (almorexant, koff = 0.005 min−1). Notably, there was a clear correlation between association rate and affinity. In the cell-based assays, fast-offset antagonists EMPA and TCS-OX2-29 displayed surmountable antagonism of orexin-A agonist activity. However, both suvorexant and particularly almorexant cause concentration-dependent depression in the maximal orexin-A response, a profile that is more evident with a shorter agonist incubation time. Analysis according to a hemi-equilibrium model suggests that antagonist dissociation is slower in a cellular system than in membrane binding; under these conditions, almorexant effectively acts as a pseudo-irreversible antagonist. Linked ArticlesThis article is part of a themed section on Orexin Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-2 PMID:23692283

  18. 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

  19. 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

  20. Adenosine A1 receptors determine effects of caffeine on total fluid intake but not caffeine appetite.

    PubMed

    Rieg, Timo; Schnermann, Jürgen; Vallon, Volker

    2007-01-26

    Adenosine A1 receptor wild-type (+/+) and knockout (-/-) mice were used to elucidate the role of adenosine A1 receptors in caffeine self-administration in a two-bottle choice test and in the effect of caffeine on total fluid intake and plasma renin concentration. With access to water only, adenosine A1 receptor -/- mice showed greater basal fluid intake and greater plasma renin concentration than +/+ mice. Free access to both water and a caffeinated solution (30 mg/100 ml) for 14 days increased total fluid intake only in adenosine A1 receptor +/+ mice (by 23+/-3%), and both total fluid intake and plasma renin concentration were no longer different between genotypes. Mean intake of water and caffeinated solution was not different between adenosine A1 receptor +/+ and -/- mice. These data reveal that adenosine A1 receptors do not contribute to caffeine consumption, but determine the effects of caffeine on fluid intake and plasma renin concentration. PMID:17126319

  1. Adenosine through the A2A adenosine receptor increases IL-1β in the brain contributing to anxiety

    PubMed Central

    Chiu, Gabriel S.; Darmody, Patrick T.; Walsh, John P.; Moon, Morgan L.; Kwakwa, Kristin A.; Bray, Julie K.; McCusker, Robert H.; Freund, Gregory G.

    2014-01-01

    Anxiety is one of the most commonly reported psychiatric conditions, but its pathogenesis is poorly understood. Ailments associated with activation of the innate immune system, however, are increasingly linked to anxiety disorders. In adult male mice, we found that adenosine doubled caspase-1 activity in brain by a pathway reliant on ATP-sensitive potassium (KATP) channels, protein kinase A (PKA) and the A2A adenosine receptor (AR). In addition, adenosine-dependent activation of caspase-1 increased interleukin (IL)-1β in the brain by two-fold. Peripheral administration of adenosine in wild-type (WT) mice led to a 2.3-fold increase in caspase-1 activity in the amygdala and to a 33% and 42% reduction in spontaneous locomotor activity and food intake, respectively, that were not observed in caspase-1 knockout (KO), IL-1 receptor type 1 (IL-1R1) KO and A2A AR KO mice or in mice administered a caspase-1 inhibitor centrally. Finally, adenosine administration increased anxiety-like behaviors in WT mice by 28% in the open field test and by 55% in the elevated zero-maze. Caspase-1 KO mice, IL-1R1 KO mice, A2A AR KO mice and WT mice treated with the KATP channel blocker, glyburide, were resistant to adenosine-induced anxiety-like behaviors. Thus, our results indicate that adenosine can act as an anxiogenic by activating caspase-1 and increasing IL-1β in the brain. PMID:24907587

  2. 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

  3. Impaired inhibitory function of presynaptic A1-adenosine receptors in SHR mesenteric arteries.

    PubMed

    Rocha-Pereira, Carolina; Arribas, Silvia Magdalena; Fresco, Paula; González, Maria Carmen; Gonçalves, Jorge; Diniz, Carmen

    2013-01-01

    In hypertension, vascular reactivity alterations have been attributed to numerous factors, including higher sympathetic innervation/adenosine. This study examined the modulation of adenosine receptors on vascular sympathetic nerves and their putative contribution to higher noradrenaline spillover in hypertension. We assessed adenosine receptors distribution in the adventitia through confocal microscopy, histomorphometry, and their regulatory function on electrically-evoked [(3)H]-noradrenaline overflow, using selective agonists/antagonists. We found that: i) A1-adenosine receptor agonist (CPA: 100 nM) inhibited tritium overflow to a lower extent in SHR (25% ± 3%, n = 14) compared to WKY (38% ± 3%, n = 14) mesenteric arteries; ii) A2A-adenosine receptor agonist (CGS 21680: 100 nM) induced a slight increase of tritium overflow that was similar in SHR (22% ± 8%, n = 8) and WKY (24% ± 5%, n = 8) mesenteric arteries; iii) A2B- and A3-adenosine receptors did not alter tritium overflow in either strain; iv) all adenosine receptors were present on mesenteric artery sympathetic nerves and/or some adventitial cells of both strains; and v) A1-adenosine receptor staining fractional area was lower in SHR than in WKY mesenteric arteries. We conclude that there is an impaired inhibitory function of vascular presynaptic A1-adenosine receptors in SHR, likely related to a reduced presence of these receptors on sympathetic innervation, which might lead to higher levels of noradrenaline in the synaptic cleft and contribute to hypertension in this strain.

  4. Medicinal Chemistry of the A3 Adenosine Receptor: Agonists, Antagonists, and Receptor Engineering

    PubMed Central

    Jacobson, Kenneth A.; Klutz, Athena M.; Tosh, Dilip K.; Ivanov, Andrei A.; Preti, Delia; Baraldi, Pier Giovanni

    2012-01-01

    A3 adenosine receptor (A3AR) ligands have been modified to optimize their interaction with the A3AR. Most of these modifications have been made to the N6 and C2 positions of adenine as well as the ribose moiety, and using a combination of these substitutions leads to the most efficacious, selective, and potent ligands. A3AR agonists such as IB-MECA and Cl-IB-MECA are now advancing into Phase II clinical trials for treatments targeting diseases such as cancer, arthritis, and psoriasis. Also, a wide number of compounds exerting high potency and selectivity in antagonizing the human (h)A3AR have been discovered. These molecules are generally characterized by a notable structural diversity, taking into account that aromatic nitrogen-containing monocyclic (thiazoles and thiadiazoles), bicyclic (isoquinoline, quinozalines, (aza)adenines), tricyclic systems (pyrazoloquinolines, triazoloquinoxalines, pyrazolotriazolopyrimidines, triazolopurines, tricyclic xanthines) and nucleoside derivatives have been identified as potent and selective A3AR antagonists. Probably due to the “enigmatic” physiological role of A3AR, whose activation may produce opposite effects (for example, concerning tissue protection in inflammatory and cancer cells) and may produce effects that are species dependent, only a few molecules have reached preclinical investigation. Indeed, the most advanced A3AR antagonists remain in preclinical testing. Among the antagonists described above, compound OT-7999 is expected to enter clinical trials for the treatment of glaucoma, while several thiazole derivatives are in development as antiallergic, antiasthmatic and/or antiinflammatory drugs. PMID:19639281

  5. 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

  6. 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

  7. 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

  8. Nucleus accumbens neurotransmission and effort-related choice behavior in food motivation: effects of drugs acting on dopamine, adenosine, and muscarinic acetylcholine receptors.

    PubMed

    Nunes, Eric J; Randall, Patrick A; Podurgiel, Samantha; Correa, Mercè; Salamone, John D

    2013-11-01

    Mesolimbic dopamine (DA) is a critical component of the brain circuitry regulating behavioral activation and effort-related processes. Although nucleus accumbens (NAc) DA depletions or antagonism leave aspects of appetite and primary food motivation intact, rats with impaired DA transmission reallocate their instrumental behavior away from food-reinforced tasks with high response requirements, and instead select less effortful food-seeking behaviors. Previous work showed that adenosine A2A antagonists can reverse the effects of DA D2 antagonists on effort-related choice, and that stimulation of adenosine A2A receptors produces behavioral effects that are similar to those induced by DA antagonism. The present review summarizes the literature on the role of NAc DA and adenosine in effort-related processes, and also presents original data on the effects of local stimulation of muscarinic acetylcholine receptors in NAc core. Local injections of the muscarinic agonist pilocarpine directly into NAc core produces shifts in effort-related choice behavior similar to those induced by DA antagonism or A2A receptor stimulation, decreasing lever pressing but increasing chow intake in rats responding on a concurrent fixed ratio/chow feeding choice task. In contrast, injections into a neostriatal control site dorsal to the NAc were ineffective. The actions of pilocarpine on this task were attenuated by co-administration of the muscarinic antagonist scopolamine. Thus, drugs that act on DA, adenosine A2A, and muscarinic receptors regulate effort-related choice behavior, which may have implications for the treatment of psychiatric symptoms such as psychomotor slowing, fatigue or anergia that can be observed in depression and other disorders.

  9. Targeting the A2B adenosine receptor during gastrointestinal ischemia and inflammation

    PubMed Central

    Eltzschig, Holger K; Rivera-Nieves, Jesus; Colgan, Sean P

    2014-01-01

    Extracellular adenosine functions as an endogenous distress signal via activation of four distinct adenosine receptors (A1, A2A, A2B and A3). Conditions of limited oxygen availability or acute inflammation lead to elevated levels of extracellular adenosine and enhanced signaling events. This relates to a combination of four mechanisms: i) increased production of adenosine via extracellular phosphohydrolysis of precursor molecules (particularly ATP and ADP); ii) increased expression and signaling via hypoxia-induced adenosine receptors, particularly the A2B adenosine receptor; iii) attenuated uptake from the extracellular towards the intracellular compartment; and iv) attenuated intracellular metabolism. Due to their large surface area, mucosal organs are particularly prone to hypoxia and ischemia associated inflammation. Therefore, it is not surprising that adenosine production and signaling plays a central role in attenuating tissue inflammation and injury during intestinal ischemia or inflammation. In fact, recent studies combining pharmacological and genetic approaches demonstrated that adenosine signaling via the A2B adenosine receptor dampens mucosal inflammation and tissue injury during intestinal ischemia or experimental colitis. This review outlines basic principles of extracellular adenosine production, signaling, uptake and metabolism. In addition, we discuss the role of this pathway in dampening hypoxia-elicited inflammation, specifically in the setting of intestinal ischemia and inflammation. PMID:19769545

  10. 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

  11. 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

  12. The resurgence of A2B adenosine receptor signaling

    PubMed Central

    Aherne, Carol M.; Kewley, Emily M.; Eltzschig, Holger K.

    2010-01-01

    Since its discovery as a low-affinity adenosine receptor (AR), the A2B receptor (A2BAR), has proven enigmatic in its function. The previous discovery of the A2AAR, which shares many similarities with the A2BAR but demonstrates significantly greater affinity for its endogenous ligand, led to the original perception that the A2BAR was not of substantial physiologic relevance. In addition, lack of specific pharmacological agents targeting the A2BAR made its initial characterization challenging. However, the importance of this receptor was reconsidered when it was observed that the A2BAR is highly transcriptionally regulated by factors implicated in inflammatory hypoxia. Moreover, the notion that during ischemia or inflammation extracellular adenosine is dramatically elevated to levels sufficient for A2BAR activation, indicated that A2BAR signaling may be important to dampen inflammation particularly during tissue hypoxia. In addition, the recent advent of techniques for murine genetic manipulation along with development of pharmacological agents with enhanced A2BAR specificity has provided invaluable tools for focused studies on the explicit role of A2BAR signaling in different disease models. Currently, studies performed with combined genetic and pharmacological approaches have demonstrated that A2BAR signaling plays a tissue protective role in many models of acute diseases e.g. myocardial ischemia, or acute lung injury. These studies indicate that the A2BAR is expressed on a wide variety of cell types and exerts tissue/cell specific effects. This is an important consideration for future studies where tissue or cell type specific targeting of the A2BAR may be used as therapeutic approach. PMID:20546702

  13. 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

  14. Biophysical Mapping of the Adenosine A2A Receptor

    PubMed Central

    2011-01-01

    A new approach to generating information on ligand receptor interactions within the binding pocket of G protein-coupled receptors has been developed, called Biophysical Mapping (BPM). Starting from a stabilized receptor (StaR), minimally engineered for thermostability, additional single mutations are then added at positions that could be involved in small molecule interactions. The StaR and a panel of binding site mutants are captured onto Biacore chips to enable characterization of the binding of small molecule ligands using surface plasmon resonance (SPR) measurement. A matrix of binding data for a set of ligands versus each active site mutation is then generated, providing specific affinity and kinetic information (KD, kon, and koff) of receptor–ligand interactions. This data set, in combination with molecular modeling and docking, is used to map the small molecule binding site for each class of compounds. Taken together, the many constraints provided by these data identify key protein–ligand interactions and allow the shape of the site to be refined to produce a high quality three-dimensional picture of ligand binding, thereby facilitating structure based drug design. Results of biophysical mapping of the adenosine A2A receptor are presented. PMID:21661720

  15. Purine derivatives as ligands for A3 adenosine receptors.

    PubMed

    Joshi, Bhalchandra V; Jacobson, Kenneth A

    2005-01-01

    Selective agonists and antagonists for A3 adenosine receptors (ARs) are being explored for the treatment of a variety of disorders, including brain and heart ischemic conditions, cancer, and rheumatoid arthritis. This review covers both the structure activity relationships of nucleoside agonist ligands and selected antagonists acting at this receptor and the routes of synthesis. Highly selective agonists have been designed, using both empirical approaches and a semi-rational approach based on molecular modeling. The prototypical A3 agonists IB-MECA 10 and the more receptor-subtype-selective Cl-IB-MECA 11, both of which have affinity in binding to the receptor of approximately 1 nM, have been used widely as pharmacological probes in the elucidation of the physiological role of this receptor. In addition to the exploration of the effects of structural modification of the adenine and ribose moieties on A3AR affinity, the effects of these structural changes on the intrinsic efficacy have also been studied in a systematic fashion. Key structural features determining A3AR interaction include the N6-benzyl group, 2-position substitution such as halo, substitution of ribose (e.g., the (N)-methanocarba ring system, various 2'- and 3'-substitutions and 4'-thio substitution of oxygen). Conformational studies of the ribose moiety and its equivalents indicate that the ring oxygen is not required and the North (N) ring conformation is preferred in binding to the A3AR. Using these observations, a series of ring constrained (N)-methanocarba 5'-uronamide derivatives was recently reported to be highly selective A3AR agonists, the most notable amongst them was MRS3558 113 having a Ki value in binding to the human A3 receptor of 0.3 nM. PMID:16305531

  16. The cardiac effects of a novel A1-adenosine receptor agonist in guinea pig isolated heart.

    PubMed

    Belardinelli, L; Lu, J; Dennis, D; Martens, J; Shryock, J C

    1994-12-01

    Adenosine increases atrioventricular (AV) nodal conduction time and is used for termination of AV nodal re-entrant tachycardias, but it is rapidly metabolized. The purposes of the present study were to characterize the cardiac actions and effects of an orally active and stable adenosine analog, N6-cyclohexyl-2-O-methyladenosine (SDZ WAG-994) and to evaluate its potential as an antiarrhythmic agent. Guinea pig hearts were isolated and perfused with oxygenated Krebs-Henseleit solution. SDZ WAG-994 slowed the atrial rate and prolonged the AV nodal conduction time of spontaneously beating hearts in a concentration-dependent manner. The EC50 values for the negative chronotropic and dromotropic effects of SDZ WAG-994 were 0.69 +/- 0.04 and 1.49 +/- 0.54 microM, respectively. The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.2 microM) significantly antagonized SDZ WAG-994-induced stimulus-to-His bundle (S-H) interval prolongation. The negative dromotropic effect of SDZ WAG-994 showed very strong frequency dependence. In hearts paced at an atrial cycle length of 300 msec (200 beats/min), the EC50 value of SDZ WAG-994 to prolong the S-H interval was 3.7-fold lower (0.40 +/- 0.02 microM) than in unpaced hearts, and at atrial pacing cycle lengths of 500 and 250 msec, 0.3 microM SDZ WAG-994 prolonged the S-H interval by 8 and 26 msec, respectively. SDZ WAG-994 also decreased coronary perfusion pressure (EC50 = 1.50 +/- 0.80 microM); this effect of SDZ WAG-994 was attenuated by adenosine deaminase and by 8-cyclopentyltheophylline (2 microM). Radioligand binding assays revealed that SDZ WAG-994 had a 280-fold greater affinity for A1- than for A2a receptors of the guinea pig brain. The marked frequency dependence of the negative dromotropic effect of SDZ WAG-994 suggests that this A1 agonist may be highly effective in the termination of AV nodal re-entrant tachycardias. PMID:7996449

  17. Critical role of hypoxia and A2A adenosine receptors in liver tissue-protecting physiological anti-inflammatory pathway.

    PubMed

    Choukèr, Alexander; Thiel, Manfred; Lukashev, Dmitriy; Ward, Jerrold M; Kaufmann, Ines; Apasov, Sergey; Sitkovsky, Michail V; Ohta, Akio

    2008-01-01

    Whole body exposure of wild type control littermates and A2A adenosine receptor (A2AR) gene deleted mice to low oxygen containing inspired gas mixture allowed the investigation of the mechanism that controls inflammatory liver damage and protects the liver using a mouse model of T cell-mediated viral and autoimmune hepatitis. We tested the hypothesis that the inflammatory tissue damage-associated hypoxia and extracellular adenosine --> A2AR signaling plays an important role in the physiological anti-inflammatory mechanism that limits liver damage during fulminant hepatitis. After induction of T cell-mediated hepatitis, mice were kept in modular chambers either under normoxic (21% oxygen) or hypoxic (10% oxygen) conditions for 8 h. It was shown that the whole body exposure to hypoxic atmosphere caused tissue hypoxia in healthy animals as evidenced by a decrease in the arterial blood oxygen tension and increase of the plasma adenosine concentration (P < 0.05). This "hypoxic" treatment resulted in significantly reduced hepatocellular damage and attenuated levels of serum cytokines in mice with acute liver inflammation. The anti-inflammatory effects of hypoxia were not observed in the absence of A2AR in studies of A2AR gene-deficient mice or when A2AR have been pharmacologically antagonized with synthetic antagonist. The presented data demonstrate that total body hypoxia-triggered pathway provides protection in acute hepatitis and that hypoxia (upstream) and A2AR (downstream) function in the same immunosuppressive and liver tissue-protecting pathway.

  18. Regulation of muscarinic acetylcholine receptor-mediated synaptic responses by adenosine receptors in the rat hippocampus.

    PubMed Central

    Morton, R A; Davies, C H

    1997-01-01

    1. Intracellular current clamp recordings were made from CA1 pyramidal neurones in rat hippocampal slices. Experiments were performed in the presence of ionotropic glutamate receptor antagonists and gamma-aminobutyric acid (GABA) receptor antagonists to block all fast excitatory and inhibitory synaptic transmission. A single stimulus, delivered extracellularly in the stratum oriens, caused a reduction in spike frequency adaptation in response to a depolarizing current step delivered 2 s after the stimulus. A 2- to 10-fold increase in stimulus intensity evoked a slow excitatory postsynaptic potential (EPSP) which was associated with a small increase in input resistance. The peak amplitude of the EPSP occurred approximately 2.5 s after the stimulus and its magnitude (up to 30 mV) and duration (10-50 s) increased with increasing stimulus intensity. 2. The slow EPSP was unaffected by the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG; 1000 microM) but was greatly enhanced by the acetylcholinesterase inhibitor physostigmine (1-5 microM). Both the slow EPSP and the stimulus-evoked reduction in spike frequency adaptation were inhibited by the muscarinic acetylcholine receptor (mAChR) antagonist atropine (1-5 microM). These results are consistent with these effects being mediated by mAChRs. 3. Both the mAChR-mediated EPSP (EPSPm) and the associated reduction in spike frequency adaptation were reversibly depressed (up to 97%) by either adenosine (100 microM) or its non-hydrolysable analogue 2-chloroadenosine (CADO; 0.1-5.0 microM). These effects were often accompanied by postsynaptic hyperpolarization (up to 8 mV) and a reduction in input resistance (up to 11%). The selective adenosine A1 receptor agonists 2-chloro-N6-cyclopentyladenosine (CCPA; 0.1-0.4 microM) and R(-)N6-(2-phenylisopropyl)-adenosine (R-PIA; 1 microM) both depressed the EPSPm. In contrast, the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5

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

    PubMed Central

    Welch, William 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

  20. 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.

  1. 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

  2. The role of adenosine receptors in regulating production of tumour necrosis factor-α and chemokines by human lung macrophages

    PubMed Central

    Buenestado, A; Delyle, S Grassin; Arnould, I; Besnard, F; Naline, E; Blouquit-Laye, S; Chapelier, A; Bellamy, JF; Devillier, P

    2010-01-01

    Background and purpose: Adenosine is a major endogenous regulator of macrophage function, and activates four specific adenosine receptors (A1, A2A, A2B and A3). Here, we have assessed in human lung macrophages the modulation of the expression of adenosine receptor mRNA by lipopolysaccharide (LPS), and the relative contributions of the different adenosine receptors to LPS-induced production of tumour necrosis factor (TNF)-α and chemokines. Experimental approach: Lung macrophages isolated from resected lungs were stimulated with LPS and treated with adenosine receptor agonists or/and antagonists. Adenosine receptor expression was assessed with qRT-PCR. Cytokines were measured in lung macrophage supernatants with elisa. Key results: LPS increased (about 400-fold) mRNA for A2A adenosine receptors, decreased mRNA for A1 and A2B, but had no effect on A3 adenosine receptor mRNA. The adenosine receptor agonist NECA inhibited TNF-α production concentration dependently, whereas the A1 receptor agonist, CCPA, and the A3 receptor agonist, AB-MECA, inhibited TNF-α production only at concentrations affecting A2A receptors. NECA also inhibited the production of CCL chemokines (CCL2, CCL3, CCL4, CCL5) and CXCL chemokines (CXCL9 and CXCL10), but not that of CXCL1, CXCL8 and CXCL5. Reversal of NECA-induced inhibition of TNF-α and chemokine production by the selective A2A adenosine receptor antagonist ZM 241385, but not the A2B receptor antagonist, MRS 1754, or the A3 receptor antagonist, MRS 1220, indicated involvement of A2A receptors. Conclusions and implications: LPS up-regulated A2A adenosine receptor gene transcription, and this receptor subtype mediated inhibition of the LPS-induced production of TNF-α and of a subset of chemokines in human lung macrophages. PMID:20136829

  3. 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...

  4. 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

  5. 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.

  6. 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

  7. Kinetic analysis of antagonist-occupied adenosine-A3 receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism

    PubMed Central

    Corriden, Ross; Kilpatrick, Laura E.; Kellam, Barrie; Briddon, Stephen J.; Hill, Stephen J.

    2014-01-01

    In our previous work, using a fluorescent adenosine-A3 receptor (A3AR) agonist and fluorescence correlation spectroscopy (FCS), we demonstrated high-affinity labeling of the active receptor (R*) conformation. In the current study, we used a fluorescent A3AR antagonist (CA200645) to study the binding characteristics of antagonist-occupied inactive receptor (R) conformations in membrane microdomains of individual cells. FCS analysis of CA200645-occupied A3ARs revealed 2 species, τD2 and τD3, that diffused at 2.29 ± 0.35 and 0.09 ± 0.03 μm2/s, respectively. FCS analysis of a green fluorescent protein (GFP)-tagged A3AR exhibited a single diffusing species (0.105 μm2/s). The binding of CA200645 to τD3 was antagonized by nanomolar concentrations of the A3 antagonist MRS 1220, but not by the agonist NECA (up to 300 nM), consistent with labeling of R. CA200645 normally dissociated slowly from the A3AR, but inclusion of xanthine amine congener (XAC) or VUF 5455 during washout markedly accelerated the reduction in the number of particles exhibiting τD3 characteristics. It is notable that this effect was accompanied by a significant increase in the number of particles with τD2 diffusion. These data show that FCS analysis of ligand-occupied receptors provides a unique means of monitoring ligand A3AR residence times that are significantly reduced as a consequence of allosteric interaction across the dimer interface.—Corriden, R., Kilpatrick, L. E., Kellam, B., Briddon, S. J., Hill, S. J. Kinetic analysis of antagonist-occupied adenosine-A3 receptors within membrane microdomains of individual cells provides evidence for receptor dimerization and allosterism. PMID:24970394

  8. Lack of tolerance to motor stimulant effects of a selective adenosine A(2A) receptor antagonist.

    PubMed

    Halldner, L; Lozza, G; Lindström, K; Fredholm, B B

    2000-10-20

    It is well known that tolerance develops to the actions of caffeine, which acts as an antagonist on adenosine A(1) and A(2A) receptors. Since selective adenosine A(2A) antagonists have been proposed as adjuncts to 3,4-dihydroxyphenylalanine (L-DOPA) therapy in Parkinson's disease we wanted to examine if tolerance also develops to the selective A(2A) receptor antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2, 4-triazolo [1,5-c]pyrimidine (SCH 58261). SCH 58261 (0.1 and 7.5 mg/kg) increased basal locomotion and the motor stimulation afforded by apomorphine. Neither effect was subject to tolerance following long-term treatment with the same doses given intraperitoneally twice daily. There were no adaptive changes in A(1) and A(2A) adenosine receptors or their corresponding messenger RNA or in dopamine D(1) or D(2) receptors. These results demonstrate that the tolerance that develops to caffeine is not secondary to its inhibition of adenosine A(2A) receptors. The results also offer hope that long-term treatment with an adenosine A(2A) receptor antagonist may be possible in man.

  9. 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.

  10. 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

  11. 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

  12. Adenosine enhances sweet taste through A2B receptors in the taste bud

    PubMed Central

    Dando, Robin; Dvoryanchikov, Gennady; Pereira, Elizabeth; Chaudhari, Nirupa; Roper, Stephen D.

    2012-01-01

    Mammalian taste buds use ATP as a neurotransmitter. Taste Receptor (Type II) cells secrete ATP via gap junction hemichannels into the narrow extracellular spaces within a taste bud. This ATP excites primary sensory afferent fibers and also stimulates neighboring taste bud cells. Here we show that extracellular ATP is enzymatically degraded to adenosine within mouse vallate taste buds and that this nucleoside acts as an autocrine neuromodulator to selectively enhance sweet taste. In Receptor cells in a lingual slice preparation, Ca2+ mobilization evoked by focally applied artificial sweeteners was significantly enhanced by adenosine (50 µM). Adenosine had no effect on bitter or umami taste responses, and the nucleoside did not affect Presynaptic (Type III) taste cells. We also used biosensor cells to measure transmitter release from isolated taste buds. Adenosine (5 µM) enhanced ATP release evoked by sweet but not bitter taste stimuli. Using single-cell RT-PCR on isolated vallate taste cells, we show that many Receptor cells express adenosine receptors, Adora2b, while Presynaptic (Type III) and Glial-like (Type I) cells seldom do. Furthermore, Adora2b receptors are significantly associated with expression of the sweet taste receptor subunit, Tas1r2. Adenosine is generated during taste stimulation mainly by the action of the ecto-5′-nucleotidase, NT5E, and to a lesser extent, prostatic acid phosphatase (ACPP). Both these ecto-nucleotidases are expressed by Presynaptic cells, as shown by single-cell RT-PCR, enzyme histochemistry and immunofluorescence. Our findings suggest that ATP released during taste reception is degraded to adenosine to exert positive modulation particularly on sweet taste. PMID:22219293

  13. Adenosine enhances sweet taste through A2B receptors in the taste bud.

    PubMed

    Dando, Robin; Dvoryanchikov, Gennady; Pereira, Elizabeth; Chaudhari, Nirupa; Roper, Stephen D

    2012-01-01

    Mammalian taste buds use ATP as a neurotransmitter. Taste Receptor (type II) cells secrete ATP via gap junction hemichannels into the narrow extracellular spaces within a taste bud. This ATP excites primary sensory afferent fibers and also stimulates neighboring taste bud cells. Here we show that extracellular ATP is enzymatically degraded to adenosine within mouse vallate taste buds and that this nucleoside acts as an autocrine neuromodulator to selectively enhance sweet taste. In Receptor cells in a lingual slice preparation, Ca(2+) mobilization evoked by focally applied artificial sweeteners was significantly enhanced by adenosine (50 μM). Adenosine had no effect on bitter or umami taste responses, and the nucleoside did not affect Presynaptic (type III) taste cells. We also used biosensor cells to measure transmitter release from isolated taste buds. Adenosine (5 μM) enhanced ATP release evoked by sweet but not bitter taste stimuli. Using single-cell reverse transcriptase (RT)-PCR on isolated vallate taste cells, we show that many Receptor cells express the adenosine receptor, Adora2b, while Presynaptic (type III) and Glial-like (type I) cells seldom do. Furthermore, Adora2b receptors are significantly associated with expression of the sweet taste receptor subunit, Tas1r2. Adenosine is generated during taste stimulation mainly by the action of the ecto-5'-nucleotidase, NT5E, and to a lesser extent, prostatic acid phosphatase. Both these ecto-nucleotidases are expressed by Presynaptic cells, as shown by single-cell RT-PCR, enzyme histochemistry, and immunofluorescence. Our findings suggest that ATP released during taste reception is degraded to adenosine to exert positive modulation particularly on sweet taste.

  14. 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.

  15. Computational study of the molecular mechanisms of caffeine action: Caffeine complexes with adenosine receptors

    NASA Astrophysics Data System (ADS)

    Poltev, V. I.; Rodríguez, E.; Grokhlina, T. I.; Deriabina, A.; Gonzalez, E.

    To understand the molecular basis of the principal biological action of the caffeine (CAF), the molecular mechanics calculations of possible complexes between CAF and the fragments of human A1 adenosine receptor were performed. The fragments were selected after considerations of the CAF molecular structure and its possible interactions, as well as after an analysis of the extensive bibliography on the structure, biological role, site-directed mutagenesis, and the modeling of the adenosine receptors. The minimum energy configurations of these complexes were obtained using two different computer programs with different force fields. The most favorable configurations correspond to the formation of two hydrogen bonds between the CAF molecule and hydrophilic amino acid residues of the fragments of transmembrane domains of the receptor. These configurations are supposed to contribute to CAF blocking of the adenosine receptors. They will be used later for the construction of model CAF complexes with two transmembrane domains simultaneously.

  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. The impact of adenosine and A(2B) receptors on glucose homoeostasis.

    PubMed

    Rüsing, D; Müller, C E; Verspohl, E J

    2006-12-01

    Adenosine and adenosine receptor antagonists are involved in glucose homoeostasis. The participating receptors are not known, mainly due to a lack of specific agonists and antagonists, but are reasonable targets for anti-diabetic therapy. The stable, albeit nonselective, adenosine analogue NECA (5'-N-ethylcarboxamidoadenosine) (10 microM) reduced glucose-stimulated insulin release from INS-1 cells. This was mimicked by A(1)-(CHA), A(2A)-(CGS-21680) and A(3)-receptor agonists (Cl-IB-MECA). Two newly synthesized A(2B)-receptor antagonists, PSB-53 and PSB-1115, counteracted the inhibitory effect of NECA. These in-vitro effects were mirrored by in-vivo data with respect to CHA, CGS and Cl-IB-MECA. Distinct concentrations of either PSB-53 or PSB-1115 reversed the decrease in plasma insulin induced by NECA. This was not mimicked by a corresponding change in blood glucose. The effect of PSB-1115 was also obvious in diabetic GotoKakizaki rats: plasma insulin was increased whereas blood glucose was unchanged. During most experiments the effects on blood glucose were not impressive probably because of the physiologically necessary homoeostasis. The adenosine levels were not different in normal Wistar rats and in diabetic GotoKakzaki rats. Altogether the A(2B)-receptor antagonists showed an anti-diabetic potential mainly by increasing plasma insulin levels under conditions when the adenosine tonus was elevated in-vivo and increased insulin release in-vitro.

  18. Sulfur-Containing 1,3-Dialkylxanthine Derivatives as Selective Antagonists at A1-Adenosine Receptors

    PubMed Central

    Kiriasis, Leonidas; Barone, Suzanne; Bradbury, Barton J.; Kammula, Udai; Campagne, Jean Michel; Secunda, Sherrie; Daly, John W.; Neumeyer, John L.; Pfleiderer, Wolfgang

    2012-01-01

    Sulfur-containing analogues of 8-substituted xanthines were prepared in an effort to increase selectivity or potency as antagonists at adenosine receptors. Either cyclopentyl or various aryl substituents were utilized at the 8-position, because of the association of these groups with high potency at A1-adenosine receptors. Sulfur was incorporated on the purine ring at positions 2 and/or 6, in the 8-position substituent in the form of 2- or 3-thienyl groups, or via thienyl groups separated from an 8-aryl substituent through an amide-containing chain. The feasibility of using the thienyl group as a prosthetic group for selective iodination via its Hg2+ derivative was explored. Receptor selectivity was determined in binding assays using membrane homogenates from rat cortex [[3H]-N6-(phenylisopropyl) adenosine as radioligand] or striatum [[3H]-5′-(N-ethylcarbamoyl)adenosine as radioligand] for A1- and A2-adenosine receptors, respectively. Generally, 2-thio-8-cycloalkylxanthines were at least as A1 selective as the corresponding oxygen analogue. 2-Thio-8-aryl derivatives tended to be more potent at A2 receptors than the oxygen analogue. 8-[4-[(Carboxymethyl)oxy]phenyl]-1,3-dipropyl-2-thioxanthine ethyl ester was >740-fold A1 selective. PMID:2754711

  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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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

  6. Caffeine promotes anti-tumor immune response during tumor initiation: Involvement of the adenosine A2A receptor.

    PubMed

    Eini, Hadar; Frishman, Valeria; Yulzari, Robert; Kachko, Leonid; Lewis, Eli C; Chaimovitz, Cidio; Douvdevani, Amos

    2015-11-01

    Epidemiologic studies depict a negative correlation between caffeine consumption and incidence of tumors in humans. The main pharmacological effects of caffeine are mediated by antagonism of the adenosine receptor, A2AR. Here, we examine whether the targeting of A2AR by caffeine plays a role in anti-tumor immunity. In particular, the effects of caffeine are studied in wild-type and A2AR knockout (A2AR(-/-)) mice. Tumor induction was achieved using the carcinogen 3-methylcholanthrene (3-MCA). Alternatively, tumor cells, comprised of 3-MCA-induced transformed cells or B16 melanoma cells, were inoculated into animal footpads. Cytokine release was determined in a mixed lymphocyte tumor reaction (MLTR). According to our findings, caffeine-consuming mice (0.1% in water) developed tumors at a lower rate compared to water-consuming mice (14% vs. 53%, respectively, p=0.0286, n=15/group). Within the caffeine-consuming mice, tumor-free mice displayed signs of autoimmune alopecia and pronounced leukocyte recruitment intocarcinogen injection sites. Similarly, A2AR(-/-) mice exhibited reduced rates of 3-MCA-induced tumors. In tumor inoculation studies, caffeine treatment resulted in inhibition of tumor growth and elevation in proinflammatory cytokine release over water-consuming mice, as depicted by MLTR. Addition of the adenosine receptor agonist, NECA, to MLTR resulted in a sharp decrease in IFNγ levels; this was reversed by the highly selective A2AR antagonist, ZM241385. Thus, immune response modulation through either caffeine or genetic deletion of A2AR leads to a Th1 immune profile and suppression of carcinogen-induced tumorigenesis. Taken together, our data suggest that the use of pharmacologic A2AR antagonists may hold therapeutic potential in diminishing the rate of cancer development.

  7. Chaperoning of the A1-Adenosine Receptor by Endogenous Adenosine—An Extension of the Retaliatory Metabolite Concept*

    PubMed Central

    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-Y288 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-Y288 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

  8. Brain stem adenosine receptors modulate centrally mediated hypotensive responses in conscious rats: A review.

    PubMed

    Nassar, Noha N; Abdel-Rahman, Abdel A

    2015-05-01

    Adenosine is implicated in the modulation of cardiovascular responses either at the peripheral or at central level in experimental animals. However, there are no dedicated reviews on the involvement of adenosine in mediating the hypotensive response of centrally administered clonidine in general and specifically in aortically barodenervated rats (ABD). The conscious ABD rat model exhibits surgically induced baroreflex dysfunction and exaggerated hypotensive response, compared with conscious sham-operated (SO) rats. The current review focuses on, the role of adenosine receptors in blood pressure (BP) regulation and their possible crosstalk with other receptors e.g. imidazoline (I1) and alpha (α2A) adrenergic receptor (AR). The former receptor is a molecular target for clonidine, whose hypotensive effect is enhanced approx. 3-fold in conscious ABD rats. We also discussed how the balance between the brain stem adenosine A1 and A2A receptors is regulated by baroreceptors and how such balance influences the centrally mediated hypotensive responses. The use of the ABD rat model yielded insight into the downstream signaling cascades following clonidine-evoked hypotension in a surgical model of baroreflex dysfunction. PMID:26257930

  9. Adenosine A2(A) receptor modulates the oxidative stress response of primed polymorphonuclear leukocytes after parabolic flight.

    PubMed

    Kaufmann, Ines; Feuerecker, Matthias; Salam, Alex; Schelling, Gustav; Thiel, Manfred; Choukèr, Alexander

    2011-07-01

    Space flight and gravitational stress can alter innate immune function. Parabolic flights (PFs) as a model for short-term gravitational changes prime the cytotoxic capability of polymorphonuclear leukocytes (PMNs). In view of the emerging role of adenosine in the regulation of innate immune responses, we examined the potency of adenosine to control the release of cytotoxic H(2)O(2) by primed PMNs via the adenosine receptor system. During PFs, microgravity conditions (<10(-2) G) are generated for approximately 22 seconds, followed by a hypergravity (1.8 G) phase resulting in gravitational stress. We studied the ex vivo effects of adenosine on the production of H(2)O(2) by stimulated PMNs and determined adenosine plasma levels and adenosine A2(A) receptor transcripts of leukocytes of PF participants (n = 15). Increasing concentrations of adenosine dose dependently reduced tissue-toxic H(2)O(2) production by PMNs with a half-maximal inhibitory concentration (IC(50)) of 19.5 nM before takeoff and 7.6 nM at 48 hours after PF. This increase in the adenosine-mediated inhibition of PMNs' H(2)O(2) production was completely reversed by addition of the A2(A) receptor antagonist ZM241385. PF induced a nonsignificant elevation in adenosine plasma levels; A2(A) receptor mRNA from leukocytes remained almost unchanged. Adenosine limits the oxidative stress response of PMNs after PFs through an upregulation of the adenosine A2(A) receptor function. This stop signal on inflammation is stronger than that under normal physiologic states and may limit further cytotoxic damage. Pharmacologic manipulation of the adenosine A2(A) receptor pathway could be a potential target for control of unwanted exacerbations of cytotoxic PMN functions.

  10. In vivo adenosine A(2B) receptor desensitization in guinea-pig airway smooth muscle: implications for asthma.

    PubMed

    Breschi, Maria Cristina; Blandizzi, Corrado; Fogli, Stefano; Martinelli, Cinzia; Adinolfi, Barbara; Calderone, Vincenzo; Camici, Marcella; Martinotti, Enrica; Nieri, Paola

    2007-12-01

    This study was aimed at characterizing the role of adenosine receptor subtypes in the contractility modulation of guinea-pig airway smooth muscle in normal and pathological settings. In vitro and in vivo experiments were performed by testing selective agonists and antagonists on isolated tracheal smooth muscle preparations and pulmonary inflation pressure, respectively, under normal conditions or following ovalbumin-induced allergic sensitization. In normal and sensitized animals, the adenosine A(2A)/A(2B) receptor agonist, NECA, evoked relaxing responses of isolated tracheal preparations precontracted with histamine, and such an effect was reversed by the adenosine A(2B) antagonist, MRS 1706, in the presence or in the absence of epithelium. The expression of mRNA coding for adenosine A(2B) receptors was demonstrated in tracheal specimens. In vitro desensitization with 100 microM NECA markedly reduced the relaxing effect of the agonist. In vivo NECA or adenosine administration to normal animals inhibited histamine-mediated bronchoconstriction, while these inhibitory effects no longer occurred in sensitized guinea-pigs. Adenosine plasma levels were significantly higher in sensitized than normal animals. In conclusion, our data demonstrate that: (i) adenosine A(2B) receptors are responsible for the relaxing effects of adenosine on guinea-pig airways; (ii) these receptors can undergo rapid adaptive changes that may affect airway smooth muscle responsiveness to adenosine; (iii) ovalbumin-induced sensitization promotes a reversible inactivation of adenosine A(2B) receptors which can be ascribed to homologous desensitization. These findings can be relevant to better understand adenosine functions in airways as well as mechanisms of action of asthma therapies targeting the adenosine system.

  11. 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

  12. 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.

  13. Molecular Determinants of CGS21680 Binding to the Human Adenosine A2A Receptor

    PubMed Central

    Edwards, Patricia C.; Leslie, Andrew G. W.

    2015-01-01

    The adenosine A2A receptor (A2AR) plays a key role in transmembrane signaling mediated by the endogenous agonist adenosine. Here, we describe the crystal structure of human A2AR thermostabilized in an active-like conformation bound to the selective agonist 2-[p-(2-carboxyethyl)phenylethyl-amino]-5′-N-ethylcarboxamido adenosine (CGS21680) at a resolution of 2.6 Å. Comparison of A2AR structures bound to either CGS21680, 5′-N-ethylcarboxamido adenosine (NECA), UK432097 [6-(2,2-diphenylethylamino)-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-tetrahydrofuran-2-yl]-N-[2-[[1-(2-pyridyl)-4-piperidyl]carbamoylamino]ethyl]purine-2-carboxamide], or adenosine shows that the adenosine moiety of the ligands binds to the receptor in an identical fashion. However, an extension in CGS21680 compared with adenosine, the (2-carboxyethyl)phenylethylamino group, binds in an extended vestibule formed from transmembrane regions 2 and 7 (TM2 and TM7) and extracellular loops 2 and 3 (EL2 and EL3). The (2-carboxyethyl)phenylethylamino group makes van der Waals contacts with side chains of amino acid residues Glu169EL2, His264EL3, Leu2677.32, and Ile2747.39, and the amine group forms a hydrogen bond with the side chain of Ser672.65. Of these residues, only Ile2747.39 is absolutely conserved across the human adenosine receptor subfamily. The major difference between the structures of A2AR bound to either adenosine or CGS21680 is that the binding pocket narrows at the extracellular surface when CGS21680 is bound, due to an inward tilt of TM2 in that region. This conformation is stabilized by hydrogen bonds formed by the side chain of Ser672.65 to CGS21680, either directly or via an ordered water molecule. Mutation of amino acid residues Ser672.65, Glu169EL2, and His264EL3, and analysis of receptor activation either in the presence or absence of ligands implicates this region in modulating the level of basal activity of A2AR. PMID:25762024

  14. 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.

  15. Effect of adenosine and adenosine receptor antagonist on Müller cell potassium channel in Rat chronic ocular hypertension models.

    PubMed

    Yang, Zijian; Huang, Ping; Liu, Xiaohong; Huang, Shouyue; Deng, Lianfu; Jin, Zhe; Xu, Shuo; Shen, Xi; Luo, Xunda; Zhong, Yisheng

    2015-01-01

    Müller cells are principal glial cells in rat retina and have attracted much attention in glaucoma studies. However, it is not clear whether adenosine and adenosine receptor (AR) antagonists play any roles in the regulation of potassium channels in Müller cells and subsequently in the promotion of glutamine synthetase (GS) and L-Glutamate/L-Aspartate Transporter (GLAST) functions. We found that chronic ocular hypertension (COH) in rat down-regulated Müller cells Kir2.1, Kir4.1, TASK-1, GS and GLAST expressions and attenuated the peak of inward potassium current. Retinal ganglion cells (RGC) count was lower in the COH rats than that in the sham operation animals. Intravitreal injection of selective A2A AR antagonist SCH442416 up-regulated Müller cell Kir4.1, TASK-1, GS and GLAST expressions and enhanced inward potassium currents compared with those in the COH rats with vehicle control. Meanwhile, the RGC count was higher following intravitreal injection of SCH442416 in the COH rats than that after vehicle injection. The fact that PKA inhibitor H-89 blocked these SCH442416 effects suggested that the PKA signaling pathway was involved in the observed ocular responses following the intravitreal SCH442416 injection. PMID:26063641

  16. Effects of endothelin ETA receptor antagonism on granulocyte and lymphocyte accumulation in LPS-induced inflammation.

    PubMed

    Sampaio, André L F; Rae, Giles A; Henriques, Maria das Graças M O

    2004-07-01

    Endothelin peptides play active roles in different aspects of inflammation. This study investigates the contribution of endogenous endothelins to lipopolysaccharide (LPS) pulmonary inflammation by assessing the influence of ET(A) receptor antagonism on leukocyte accumulation, granulocyte adhesion molecule expression, and chemokine/cytokine modulation. Local pretreatment with BQ-123 or A-127722 (150 pmol), two selective and chemically unrelated endothelin ET(A) receptor antagonists, inhibits neutrophil and eosinophil accumulation in LPS-induced pleurisy at 24 h but not neutrophil migration at 4 h. The effect of endothelin antagonism on neutrophil accumulation at 24 h was concomitant with inhibition of eosinophil and CD4 and CD8 T lymphocyte influx. It is surprising that the ET(A) receptor blockade did not inhibit the accumulation of gammadelta T lymphocytes, cells that are important for granulocyte recruitment in this model. Blockade of ET(A) receptors did not influence the expression of adhesion molecules (CD11b, CD49d) on granulocytes but abrogated the increase in tumor necrosis factor alpha levels 4 h after LPS stimulation and also markedly inhibited increases in levels of interleukin-6 and keratinocyte-derived chemokine/CXC chemokine ligand 1 but not eotaxin/chemokine ligand 11. Thus, acting via ET(A) receptors, endogenous endothelins play an important role in early cytokine/chemokine production and on granulocyte and lymphocyte mobilization in LPS-induced pleurisy.

  17. 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

  18. The Effect of Mineralocorticoid and Glucocorticoid Receptor Antagonism on Autobiographical Memory Recall and Amygdala Response to Implicit Emotional Stimuli

    PubMed Central

    Preskorn, Sheldon H.; Victor, Teresa; Misaki, Masaya; Bodurka, Jerzy; Drevets, Wayne C.

    2016-01-01

    Background: Acutely elevated cortisol levels in healthy humans impair autobiographical memory recall and alter hemodynamic responses of the amygdala to emotionally valenced stimuli. It is hypothesized that the effects of the cortisol on cognition are influenced by the ratio of mineralocorticoid receptor to glucocorticoid receptor occupation. The current study examined the effects of acutely blocking mineralocorticoid receptors and glucocorticoid receptors separately on 2 processes known to be affected by altering levels of cortisol: the specificity of autobiographical memory recall, and the amygdala hemodynamic response to sad and happy faces. Methods: We employed a within-subjects design in which 10 healthy male participants received placebo, the mineralocorticoid receptor antagonist spironolactone (600mg) alone, and the glucocorticoid receptor antagonist mifepristone (600mg) alone in a randomized, counter-balanced order separated by 1-week drug-free periods. Results: On autobiographical memory testing, mineralocorticoid receptor antagonism impaired, while glucocorticoid receptor antagonism improved, recall relative to placebo, as evinced by changes in the percent of specific memories recalled. During fMRI, the amygdala hemodynamic response to masked sad faces was greater under both mineralocorticoid receptor and glucocorticoid receptor antagonism relative to placebo, while the response to masked happy faces was attenuated only during mineralocorticoid receptor antagonism relative to placebo. Conclusions: These data suggest both mineralocorticoid receptor and glucocorticoid receptor antagonism (and potentially any deviation from the normal physiological mineralocorticoid receptor/glucocorticoid receptor ratio achieved under the circadian pattern) enhances amygdala-based processing of sad stimuli and may shift the emotional processing bias away from the normative processing bias and towards the negative valence. In contrast, autobiographical memory was enhanced by

  19. Pyrazolo Derivatives as Potent Adenosine Receptor Antagonists: An Overview on the Structure-Activity Relationships

    PubMed Central

    Cheong, Siew Lee; Venkatesan, Gopalakrishnan; Paira, Priyankar; Jothibasu, Ramasamy; Mandel, Alexander Laurence; Federico, Stephanie; Spalluto, Giampiero; Pastorin, Giorgia

    2011-01-01

    In the past few decades, medicinal chemistry research towards potent and selective antagonists of human adenosine receptors (namely, A1, A2A, A2B, and A3) has been evolving rapidly. These antagonists are deemed therapeutically beneficial in several pathological conditions including neurological and renal disorders, cancer, inflammation, and glaucoma. Up to this point, many classes of compounds have been successfully synthesized and identified as potent human adenosine receptor antagonists. In this paper, an overview of the structure-activity relationship (SAR) profiles of promising nonxanthine pyrazolo derivatives is reported and discussed. We have emphasized the SAR for some representative structures such as pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidines; pyrazolo-[3,4-c] or -[4,3-c]quinolines; pyrazolo-[4,3-d]pyrimidinones; pyrazolo-[3,4-d]pyrimidines and pyrazolo-[1,5-a]pyridines. This overview not only clarifies the structural requirements deemed essential for affinity towards individual adenosine receptor subtypes, but it also sheds light on the rational design and optimization of existing structural templates to allow us to conceive new, more potent adenosine receptor antagonists. PMID:25954519

  20. Antagonism effects of cypermethrin on interleukin-6-induced androgen receptor activation.

    PubMed

    Wang, Qi; Xu, Li-Fang; Zhou, Ji-Long; Zhou, Xiao-Long; Wang, Hui; Ju, Qiang; Pan, Chen; Zhang, Jin-Peng; Zhang, Mei-Rong; Yu, Hong-Min; Xu, Li-Chun

    2015-07-01

    To identify whether androgen receptor (AR) antagonism by cypermethrin involves interleukin-6 (IL-6)-induced ligand-independent AR signaling, we have developed the AR reporter gene assay. The reporter gene plasmid pMMTV-chloramphenicol transferase (CAT) was transfected into LNCaP cells. IL-6 increased expression of MMTV-CAT significantly (P<0.05). Cypermethrin decreased CAT reporter expression induced by IL-6 (50 ng/ml), and the significant inhibition was detected at 10(-5)M (P<0.05). IL-6 induces ligand-independent activation of AR. Cypermethrin exhibits inhibitory effects on IL-6-induced ligand-independent AR signaling. We provide a novel insight into cypermethrin-mediated antagonism of the IL-6-mediated ligand-independent activation of the AR.

  1. Binding of adenosine and receptor-specific analogues to lymphocytes from control subjects and patients with common variable immunodeficiency.

    PubMed Central

    Shah, T; Simpson, R J; Webster, A D; Peters, T J

    1987-01-01

    Studies were performed on the binding of tritiated adenosine and its analogues, 5'-N-ethylcarboxamide adenosine (NECA) and N6-phenylisopropyladenosine (PIA), to human peripheral blood lymphocytes. These revealed binding only of adenosine (Kd, 1-10 microM, 14,000 binding sites/cell), which was abolished by dipyridamole, a specific adenosine transport inhibitor, suggesting that the binding is to the nucleoside transporter. The absence of high affinity (Kd less than or equal to 1 microM) binding of adenosine or of the two analogues. NECA and PIA suggests that the previously reported effects of adenosine on cAMP formation are not mediated by cell surface specific nucleoside receptors. Binding of adenosine to the carrier in lymphocytes from patients with common variable immunodeficiency was similar to those from control subjects. PMID:2958197

  2. Differential expression of adenosine A3 receptors controls adenosine A2A receptor-mediated inhibition of TLR responses in microglia.

    PubMed

    van der Putten, Céline; Zuiderwijk-Sick, Ella A; van Straalen, Linda; de Geus, Eveline D; Boven, Leonie A; Kondova, Ivanela; IJzerman, Ad P; Bajramovic, Jeffrey J

    2009-06-15

    Microglia activation is a prominent feature in many neuroinflammatory disorders. Unrestrained activation can generate a chronic inflammatory environment that might lead to neurodegeneration and autoimmunity. Extracellular adenosine modulates cellular activation through adenosine receptor (ADORA)-mediated signaling. There are four ADORA subtypes that can either increase (A(2A) and A(2B) receptors) or decrease (A(1) and A(3) receptors) intracellular cyclic AMP levels. The expression pattern of the subtypes thus orchestrates the cellular response to extracellular adenosine. We have investigated the expression of ADORA subtypes in unstimulated and TLR-activated primary rhesus monkey microglia. Activation induced an up-regulation of A(2A) and a down-regulation of A(3) receptor (A(3)R) levels. The altered ADORA-expression pattern sensitized microglia to A(2A) receptor (A(2A)R)-mediated inhibition of subsequent TLR-induced cytokine responses. By using combinations of subtype-specific agonists and antagonists, we revealed that in unstimulated microglia, A(2A)R-mediated inhibitory signaling was effectively counteracted by A(3)R-mediated signaling. In activated microglia, the decrease in A(3)R-mediated signaling sensitized them to A(2A)R-mediated inhibitory signaling. We report a differential, activation state-specific expression of ADORA in microglia and uncover a role for A(3)R as dynamically regulated suppressors of A(2A)R-mediated inhibition of TLR-induced responses. This would suggest exploration of combinations of A(2A)R agonists and A(3)R antagonists to dampen microglial activation during chronic neuroinflammatory conditions.

  3. 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.

  4. Lipopolysaccharide rapidly modifies adenosine receptor transcripts in murine and human macrophages: role of NF-kappaB in A(2A) adenosine receptor induction.

    PubMed

    Murphree, Lauren J; Sullivan, Gail W; Marshall, Melissa A; Linden, Joel

    2005-11-01

    The A(2A) adenosine receptor (A(2A)AR) mediates anti-inflammatory actions of adenosine in a variety of cell types. LPS (lipopolysaccharide) was reported to induce a small (<2-fold) increase in the expression of A(2A)AR mRNA in human monocytes and monocytic cell lines. We investigated the effects of LPS on the expression of adenosine receptor mRNAs in primary mouse IPMPhi (intraperitoneal macrophages), human macrophages and Wehi-3 cells. Treatment with 10 ng/ml LPS for 4 h produced a >100-fold increase in A(2A)AR mRNA. LPS-induced increases in mRNA for A(2A)AR and TNFalpha (tumour necrosis factor alpha) are reduced by 90% in IPMPhi pretreated with the NF-kappaB (nuclear factor kappaB) inhibitor, BAY 11-7082 {(E)3-[(4-methylphenyl)sulphonyl]-2-propenenitrile; 10 microM}. In Wehi-3 cells exposed to LPS, A(2A)AR and A(2B)AR transcripts are elevated by 290- and 10-fold respectively, the A(1)AR transcript is unchanged and the A(3)AR transcript is decreased by 67%. The induction of A(2A)AR mRNA by LPS is detectable after 1 h, reaches a peak at 6 h at 600 times control and remains elevated beyond 24 h. The ED50 (effective dose) of LPS is 2.3 ng/ml. A(2A)AR receptor number, measured by 125I-ZM241385 binding to whole cells, is undetectable in naïve cells and increases linearly at a rate of 23 receptors x cell(-1) x min(-1) to a B(max) of 348 fmol/mg (28000 receptors/cell) in 20 h. The increase in receptor number is correlated with an increase in the potency of an A(2A) agonist (4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester; referred to as ATL146e) to stimulate cAMP in these cells. After LPS pretreatment, the potency of the A(2A) agonist, ATL146e, to reduce TNFalpha release from IPMPhi was increased by 200-fold. The results support the hypothesis that regulation of adenosine receptor expression, especially up-regulation of the A(2A)AR, is part of a delayed feedback mechanism

  5. Functional studies but not receptor binding can distinguish surmountable from insurmountable AT1 antagonism.

    PubMed

    Panek, R L; Lu, G H; Overhiser, R W; Major, T C; Hodges, J C; Taylor, D G

    1995-05-01

    Our study demonstrated that inhibition of angiotensin II- (Ang II) mediated contractions of rabbit aorta by structurally diverse nonpeptide AT1 antagonists could distinguish surmountable from insurmountable AT1 antagonism. CI-996, L158809, EXP 3174 and SKF 108834 produced concentration-related rightward shifts in Ang II response curves and reduced the maximal contraction to Ang II, characteristic of insurmountable antagonism. In contrast, DuP 753 and SKF 108566, produced parallel rightward shifts in Ang II contractile curves without affecting the maximal response which is consistent with the definition of surmountable or competitive antagonism. In addition, CI-996 demonstrated potent inhibition of Ang II-stimulated inositol phosphate accumulation in rat aortic smooth muscle cells, behaving as an insurmountable antagonist. However, DuP 753 was a surmountable antagonist of Ang II-stimulated inositol phosphate accumulation. Repeated washing of rabbit aorta preincubated with either CI-996 or EXP 3174 did not restore the blunted Ang II contractions. In contrast, both DuP 753 and the structurally dissimilar SKF 108566 at a concentration of 100 nM showed complete recovery of Ang II responses within 2 hr of repeated washing. Surprisingly, repeated rinsing of rabbit aorta for up to 5 hr after incubation with 1 microM DuP 753 failed to restore responses to Ang II. In addition, Scatchard analysis of [125I] Ang II saturation binding experiments revealed a competitive and rapidly reversible nature of AT1 receptor antagonism for all the AT1 antagonists examined. Taken together, the results of this study provide evidence for a competitive and rapidly reversible binding interaction of structurally diverse non-peptide antagonists at the AT1 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. 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

  7. Adenosine A2B receptor: from cell biology to human diseases

    NASA Astrophysics Data System (ADS)

    Sun, Ying; Huang, Pingbo

    2016-08-01

    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.

  8. 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.

  9. 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

  10. Activation of adenosine A1 receptors reduces anxiety-like behavior during acute ethanol withdrawal (hangover) in mice.

    PubMed

    Prediger, Rui D S; da Silva, George E; Batista, Luciano C; Bittencourt, Alvorita L; Takahashi, Reinaldo N

    2006-10-01

    Elevated signs of anxiety are observed in both humans and rodents during withdrawal from chronic as well as acute ethanol exposure, and it represents an important motivational factor for ethanol relapse. Several reports have suggested the involvement of brain adenosine receptors in different actions produced by ethanol such as motor incoordination and hypnotic effects. In addition, we have recently demonstrated that adenosine A1 receptors modulate the anxiolytic-like effect induced by ethanol in mice. In the present study, we evaluated the potential of adenosine A1 and A2A receptor agonists in reducing the anxiety-like behavior during acute ethanol withdrawal (hangover) in mice. Animals received a single intraperitoneal administration of saline or ethanol (4 g/kg) and were tested in the elevated plus maze after an interval of 0.5-24 h. The results indicated that hangover-induced anxiety was most pronounced between 12 and 18 h after ethanol administration, as indicated by a significant reduction in the exploration of the open arms of the maze. At this time interval, ethanol was completely cleared. The acute administration of 'nonanxiolytic' doses of adenosine and the selective adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), but not the adenosine A2A receptor agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine (DPMA), at the onset of peak withdrawal (18 h), reduced this anxiogenic-like response. In addition, the effect of CCPA on the anxiety-like behavior of ethanol hangover was reversed by pretreatment with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). These results reinforce the notion of the involvement of adenosine receptors in the anxiety-like responses and indicate the potential of adenosine A1 receptor agonists to reduce the anxiogenic effects during ethanol withdrawal.

  11. Modulation of audiogenic seizures by histamine and adenosine receptors in the inferior colliculus.

    PubMed

    Feng, H J; Faingold, C L

    2000-05-01

    Susceptibility to behaviorally similar audiogenic seizures (AGS) occurs genetically and is inducible during ethanol withdrawal (ETX). Comparisons between AGS mechanisms of genetically epilepsy-prone rats (GEPR-9s) and ethanol-withdrawn rats (ETX-Rs) are yielding information about general pathophysiological mechanisms of epileptogenesis. The inferior colliculus (IC) is the AGS initiation site. Excitatory amino acid (EAA) abnormalities in the IC are implicated in AGS, and histamine and adenosine receptor activation each reduce EAA release and inhibit several seizure types. Previous studies indicate that focal infusion of an adenosine receptor agonist into the IC blocked AGS in GEPR-9s, but the effects of adenosine receptor activation in the IC on AGS in ETX-Rs are unknown. The effects of histamine receptor activation on either form of AGS are also unexamined. The present study evaluated effects of histamine or a nonselective adenosine A(1) agonist, 2-chloroadenosine, on AGS by focal microinjection into the IC. Ethanol dependence and AGS susceptibility were induced in normal rats by intragastric ethanol. Histamine (40 or 60 nmol/side) significantly reduced AGS in GEPR-9s, but histamine in doses up to 120 nmol/side did not affect AGS in ETX-Rs. 2-Chloroadenosine (5 or 10 nmol/side) did not affect AGS in ETX-Rs, despite the effectiveness of lower doses of this agent in GEPR-9s reported previously. Thus, histamine and adenosine receptors in the IC modulate AGS of GEPR-9s, but do not modulate ETX-induced AGS. The reasons for this difference may involve the chronicity of AGS susceptibility in GEPR-9s, which may lead to more extensive neuromodulation as compensatory mechanisms to limit the seizures compared to the acute AGS of ETX-Rs.

  12. Activation of two sites by adenosine receptor agonists to cause relaxation in rat isolated mesenteric artery

    PubMed Central

    Prentice, D J; Payne, S L; Hourani, S M O

    1997-01-01

    In this study we have characterized the receptor(s) in the rat mesenteric artery mediating relaxant responses to adenosine and a number of adenosine analogues, N6 -R-phenylisopropyladenosine (R-PIA), N6-cyclopentyladenosine (CPA), N6-(3-iodo-benzyl)-adenosine-5′-N-methyluronamide (IB-MECA) and 5′-N-ethylcarboxamidoadenosine (NECA), by use of the non-selective antagonist 8-sulphophenyltheophylline (8-SPT) and the A2A selective ligands 2-[p-(2-carbonylethyl)-phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680) 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). We have also studied the effects of endothelial removal and uptake inhibition by nitrobenzylthioinosine (NBTI) and the effects of the A3 receptor antagonist 1,3-dipropyl-8-(4-acrylate)phenylxanthine (BWA1433).Adenosine, NECA, CPA and R-PIA all elicited relaxant responses in tissues precontracted with phenylephrine (1 μM) with the following potency order: NECA>R-PIA>adenosine=CPA. However, E/[A] curves to NECA were biphasic. CGS 21680 was inactive at concentrations up to 30 μM and IB-MECA elicited relaxant responses which were resistant to blockade by 8-SPT and BWA1433 (100 μM).Removal of the endothelium produced a small but significant decrease in the asymptote of the high potency phase of E/[A] curves to NECA with no change in p[A]50. E/[A] curves to adenosine were not altered by removal of the endothelium. However, there were small rightward shifts of E/[A] curves to CPA and R-PIA in the absence of endothelium.Inhibition of uptake by NBTI (1 μM) had no effect on E/[A] curves to NECA, CPA or R-PIA, but E/[A] curves to adenosine were significantly left-shifted in the presence of NBTI.8-SPT (10–100 μM) caused significant rightward shifts of the high potency phase of the E/[A] curves to NECA (pA2=5.63±0.26). The second phase of the concentration-response curve to NECA appeared to be resistant to blockade by 8-SPT, as were E

  13. 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

  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. Binary Drugs: Conjugates of Purines and a Peptide That Bind to Both Adenosine and Substance P Receptors

    PubMed Central

    Jacobson, Kenneth A.; Lipkowski, Andrzej W.; Moody, Terry W.; Padgett, William; Pijl, Evelyn; Kirk, Kenneth L.; Daly, John W.

    2012-01-01

    A “functionalized congener” approach to adenosine receptor antagonists has provided a means to synthesize highly potent peptide conjugates of 1,3-dialkylxanthines. The antagonist XAC, such a functionalized xanthine amine congener, has been attached to a segment derived from the neurotransmitter peptide substance P (SP) to form a binary drug that binds to both receptors with Ki values of 35 nM (central A1-adenosine) and 300 nM (striatal SP). Coupling of the functionalized adenosine agonist N6-[p-(carboxymethyl)phenyl]adenosine to an SP C-terminal peptide also resulted in a binary drug that binds to both receptors. The demonstration that the biochemical properties of two unrelated drugs, both of which act through binding at extracellular receptors, may be combined in the same molecule suggests a novel strategy for drug design. In principle, a combined effect of the two different substances that produce the same final effect (e.g., hypotension by adenosine agonists and by SP analogues) might occur in vivo. Adenosine analogues have analgesic properties, and the binary drug derived from substance P and adenosine agonists or antagonists might provide useful tools for probing interrelationships of SP pathways and sites for the antinociceptive action of adenosine. PMID:2441057

  16. Homology modelling of the human adenosine A2B receptor based on X-ray structures of bovine rhodopsin, the beta2-adrenergic receptor and the human adenosine A2A receptor.

    PubMed

    Sherbiny, Farag F; Schiedel, Anke C; Maass, Astrid; Müller, Christa E

    2009-11-01

    A three-dimensional model of the human adenosine A2B receptor was generated by means of homology modelling, using the crystal structures of bovine rhodopsin, the beta2-adrenergic receptor, and the human adenosine A2A receptor as templates. In order to compare the three resulting models, the binding modes of the adenosine A2B receptor antagonists theophylline, ZM241385, MRS1706, and PSB601 were investigated. The A2A-based model was much better able to stabilize the ligands in the binding site than the other models reflecting the high degree of similarity between A2A and A2B receptors: while the A2B receptor shares about 21% of the residues with rhodopsin, and 31% with the beta2-adrenergic receptor, it is 56% identical to the adenosine A2A receptor. The A2A-based model was used for further studies. The model included the transmembrane domains, the extracellular and the intracellular hydrophilic loops as well as the terminal domains. In order to validate the usefulness of this model, a docking analysis of several selective and nonselective agonists and antagonists was carried out including a study of binding affinities and selectivities of these ligands with respect to the adenosine A2A and A2B receptors. A common binding site is proposed for antagonists and agonists based on homology modelling combined with site-directed mutagenesis and a comparison between experimental and calculated affinity data. The new, validated A2B receptor model may serve as a basis for developing more potent and selective drugs.

  17. 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

  18. Ligand Binding and Subtype Selectivity of the Human A2A Adenosine Receptor

    PubMed Central

    Jaakola, Veli-Pekka; Lane, J. Robert; Lin, Judy Y.; Katritch, Vsevolod; IJzerman, Adriaan P.; Stevens, Raymond C.

    2010-01-01

    The crystal structure of the human A2A adenosine receptor bound to the A2A receptor-specific antagonist, ZM241385, was recently determined at 2.6-Å resolution. Surprisingly, the antagonist binds in an extended conformation, perpendicular to the plane of the membrane, and indicates a number of interactions unidentified before in ZM241385 recognition. To further understand the selectivity of ZM241385 for the human A2A adenosine receptor, we examined the effect of mutating amino acid residues within the binding cavity likely to have key interactions and that have not been previously examined. Mutation of Phe-168 to Ala abolishes both agonist and antagonist binding as well as receptor activity, whereas mutation of this residue to Trp or Tyr had only moderate effects. The Met-177 → Ala mutation impeded antagonist but not agonist binding. Finally, the Leu-249 → Ala mutant showed neither agonist nor antagonist binding affinity. From our results and previously published mutagenesis data, we conclude that conserved residues Phe-168(5.29), Glu-169(5.30), Asn-253(6.55), and Leu-249(6.51) play a central role in coordinating the bicyclic core present in both agonists and antagonists. By combining the analysis of the mutagenesis data with a comparison of the sequences of different adenosine receptor subtypes from different species, we predict that the interactions that determine subtype selectivity reside in the more divergent “upper” region of the binding cavity while the “lower” part of the binding cavity is conserved across adenosine receptor subtypes. PMID:20147292

  19. GLUCOSE-INDUCED INTESTINAL VASODILATION VIA ADENOSINE A1 RECEPTORS REQUIRES NITRIC OXIDE BUT NOT K+ATP CHANNELS

    PubMed Central

    Matheson, Paul J.; Li, Na; Harris, Patrick D.; Zakaria, El Rasheid; Garrison, R. Neal

    2010-01-01

    INTRODUCTION Both nitric oxide (NO) and adenosine A1 receptor activation mediate microvascular vasodilation during intestinal glucose absorption. Our overall hypothesis is that ATP utilization during glucose absorption would increase adenosine metabolite release, which acts on adenosine A1 receptors to alter endothelial production of NO and/or activate ATP-dependent potassium channels (K+ATP) to dilate intestinal microvessels. METHODS Intravital videomicroscopy of the rat jejunum was used to record the vascular responses of inflow (termed 1A) arterioles and proximal (p3A) and distal (d3A) premucosal arterioles during exposure to isotonic glucose or mannitol solutions alone or in the presence of the selective nitric oxide synthase (NOS) inhibitor (L-NMMA), an adenosine A1 receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine (DPCPX)), or a K+ATP channel inhibitor (glibenclamide). RESULTS As expected, glucose exposure caused rapid dilation of both p3A and d3A arterioles, while mannitol exposure had no effect on microvascular diameters. Adenosine A1 receptor blockade completely prevented glucose-induced dilation of the premucosal arterioles. NOS inhibition significantly blunted the glucose-induced vasodilation of the premucosal arterioles, but had little effect in the mannitol group. Simultaneous application of both the NOS inhibitor and the adenosine A1 receptor antagonist gave the same reduction in glucose-induced dilation of the premucosal arterioles as the adenosine A1 receptor antagonist alone. Blockade of K+ATP channels with glibenclamide did not attenuate glucose-induced vasodilation of the premucosal arterioles. CONCLUSIONS These data suggest that glucose-induced vasodilation of premucosal jejunal arterioles is mediated through adenosine A1 receptors, and NO at least partially mediates the adenosine A1 receptor-induced vasodilation. In addition, K+ATP channels are not involved in premucosal arteriolar vasodilation during intestinal glucose exposure. PMID

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. Crystal structures of the A2A adenosine receptor and their use in medicinal chemistry.

    PubMed

    Jacobson, Kenneth A

    2013-12-20

    New insights into drug design are derived from the X-ray crystallographic structures of G protein-coupled receptors (GPCRs), and the adenosine receptors (ARs) are at the forefront of this effort. The 3D knowledge of receptor binding and activation promises to enable drug discovery for GPCRs in general, and specifically for the ARs. The predictability of modeling based on the X-ray structures of the A2AAR has been well demonstrated in the identification, design and modification of both known and novel AR agonists and antagonists. It is expected that structure-based design of drugs acting through ARs will provide new avenues to clinically useful agents.

  5. [3H]-SCH 58261 labelling of functional A2A adenosine receptors in human neutrophil membranes

    PubMed Central

    Varani, Katia; Gessi, Stefania; Dionisotti, Silvio; Ongini, Ennio; Andrea Borea, Pier

    1998-01-01

    The present study describes the direct labelling of A2A adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4 triazolo[1,5-c]pyrimidine, ([3H]-SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays.Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 1.34 nM and 75 fmol mg−1 protein, respectively. Adenosine receptor ligands competed for the binding of 1 nM [3H]-SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A2A adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments.Thermodynamic data indicated that [3H]-SCH 58261 binding to human neutrophils is entropy and enthalpy-driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A2A adenosine receptors.It was concluded that in human neutrophil membranes, [3H]-SCH 58261 directly labels binding sites with pharmacological properties similar to those of A2A adenosine receptors of other tissues. The receptors labelled by [3H]-SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils. PMID:9605581

  6. 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

  7. Myocardial blood flow and adenosine A2A receptor density in endurance athletes and untrained men

    PubMed Central

    Heinonen, Ilkka; Nesterov, Sergey V; Liukko, Kaisa; Kemppainen, Jukka; Någren, Kjell; Luotolahti, Matti; Virsu, Pauliina; Oikonen, Vesa; Nuutila, Pirjo; Kujala, Urho M; Kainulainen, Heikki; Boushel, Robert; Knuuti, Juhani; Kalliokoski, Kari K

    2008-01-01

    Previous human studies have shown divergent results concerning the effects of exercise training on myocardial blood flow (MBF) at rest or during adenosine-induced hyperaemia in humans. We studied whether these responses are related to alterations in adenosine A2A receptor (A2AR) density in the left-ventricular (LV) myocardium, size and work output of the athlete's heart, or to fitness level. MBF at baseline and during intravenous adenosine infusion, and A2AR density at baseline were measured using positron emission tomography, and by a novel A2AR tracer in 10 healthy male endurance athletes (ET) and 10 healthy untrained (UT) men. Structural LV parameters were measured with echocardiography. LV mass index was 71% higher in ET than UT (193 ± 18 g m−2versus 114 ± 13 g m−2, respectively). MBF per gram of tissue was significantly lower in the ET than UT at baseline, but this was only partly explained by reduced LV work load since MBF corrected for LV work was higher in ET than UT, as well as total MBF. The MBF during adenosine-induced hyperaemia was reduced in ET compared to UT, and the fitter the athlete was, the lower was adenosine-induced MBF. A2AR density was not different between the groups and was not coupled to resting or adenosine-mediated MBF. The novel findings of the present study show that the adaptations in the heart of highly trained endurance athletes lead to relative myocardial ‘overperfusion’ at rest. On the other hand hyperaemic perfusion is reduced, but is not explained by A2AR density. PMID:18772204

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. An efficient route to xanthine based A(2A) adenosine receptor antagonists and functional derivatives.

    PubMed

    Labeaume, Paul; Dong, Ma; Sitkovsky, Michail; Jones, Elizabeth V; Thomas, Rhiannon; Sadler, Sara; Kallmerten, Amy E; Jones, Graham B

    2010-09-21

    A one-pot route to 8-substituted xanthines has been developed from 5,6-diaminouracils and carboxaldehydes. Yields are good and the process applicable to a range of substrates including a family of A(2A) adenosine receptor antagonists. A new route to the KW-6002 family of antagonists is presented including a pro-drug variant, and application to related image contrast agents developed.

  14. 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

  15. 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

  16. Blockade of adenosine A1 receptors in the posterior cingulate cortex facilitates memory in rats.

    PubMed

    Pereira, Grace S; Mello e Souza, Tadeu; Vinadé, Elsa R C; Choi, Humberto; Rodrigues, Cristina; Battastini, Ana M O; Izquierdo, Iván; Sarkis, João J F; Bonan, Carla D

    2002-02-22

    Male Wistar rats were bilaterally implanted with indwelling cannulae in the caudal region of the posterior cingulate cortex. After recovery, animals were trained in a step-down inhibitory avoidance task (3.0-s, 0.4-mA foot shock) and received, immediately after training, a 0.5-microl infusion of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA; 1, 50 or 100 nM) or of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1, 25 or 50 nM). Animals were tested twice, 1.5 h and, again, 24 h after training, in order to examine the effects of these agents on short- and long-term memory, respectively. Only 50-nM DPCPX was effective in altering memory, promoting a facilitation. These results suggest that adenosine A1 receptors in the posterior cingulate cortex inhibit memory consolidation in a way that their blockade facilitates memory for inhibitory avoidance in rats.

  17. 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

  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

    2015-08-04

    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.

  19. 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.

  20. 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

  1. 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

  2. Heteromerization of the μ- and δ-opioid receptors produces ligand-biased antagonism and alters μ-receptor trafficking.

    PubMed

    Milan-Lobo, Laura; Whistler, Jennifer L

    2011-06-01

    Heteromerization of opioid receptors has been shown to alter opioid receptor pharmacology. However, how receptor heteromerization affects the processes of endocytosis and postendocytic sorting has not been closely examined. This question is of particular relevance for heteromers of the μ-opioid receptor (MOR) and δ-opioid receptor (DOR), because the MOR is recycled primarily after endocytosis and the DOR is degraded in the lysosome. Here, we examined the endocytic and postendocytic fate of MORs, DORs, and DOR/MOR heteromers in human embryonic kidney 293 cells stably expressing each receptor alone or coexpressing both receptors. We found that the clinically relevant MOR agonist methadone promotes endocytosis of MOR but also the DOR/MOR heteromer. Furthermore, we show that DOR/MOR heteromers that are endocytosed in response to methadone are targeted for degradation, whereas MORs in the same cell are significantly more stable. It is noteworthy that we found that the DOR-selective antagonist naltriben mesylate could block both methadone- and [D-Ala2,NMe-Phe4,Gly-ol5]-enkephalin-induced endocytosis of the DOR/MOR heteromers but did not block signaling from this heteromer. Together, our results suggest that the MOR adopts novel trafficking properties in the context of the DOR/MOR heteromer. In addition, they suggest that the heteromer shows "biased antagonism," whereby DOR antagonist can inhibit trafficking but not signaling of the DOR/MOR heteromer.

  3. Adenosine A2A receptor antagonists: blockade of adenosinergic effects and T regulatory cells

    PubMed Central

    Sitkovsky, M; Lukashev, D; Deaglio, S; Dwyer, K; Robson, S C; Ohta, A

    2008-01-01

    The intensity and duration of host responses are determined by protective mechanisms that control tissue injury by dampening down inflammation. Adenosine generation and consequent effects, mediated via A2A adenosine receptors (A2AR) on effector cells, play a critical role in the pathophysiological modulation of these responses in vivo. Adenosine is both released by hypoxic cells/tissues and is also generated from extracellular nucleotides by ecto-enzymes e.g. CD39 (ENTPD1) and CD73 that are expressed by the vasculature and immune cells, in particular by T regulatory cell. In general, these adenosinergic mechanisms minimize the extent of collateral damage to host tissues during the course of inflammatory reactions. However, induction of suppressive pathways might also cause escape of pathogens and permit dissemination. In addition, adenosinergic responses may inhibit immune responses while enhancing vascular angiogenic responses to malignant cells that promote tumor growth. Novel drugs that block A2AR-adenosinergic effects and/or adenosine generation have the potential to boost pathogen destruction and to selectively destroy malignant tissues. In the latter instance, future treatment modalities might include novel ‘anti-adenosinergic' approaches that augment immune clearance of malignant cells and block permissive angiogenesis. This review addresses several possible pharmacological modalities to block adenosinergic pathways and speculates on their future application together with impacts on human disease. PMID:18311159

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

    PubMed

    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; Salvemini, Daniela

    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

  5. Adenosine Receptor Blockade by Caffeine Inhibits Carotid Sinus Nerve Chemosensory Activity in Chronic Intermittent Hypoxic Animals.

    PubMed

    Sacramento, J F; Gonzalez, C; Gonzalez-Martin, M C; Conde, S V

    2015-01-01

    Adenosine is a key excitatory neurotransmitter at the synapse between O(2)-sensing chemoreceptor cells-carotid sinus nerve (CSN) endings in the carotid body (CB). Herein, we have investigated the significance of adenosine, through the blockade of its receptors with caffeine, on the CB hypoxic sensitization induced by chronic intermittent hypoxia (CIH) in the rat. CIH animals were obtained by submitting rats during 15 days from 8:00 to 16:00 to 10 %O(2) for 40 s and 20 % O(2) for 80 s (i.e., 30 episodes/h). Caffeine (1 mM) was tested in spontaneous and 5 %O(2) evoked-CSN chemosensory activity in normoxic and CIH animals. CIH decreased basal spontaneous activity but increased significantly CSN activity evoked by acute hypoxia. Caffeine did not modify basal spontaneous activity in normoxic rats, but decreased significantly by 47.83 % basal activity in CIH animals. In addition, acute application of caffeine decreased 49.31 % and 56.01 % the acute hypoxic response in normoxic and CIH animals, respectively. We demonstrate that adenosine contributes to fix CSN basal activity during CIH, being also involved in hypoxic CB chemotransduction. It is concluded that adenosine participates in CB sensitization during CIH. PMID:26303475

  6. Activation of NTS A(1) adenosine receptors inhibits regional sympathetic responses evoked by activation of cardiopulmonary chemoreflex.

    PubMed

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

    2012-09-01

    Previously we have shown that adenosine operating via the A(1) receptor subtype may inhibit glutamatergic transmission in the baroreflex arc within the nucleus of the solitary tract (NTS) and differentially increase renal (RSNA), preganglionic adrenal (pre-ASNA), and lumbar (LSNA) sympathetic nerve activity (ASNA>RSNA≥LSNA). Since the cardiopulmonary chemoreflex and the arterial baroreflex are mediated via similar medullary pathways, and glutamate is a primary transmitter in both pathways, it is likely that adenosine operating via A(1) receptors in the NTS may differentially inhibit regional sympathetic responses evoked by activation of cardiopulmonary chemoreceptors. Therefore, in urethane-chloralose-anesthetized rats (n = 37) we compared regional sympathoinhibition evoked by the cardiopulmonary chemoreflex (activated with right atrial injections of serotonin 5HT(3) receptor agonist phenylbiguanide, PBG, 1-8 μg/kg) before and after selective stimulation of NTS A(1) adenosine receptors [microinjections of N(6)-cyclopentyl adenosine (CPA), 0.033-330 pmol/50 nl]. Activation of cardiopulmonary chemoreceptors evoked differential, dose-dependent sympathoinhibition (RSNA>ASNA>LSNA), and decreases in arterial pressure and heart rate. These differential sympathetic responses were uniformly attenuated in dose-dependent manner by microinjections of CPA into the NTS. Volume control (n = 11) and blockade of adenosine receptor subtypes in the NTS via 8-(p-sulfophenyl)theophylline (8-SPT, 1 nmol in 100 nl) (n = 9) did not affect the reflex responses. We conclude that activation of NTS A(1) adenosine receptors uniformly inhibits neural and cardiovascular cardiopulmonary chemoreflex responses. A(1) adenosine receptors have no tonic modulatory effect on this reflex under normal conditions. However, when adenosine is released into the NTS (i.e., during stress or severe hypotension/ischemia), it may serve as negative feedback regulator for depressor and sympathoinhibitory reflexes

  7. Fragment Screening at Adenosine-A3 Receptors in Living Cells Using a Fluorescence-Based Binding Assay

    PubMed Central

    Stoddart, Leigh A.; Vernall, Andrea J.; Denman, Jessica L.; Briddon, Stephen J.; Kellam, Barrie; Hill, Stephen J.

    2012-01-01

    Summary G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane proteins. For GPCR drug discovery, it is important that ligand affinity is determined in the correct cellular environment and preferably using an unmodified receptor. We developed a live cell high-content screening assay that uses a fluorescent antagonist, CA200645, to determine binding affinity constants of competing ligands at human adenosine-A1 and -A3 receptors. This method was validated as a tool to screen a library of low molecular weight fragments, and identified a hit with submicromolar binding affinity (KD). This fragment was structurally unrelated to substructures of known adenosine receptor antagonists and was optimized to show selectivity for the adenosine-A3 receptor. This technology represents a significant advance that will allow the determination of ligand and fragment affinities at receptors in their native membrane environment. PMID:22999879

  8. Novel fluorescent antagonist as a molecular probe in A(3) adenosine receptor binding assays using flow cytometry.

    PubMed

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

    2012-06-01

    The physiological role of the A(3) adenosine receptor (AR) was explored in cardiac ischaemia, inflammatory diseases and cancer. We report a new fluorophore-conjugated human (h) A(3)AR 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 K(i) value of 6.4±2.5nM in hA(3)AR-expressing CHO cell membranes. MRS5449 antagonized hA(3)AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner (K(B)=4.8nM). Using flow cytometry (FCM), MRS5449 saturated hA(3)ARs with very high specific-to-nonspecific binding ratio with an equilibrium binding constant 5.15nM, comparable to the K(d) value of 6.65nM calculated from kinetic experiments. K(i) 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 [(125)I]I-AB-MECA. Further binding analysis of MRS5549 suggested multiple agonist binding states of the A(3)AR. Molecular docking predicted binding modes of these fluorescent antagonists. Thus, MRS5449 is a useful tool for hA(3)AR characterization.

  9. 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

  10. 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

  11. 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

  12. Deletion of adenosine A1 or A2A receptors reduces L-3,4-dihydroxyphenylalanine-induced dyskinesia in a model of Parkinson’s disease

    PubMed Central

    Xiao, Danqing; Cassin, Jared J.; Healy, Brian; Burdett, Thomas C.; Chen, Jiang-Fan; Fredholm, Bertil B.; Schwarzschild, Michael A.

    2010-01-01

    Adenosine A2A receptor antagonism provides a promising approach to developing nondopaminergic therapy for Parkinson’s disease (PD). Clinical trials of A2A antagonists have targeted PD patients with L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID) in an effort to improve parkinsonian symptoms. The role of adenosine in the development of LID is little known, especially regarding its actions via A1 receptors. We aimed to examine the effects of genetic deletion and pharmacological blockade of A1 and/or A2A receptors on the development of LID, on the induction of molecular markers of LID including striatal preprodynorphin and preproenkephalin (PPE), and on the integrity of dopaminergic nigrostriatal neurons in hemiparkinsonian mice. Following a unilateral 6-hydroxydopamine lesion A1, A2A and double A1-A2A knockout (KO) and wild-type littermate mice, and mice pretreated with caffeine (an antagonist of both A1 and A2A receptors) or saline were treated daily for 18–21 days with a low dose of L-DOPA. Total abnormal involuntary movements (AIMs, a measure of LID) were significantly attenuated (p<0.05) in A1 and A2A KOs, but not in A1-A2A KOs and caffeine-pretreated mice. An elevation of PPE mRNA ipsilateral to the lesion in WT mice was reduced in all KO mice. In addition, neuronal integrity assessed by striatal dopamine content was similar in all KOs and caffeine-pretreated mice following 6-hydroxydopamine lesioning. Our findings raise the possibility that A1 or A2A receptors blockade might also confer a disease-modifying benefit of reduced risk of disabling LID, whereas the effect of their combined inactivation is less clear. PMID:20828543

  13. Effect of Caffeine Chronically Consumed During Pregnancy on Adenosine A1 and A2A Receptors Signaling in Both Maternal and Fetal Heart from Wistar Rats

    PubMed Central

    Iglesias, Inmaculada; Albasanz, Jose Luis

    2014-01-01

    Background: Caffeine is the most widely consumed psychoactive substance in the world, even during pregnancy. Its stimulatory effects are mainly due to antagonism of adenosine actions by blocking adenosine A1 and A2A receptors. Previous studies have shown that caffeine can cross the placenta and therefore modulate these receptors not only in the fetal brain but also in the heart. Methods: In the present work, the effect of caffeine chronically consumed during pregnancy on A1 and A2A receptors in Wistar rat heart, from both mothers and their fetuses, were studied using radioligand binding, Western-blotting, and adenylyl cyclase activity assays, as well as reverse transcription polymerase chain reaction. Results: Caffeine did not significantly alter A1R neither at protein nor at gene expression level in both the maternal and fetal heart. On the contrary, A2AR significantly decreased in the maternal heart, although mRNA was not affected. Gi and Gs proteins were also preserved. Finally, A1R-mediated inhibition of adenylyl cyclase activity did not change in the maternal heart, but A2AR mediated stimulation of this enzymatic activity significantly decreased according to the detected loss of this receptor. Conclusions: Opposite to the downregulation and desensitization of the A1R/AC pathway previously reported in the brain, these results show that this pathway is not affected in rat heart after caffeine exposure during pregnancy. In addition, A2AR is downregulated and desensitized in the maternal heart, suggesting a differential modulation of these receptor-mediated pathways by caffeine. PMID:25538864

  14. Discovery of LAS101057: A Potent, Selective, and Orally Efficacious A2B Adenosine Receptor Antagonist

    PubMed Central

    2010-01-01

    The structure−activity relationships for a series of pyrazine-based A2B adenosine receptor antagonists are described. From this work, LAS101057 (17), a potent, selective, and orally efficacious A2B receptor antagonist, was identified as a clinical development candidate. LAS101057 inhibits agonist-induced IL-6 production in human fibroblasts and is active in an ovalbumin (OVA)-sensitized mouse model after oral administration, reducing airway hyperresponsiveness to methacholine, Th2 cytokine production, and OVA-specific IgE levels. PMID:24900298

  15. 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

  16. Characterization of A2A adenosine receptors in human lymphocyte membranes by [3H]-SCH 58261 binding

    PubMed Central

    Varani, Katia; Gessi, Stefania; Dalpiaz, Alessandro; Ongini, Ennio; Andrea Borea, Pier

    1997-01-01

    The present study describes for the first time the characterization of the adenosine A2A receptor in human lymphocyte membranes with the new potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine, ([3H]-SCH 58261). In addition, both receptor affinity and potency of reference adenosine receptor agonists and antagonists were determined in binding and adenylyl cyclase studies. Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 0.85 nM and 35 fmol mg−1 protein, respectively. A series of adenosine receptor ligands were found to compete for the binding of 0.8 nM [3H]-SCH 58261 to human lymphocyte membranes with a rank order of potency consistent with that typically found for interactions with the A2A-adenosine receptor. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency similar to that observed in binding experiments. Thermodynamic data indicate that [3H]-SCH 58261 binding to human lymphocytes is entropy and enthalpy-driven, a finding in agreement with the thermodynamic behaviour of antagonists for rat striatal A2A-adenosine receptors. It is concluded that in human lymphocyte membranes [3H]-SCH 58261 directly labels binding sites showing the characteristic properties of the adenosine A2A-receptor. The presence of A2A-receptors in peripheral tissue such as human lymphocytes strongly suggests an important role for adenosine in modulating immune and inflammatory responses. PMID:9313951

  17. Mechanisms involved in increased sensitivity to adenosine A(2A) receptor activation and hypoxia-induced vasodilatation in porcine coronary arteries.

    PubMed

    Hedegaard, Elise R; Nielsen, Berit D; Mogensen, Susie; Rembold, Christopher M; Frøbert, Ole; Simonsen, Ulf

    2014-01-15

    Hypoxia-induced coronary vasorelaxation is a compensatory mechanism increasing blood flow. We hypothesized that hypoxia shares pathways with adenosine and causes vasorelaxation through the adenosine A(2A) receptor and force suppression by increasing cAMP and phosphorylated heat shock protein (HSP)20. Adenosine receptors in porcine left anterior descending coronary arteries (LAD) were examined by RT-PCR and isometric tension recording in myographs. Vasorelaxation was induced by adenosine, 1% oxygen, or both in the absence or presence of ZM241385, an adenosine A(2A) receptor antagonist. cAMP was determined by ELISA and p-HSP20/HSP20 and p-MLC/MLC were determined by immunoblotting and densitometric analyses. In coronary arteries exposed to 1% oxygen, there was increased sensitivity to adenosine, the adenosine A2 selective agonist NECA, and the adenosine A(2A) selective receptor agonist CGS21680. ZM241385 shifted concentration-response curves for CGS21680 to the right, whereas the adenosine A1 antagonist DPCPX, the adenosine A2B receptor antagonist MRS1754 and the adenosine A3 receptor antagonist MRS1523 failed to reduce vasodilatation induced by CGS21680. 1% oxygen or adenosine increased cAMP accumulation and HSP20 phosphorylation without changing T850-MYPT1 and MLC phosphorylation. ZM241385 failed to change 1% oxygen-induced vasodilation, cAMP accumulation, HSP20 phosphorylation and MLC phosphorylation. The PKA inhibitor Rp-8-CPT-cAMPS significantly reduced vasorelaxation induced by 1% oxygen or CGS21680. Our findings suggest that the increased sensitivity to adenosine, NECA, and CGS21680 at 1% oxygen involves adenosine A(2A) receptors. Adenosine and 1% oxygen induce vasorelaxation in PGF2α-contracted porcine coronary arteries partly by force suppression caused by increased cAMP and phosphorylation of HSP20.

  18. Adenosine A2A receptors are necessary and sufficient to trigger memory impairment in adult mice

    PubMed Central

    Pagnussat, N; Almeida, A S; Marques, D M; Nunes, F; Chenet, G C; Botton, P H S; Mioranzza, S; Loss, C M; Cunha, R A; Porciúncula, L O

    2015-01-01

    Background and Purpose Caffeine (a non-selective adenosine receptor antagonist) prevents memory deficits in aging and Alzheimer’s disease, an effect mimicked by adenosine A2A receptor, but not A1 receptor, antagonists. Hence, we investigated the effects of adenosine receptor agonists and antagonists on memory performance and scopolamine-induced memory impairment in mice. Experimental Approach We determined whether A2A receptors are necessary for the emergence of memory impairments induced by scopolamine and whether A2A receptor activation triggers memory deficits in naïve mice, using three tests to assess short-term memory, namely the object recognition task, inhibitory avoidance and modified Y-maze. Key Results Scopolamine (1.0 mg·kg−1, i.p.) impaired short-term memory performance in all three tests and this scopolamine-induced amnesia was prevented by the A2A receptor antagonist (SCH 58261, 0.1–1.0 mg·kg−1, i.p.) and by the A1 receptor antagonist (DPCPX, 0.2–5.0 mg·kg−1, i.p.), except in the modified Y-maze where only SCH58261 was effective. Both antagonists were devoid of effects on memory or locomotion in naïve rats. Notably, the activation of A2A receptors with CGS 21680 (0.1–0.5 mg·kg−1, i.p.) before the training session was sufficient to trigger memory impairment in the three tests in naïve mice, and this effect was prevented by SCH 58261 (1.0 mg·kg−1, i.p.). Furthermore, i.c.v. administration of CGS 21680 (50 nmol) also impaired recognition memory in the object recognition task. Conclusions and Implications These results show that A2A receptors are necessary and sufficient to trigger memory impairment and further suggest that A1 receptors might also be selectively engaged to control the cholinergic-driven memory impairment. PMID:25939452

  19. 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.

  20. Sleep deprivation increases A1 adenosine receptor density in the rat brain

    PubMed Central

    Elmenhorst, David; Basheer, Radhika; McCarley, Robert W.; Bauer, Andreas

    2013-01-01

    Adenosine, increasing after sleep deprivation and acting via the A1 adenosine receptor (A1AR), is likely a key factor in the homeostatic control of sleep. This study examines the impact of sleep deprivation on A1AR density in different parts of the rat brain with [3H]CPFPX autoradiography. Binding of [3H]CPFPX was significantly increased in parietal cortex (PAR) (7%), thalamus (11%) and caudate-putamen (9%) after 24 h of sleep deprivation compared to a control group with an undisturbed circadian sleep-wake rhythm. Sleep deprivation of 12 h changed receptor density regionally between −5% and +9% (motor cortex (M1), statistically significant) compared to the circadian control group. These results suggest cerebral A1ARs are involved in effects of sleep deprivation and the regulation of sleep. The increase of A1AR density could serve the purpose of not only maintaining the responsiveness to increased adenosine levels but also amplifying the effect of sleep deprivation and is in line with a sleep-induced homoeostatic reorganization at the synaptic level. PMID:19146833

  1. Synthesis and Evaluation of N6-Substituted Apioadenosines as Potential Adenosine A3 Receptor Modulators

    PubMed Central

    Toti, Kiran S.; Moss, Steven M.; Paoletta, Silvia; Gao, Zhan-Guo; Jacobson, Kenneth A.; Van Calenbergh, Serge

    2014-01-01

    Adenosine receptors (ARs) trigger signal transduction pathways inside the cell when activated by extracellular adenosine. Selective modulation of the A3AR subtype may be beneficial in controlling diseases such as colorectal cancer and rheumatoid arthritis. Here, we report the synthesis and evaluation of β-D-apio-D-furano- and α-D-apio-L-furanoadenosines and derivatives thereof. Introduction of a 2-methoxy-5-chlorobenzyl group at N6 of β-D-apio-D-furanoadenosine afforded an A3AR antagonist (10c, Ki = 0.98 μM), while a similar modification of an α-D-apio-L-furanoadenosine gave rise to a partial agonist (11c, Ki = 3.07 μM). The structural basis for this difference was examined by docking to an A3AR model; the antagonist lacked a crucial interaction with Thr94. PMID:24931275

  2. Synthesis and evaluation of N⁶-substituted apioadenosines as potential adenosine A₃ receptor modulators.

    PubMed

    Toti, Kiran S; Moss, Steven M; Paoletta, Silvia; Gao, Zhan-Guo; Jacobson, Kenneth A; Van Calenbergh, Serge

    2014-08-01

    Adenosine receptors (ARs) trigger signal transduction pathways inside the cell when activated by extracellular adenosine. Selective modulation of the A₃AR subtype may be beneficial in controlling diseases such as colorectal cancer and rheumatoid arthritis. Here, we report the synthesis and evaluation of β-D-apio-D-furano- and α-D-apio-L-furanoadenosines and derivatives thereof. Introduction of a 2-methoxy-5-chlorobenzyl group at N(6) of β-D-apio-D-furanoadenosine afforded an A₃AR antagonist (10c, Ki=0.98 μM), while a similar modification of an α-D-apio-L-furanoadenosine gave rise to a partial agonist (11c, Ki=3.07 μM). The structural basis for this difference was examined by docking to an A₃AR model; the antagonist lacked a crucial interaction with Thr94. PMID:24931275

  3. 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

  4. Discovery of Potent and Highly Selective A2B Adenosine Receptor Antagonist Chemotypes.

    PubMed

    El Maatougui, Abdelaziz; Azuaje, Jhonny; González-Gómez, Manuel; Miguez, Gabriel; Crespo, Abel; Carbajales, Carlos; Escalante, Luz; García-Mera, Xerardo; Gutiérrez-de-Terán, Hugo; Sotelo, Eddy

    2016-03-10

    Three novel families of A2B adenosine receptor antagonists were identified in the context of the structural exploration of the 3,4-dihydropyrimidin-2(1H)-one chemotype. The most appealing series contain imidazole, 1,2,4-triazole, or benzimidazole rings fused to the 2,3-positions of the parent diazinone core. The optimization process enabled identification of a highly potent (3.49 nM) A2B ligand that exhibits complete selectivity toward A1, A2A, and A3 receptors. The results of functional cAMP experiments confirmed the antagonistic behavior of representative ligands. The main SAR trends identified within the series were substantiated by a molecular modeling study based on a receptor-driven docking model constructed on the basis of the crystal structure of the human A2A receptor.

  5. 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.

  6. A1 and A2a receptors mediate inhibitory effects of adenosine on the motor activity of human colon.

    PubMed

    Fornai, M; Antonioli, L; Colucci, R; Ghisu, N; Buccianti, P; Marioni, A; Chiarugi, M; Tuccori, M; Blandizzi, C; Del Tacca, M

    2009-04-01

    Experimental evidence in animal models suggests that adenosine is involved in the regulation of digestive functions. This study examines the influence of adenosine on the contractile activity of human colon. Reverse transcription-polymerase chain reaction revealed A(1) and A(2a) receptor expression in colonic neuromuscular layers. Circular muscle preparations were connected to isotonic transducers to determine the effects of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; A(1) receptor antagonist), ZM 241385 (A(2a) receptor antagonist), CCPA (A(1) receptor agonist) and 2-[(p-2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamide-adenosine (CGS 21680; A(2a) receptor agonist) on motor responses evoked by electrical stimulation or carbachol. Electrically evoked contractions were enhanced by DPCPX and ZM 241385, and reduced by CCPA and CGS 21680. Similar effects were observed when colonic preparations were incubated with guanethidine (noradrenergic blocker), L-732,138, GR-159897 and SB-218795 (NK receptor antagonists). However, in the presence of guanethidine, NK receptor antagonists and N(omega)-propyl-L-arginine (NPA; neuronal nitric oxide synthase inhibitor), the effects of DPCPX and CCPA were still evident, while those of ZM 241385 and CGS 21680 no longer occurred. Carbachol-induced contractions were unaffected by A(2a) receptor ligands, but they were enhanced or reduced by DPCPX and CCPA, respectively. When colonic preparations were incubated with guanethidine, NK antagonists and atropine, electrically induced relaxations were partly reduced by ZM 241385 or NPA, but unaffected by DPCPX. Dipyridamole or application of exogenous adenosine reduced electrically and carbachol-evoked contractions, whereas adenosine deaminase enhanced such motor responses. In conclusion, adenosine exerts an inhibitory control on human colonic motility. A(1) receptors mediate direct modulating actions on smooth muscle, whereas A(2a) receptors operate through inhibitory nitrergic nerve pathways.

  7. Adenosine restores angiotensin II-induced contractions by receptor-independent enhancement of calcium sensitivity in renal arterioles.

    PubMed

    Lai, En Yin; Martinka, Peter; Fähling, Michael; Mrowka, Ralf; Steege, Andreas; Gericke, Adrian; Sendeski, Mauricio; Persson, P B; Persson, A Erik G; Patzak, Andreas

    2006-11-10

    Adenosine is coupled to energy metabolism and regulates tissue blood flow by modulating vascular resistance. In this study, we investigated isolated, perfused afferent arterioles of mice, which were subjected to desensitization during repeated applications of angiotensin II. Exogenously applied adenosine restores angiotensin II-induced contractions by increasing calcium sensitivity of the arterioles, along with augmented phosphorylation of the regulatory unit of the myosin light chain. Adenosine restores angiotensin II-induced contractions via intracellular action, because inhibition of adenosine receptors do not prevent restoration, but inhibition of NBTI sensitive adenosine transporters does. Restoration was prevented by inhibition of Rho-kinase, protein kinase C, and the p38 mitogen-activated protein kinase, which modulate myosin light chain phosphorylation and thus calcium sensitivity in the smooth muscle. Furthermore, adenosine application increased the intracellular ATP concentration in LuciHEK cells. The results of the study suggest that restoration of the angiotensin II-induced contraction by adenosine is attributable to the increase of the calcium sensitivity by phosphorylation of the myosin light chain. This can be an important component of vascular control during ischemic and hypoxic conditions. Additionally, this mechanism may contribute to the mediation of the tubuloglomerular feedback by adenosine in the juxtaglomerular apparatus of the kidney. PMID:17038642

  8. Transmembrane AMPA receptor regulatory protein regulation of competitive antagonism: a problem of interpretation.

    PubMed

    Maclean, David M; Bowie, Derek

    2011-11-15

    Synaptic AMPA receptors are greatly influenced by a family of transmembrane AMPA receptor regulatory proteins (TARPs) which control trafficking, channel gating and pharmacology. The prototypical TARP, stargazin (or γ2), shifts the blocking ability of several AMPAR-selective compounds including the commonly used quinoxalinedione antagonists, CNQX and NBQX. Stargazin's effect on CNQX is particularly intriguing as it not only apparently lowers the potency of block, as with NBQX, but also renders it a partial agonist. Given this, agonist behaviour by CNQX has been speculated to account for its weaker blocking effect on AMPAR-TARP complexes. Here we show that this is not the case. The apparent effect of stargazin on CNQX antagonism can be almost entirely explained by an increase in the apparent affinity for l-glutamate (l-Glu), a full agonist and neurotransmitter at AMPAR synapses. Partial agonism at best plays a minor role but not through channel gating per se but rather because CNQX elicits AMPAR desensitization. Our study reveals that CNQX is best thought of as a non-competitive antagonist at glutamatergic synapses due to the predominance of non-equilibrium conditions. Consequently, CNQX primarily reports the proportion of AMPARs available for activation but may also impose additional block by receptor desensitization.

  9. (/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.

  10. 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

  11. 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

  12. Metabolic mapping of A3 adenosine receptor agonist MRS5980

    PubMed Central

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

    2015-01-01

    (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 of 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 group in feces, urine, and liver samples, but not in bile and serum. The major ions mainly contributing to the separation for 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 major 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

  13. 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

  14. 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

  15. Stimulation of NTS A1 adenosine receptors differentially resets baroreflex control of regional sympathetic outputs.

    PubMed

    Scislo, Tadeusz J; Ichinose, Tomoko K; O'Leary, Donal S

    2008-01-01

    Previously we showed that pressor and differential regional sympathoexcitatory responses (adrenal > renal >/= lumbar) evoked by stimulation of A(1) adenosine receptors located in the nucleus of the solitary tract (NTS) were attenuated/abolished by baroreceptor denervation or blockade of glutamatergic transmission in the NTS, suggesting A(1) receptor-elicited inhibition of glutamatergic transmission in baroreflex pathways. Therefore we tested the hypothesis that stimulation of NTS A(1) adenosine receptors differentially inhibits/resets baroreflex responses of preganglionic adrenal (pre-ASNA), renal (RSNA), and lumbar (LSNA) sympathetic nerve activity. In urethane-chloralose-anesthetized male Sprague-Dawley rats (n = 65) we compared baroreflex-response curves (iv nitroprusside and phenylephrine) evoked before and after bilateral microinjections into the NTS of A(1) adenosine receptor agonist (N(6)-cyclopentyladenosine, CPA; 0.033-330 pmol/50 nl). CPA evoked typical dose-dependent pressor and differential sympathoexcitatory responses and similarly shifted baroreflex curves for pre-ASNA, RSNA, and LSNA toward higher mean arterial pressure (MAP) in a dose-dependent manner; the maximal shifts were 52.6 +/- 2.8, 48.0 +/- 3.6, and 56.8 +/- 6.7 mmHg for pre-ASNA, RSNA, and LSNA, respectively. These shifts were not a result of simple baroreceptor resetting because they were two to three times greater than respective increases in baseline MAP evoked by CPA. Baroreflex curves for pre-ASNA were additionally shifted upward: the maximal increases of upper and lower plateaus were 41.8 +/- 16.4% and 45.3 +/- 8.7%, respectively. Maximal gain (%/mmHg) measured before vs. after CPA increased for pre-ASNA (3.0 +/- 0.6 vs. 4.9 +/- 1.3), decreased for RSNA (4.1 +/- 0.6 vs. 2.3 +/- 0.3), and remained unaltered for LSNA (2.1 +/- 0.2 vs. 2.0 +/- 0.1). Vehicle control did not alter the baroreflex curves. We conclude that the activation of NTS A(1) adenosine receptors differentially inhibits

  16. Treadmill running frequency on anxiety and hippocampal adenosine receptors density in adult and middle-aged rats.

    PubMed

    Costa, Marcelo S; Ardais, Ana Paula; Fioreze, Gabriela T; Mioranzza, Sabrina; Botton, Paulo Henrique S; Portela, Luis Valmor; Souza, Diogo O; Porciúncula, Lisiane O

    2012-01-10

    Physical exercise protocols have varied widely across studies raising the question of whether there is an optimal intensity, duration and frequency that would produce maximal benefits in attenuating symptoms related to anxiety disorders. Although physical exercise causes modifications in neurotransmission systems, the involvement of neuromodulators such as adenosine has not been investigated after chronic exercise training. Anxiety-related behavior was assessed in the elevated plus-maze in adult and middle-aged rats submitted to 8 weeks of treadmill running 1, 3 or 7 days/week. The speed of running was weekly adjusted to maintain moderate intensity. The hippocampal adenosine A1 and A2A receptors densities were also assessed. Treadmill running protocol was efficient in increasing physical exercise capacity in adult and middle-aged rats. All frequencies of treadmill running equally decreased the time spent in the open arms in adult animals. Middle-aged treadmill control rats presented lower time spent in the open arms than adult treadmill control rats. However, treadmill running one day/week reversed this age effect. Adenosine A1 receptor was not changed between groups, but treadmill running counteracted the age-related increase in adenosine A2A receptors. Although treadmill running, independent from frequency, triggered anxiety in adult rats and treadmill running one day/week reversed the age-related anxiety, no consistent relationship was found with hippocampal adenosine receptors densities. Thus, our data suggest that as a complementary therapy in the management of mental disturbances, the frequency and intensity of physical exercise should be taken into account according to age. Besides, this is the first study reporting the modulation of adenosine receptors after chronic physical exercise, which could be important to prevent neurological disorders associated to increase in adenosine A2A receptors.

  17. The effect of cannabidiol on ischemia/reperfusion-induced ventricular arrhythmias: the role of adenosine A1 receptors.

    PubMed

    Gonca, Ersöz; Darıcı, Faruk

    2015-01-01

    Cannabidiol (CBD) is a nonpsychoactive phytocannabinoid with anti-inflammatory activity mediated by enhancing adenosine signaling. As the adenosine A1 receptor activation confers protection against ischemia/reperfusion (I/R)-induced ventricular arrhythmias, we hypothesized that CBD may have antiarrhythmic effect through the activation of adenosine A1 receptor. Cannabidiol has recently been shown to suppress ischemia-induced ventricular arrhythmias. We aimed to research the effect of CBD on the incidence and the duration of I/R-induced ventricular arrhythmias and to investigate the role of adenosine A1 receptor activation in the possible antiarrhythmic effect of CBD. Myocardial ischemia and reperfusion was induced in anesthetized male rats by ligating the left anterior descending coronary artery for 6 minutes and by loosening the bond at the coronary artery, respectively. Cannabidiol alone was given in a dose of 50 µg/kg, 10 minutes prior to coronary artery occlusion and coadministrated with adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) in a dose of 100 µg/kg, 15 minutes prior to coronary artery occlusion to investigate whether the antiarrhythmic effect of CBD is modified by the activation of adenosine A1 receptors. The experimental groups were as follows: (1) vehicle control (n = 10), (2) CBD (n = 9), (3) DPCPX (n = 7), and (4) CBD + DPCPX group (n = 7). Cannabidiol treatment significantly decreased the incidence and the duration of ventricular tachycardia, total length of arrhythmias, and the arrhythmia scores compared to control during the reperfusion period. The DPCPX treatment alone did not affect the incidence and the duration of any type of arrhythmias. However, DPCPX aborted the antiarrhythmic effect of CBD when it was combined with it. The present results demonstrated that CBD has an antiarrhythmic effect against I/R-induced arrhythmias, and the antiarrhythmic effect of CBD may be mediated through the activation of adenosine

  18. Evaluation of neuronal phosphoproteins as effectors of caffeine and mediators of striatal adenosine A2A receptor signaling

    PubMed Central

    Sahin, Bogachan; Galdi, Stacey; Hendrick, Joseph; Greene, Robert W.; Snyder, Gretchen L.; Bibb, James A.

    2007-01-01

    Adenosine A2A receptors are predominantly expressed in the dendrites of enkephalin-positive γ-aminobutyric acidergic medium spiny neurons in the striatum. Evidence indicates that these receptors modulate striatal dopaminergic neurotransmission and regulate motor control, vigilance, alertness, and arousal. Although the physiological and behavioral correlates of adenosine A2A receptor signaling have been extensively studied using a combination of pharmacological and genetic tools, relatively little is known about the signal transduction pathways that mediate the diverse biological functions attributed to this adenosine receptor subtype. Using a candidate approach based on the coupling of these receptors to adenylate cyclase-activating G proteins, a number of membranal, cytosolic, and nuclear phosphoproteins regulated by PKA were evaluated as potential mediators of adenosine A2A receptor signaling in the striatum. Specifically, the adenosine A2A receptor agonist, CGS 21680, was used to determine whether the phosphorylation state of each of the following PKA targets is responsive to adenosine A2A receptor stimulation in this tissue: Ser40 of tyrosine hydroxylase, Ser9 of synapsin, Ser897 of the NR1 subunit of the N-methyl-D-aspartate-type glutamate receptor, Ser845 of the GluR1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptor, Ser94 of spinophilin, Thr34 of the dopamine- and cAMP-regulated phosphoprotein, Mr32,000, Ser133 of the cAMP-response element-binding protein, Thr286 of Ca2+/calmodulin-dependent protein kinase II, and Thr202/Tyr204 and Thr183/Tyr185 of the p44 and p42 isoforms, respectively, of mitogen-activated protein kinase. Although the substrates studied differed considerably in their responsiveness to selective adenosine A2A receptor activation, the phosphorylation state of all postsynaptic PKA targets was up-regulated in a time- and dose-dependent manner by treatment with CGS 21680, whereas presynaptic PKA

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

    PubMed

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

    2005-11-01

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

  20. Evaluation of neuronal phosphoproteins as effectors of caffeine and mediators of striatal adenosine A2A receptor signaling.

    PubMed

    Sahin, Bogachan; Galdi, Stacey; Hendrick, Joseph; Greene, Robert W; Snyder, Gretchen L; Bibb, James A

    2007-01-19

    Adenosine A(2A) receptors are predominantly expressed in the dendrites of enkephalin-positive gamma-aminobutyric acidergic medium spiny neurons in the striatum. Evidence indicates that these receptors modulate striatal dopaminergic neurotransmission and regulate motor control, vigilance, alertness, and arousal. Although the physiological and behavioral correlates of adenosine A(2A) receptor signaling have been extensively studied using a combination of pharmacological and genetic tools, relatively little is known about the signal transduction pathways that mediate the diverse biological functions attributed to this adenosine receptor subtype. Using a candidate approach based on the coupling of these receptors to adenylate cyclase-activating G proteins, a number of membranal, cytosolic, and nuclear phosphoproteins regulated by PKA were evaluated as potential mediators of adenosine A(2A) receptor signaling in the striatum. Specifically, the adenosine A(2A) receptor agonist, CGS 21680, was used to determine whether the phosphorylation state of each of the following PKA targets is responsive to adenosine A(2A) receptor stimulation in this tissue: Ser40 of tyrosine hydroxylase, Ser9 of synapsin, Ser897 of the NR1 subunit of the N-methyl-d-aspartate-type glutamate receptor, Ser845 of the GluR1 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptor, Ser94 of spinophilin, Thr34 of the dopamine- and cAMP-regulated phosphoprotein, M(r) 32,000, Ser133 of the cAMP-response element-binding protein, Thr286 of Ca(2+)/calmodulin-dependent protein kinase II, and Thr202/Tyr204 and Thr183/Tyr185 of the p44 and p42 isoforms, respectively, of mitogen-activated protein kinase. Although the substrates studied differed considerably in their responsiveness to selective adenosine A(2A) receptor activation, the phosphorylation state of all postsynaptic PKA targets was up-regulated in a time- and dose-dependent manner by treatment with CGS 21680

  1. 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

  2. Histamine receptors on adult rat cardiomyocytes: antagonism of alpha/sub 1/-receptor stimulation of cAMP degradation

    SciTech Connect

    Buxton, I.L.O.; Bowen, S.M.

    1986-03-01

    Incubation of intact cardiomyocytes with the histamine antagonist (/sup 3/H)mepyramine results in rapid reversible binding to a single class of high affinity sites (K/sub D/ = 1.2nM; 50,000 sites/myocyte). In membranes from purified myocytes histamine competition of (/sup 3/H)mepyramine binding (K/sub D/ = 300nM) is not altered by GTP (10..mu..M). Competition of (/sup 3/H)mepyramine binding by H-receptor subtype-selective antagonists suggests the presence of a single class of H/sub 1/-receptors. Incubation of intact myocytes with histamine (luM, H/sub 1/ receptor activation) plus norepinephrine (NE 1uM, alpha/sub 1/ + beta/sub 1/ receptor activation) for 3 min leads to significantly more cAMP accumulation (36.5 pmol/10/sup 6/ myocytes) than NE alone (30 pmol/10/sup 6/ myocytes). Histamine alone does not alter basal cAMP = 10.4 pmol/10/sup 6/ myocytes, or beta/sub 1/ stimulation (isoproternol, 1uM) = 39.6 pmol/10/sup 6/ myocytes. Cyclic AMP accumulation with NE plus prazosin 10nM, (alpha/sub 1/ + beta/sub 1/ + alpha/sub 1/ blockade) is indistinguishable from NE + histamine, (alpha/sub 1/ + beta/sub 1/ + H/sub 1/) stimulation. Histamine competition for (/sup 3/H)prazosin binding suggests that histamine does not block alpha/sub 1/ receptors on the myocyte. These data suggest that H/sub 1/ receptor activation leads to antagonism of the alpha/sub 1/ receptor mediated activation of cAMP phosphodiesterase the authors have recently described.

  3. 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

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

    PubMed

    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

    2015-03-01

    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 (also known as Arg3.1), an immediate-early gene that is required for long-term memory. Chemogenetic activation of astrocytic Gs-coupled signaling reduced long-term memory in mice without affecting learning. Like 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

  5. Stabilizing effects of G protein on the active conformation of adenosine A1 receptor differ depending on G protein type.

    PubMed

    Tateyama, Michihiro; Kubo, Yoshihiro

    2016-10-01

    G protein coupled receptors (GPCRs) trigger various cellular and physiological responses upon the ligand binding. The ligand binding induces conformational change in GPCRs which allows G protein to interact with the receptor. The interaction of G protein also affects the active conformation of GPCRs. In this study, we have investigated the effects of Gαi1, Gαo and chimeric Gαqi5 on the active conformation of the adenosine A1 receptor, as each Gα showed difference in the interaction with adenosine A1 receptor. The conformational changes in the adenosine A1 receptor were detected as the agonist-induced decreases in efficiency of Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) fused at the two intracellular domains of the adenosine A1 receptor. Amplitudes of the agonist-induced FRET decreases were subtle when the FP-tagged adenosine A1 receptor was expressed alone, whereas they were significantly enhanced when co-expressed with Gαi1Gβ1Gγ22 (Gi1) or Gαqi5Gβ1Gγ22 (Gqi5) but not with GαοGβ1Gγ22 (Go). The enhancement of the agonist-induced FRET decrease in the presence of Gqi5 was significantly larger than that of Gi1. Furthermore, the FRET recovery upon the agonist removal in the presence of Gqi5 was significantly slower than that of Gi1. From these results it was revealed that the agonist-bound active conformation of adenosine A1 receptor is unstable without the binding of G protein and that the stabilizing effects of G protein differ depending on the types of G protein.

  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. 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.

  8. Contribution of Adenosine A2B Receptors in Clostridium difficile Intoxication and Infection

    PubMed Central

    Li, Yuesheng; Calabrese, Gina M.; Freire, Rosemayre S.; Zaja-Milatovic, Snjezana; van Opstal, Edward; Figler, Robert A.; Linden, Joel; Guerrant, Richard L.

    2012-01-01

    Clostridium difficile toxins A (TcdA) and B (TcdB) induce a pronounced systemic and intestinal inflammatory response. A2B adenosine receptors (A2BARs) are the predominant adenosine receptors in the intestinal epithelium. We investigated whether A2BARs are upregulated in human intestinal cells by TcdA or TcdB and whether blockade of A2BARs can ameliorate C. difficile TcdA-induced enteritis and alter the outcome of C. difficile infection (CDI). Adenosine receptor subtype (A1, A2A, A2B, and A3) mRNAs were assayed in HCT-8 cells. Ileal loops from wild-type rabbits and mice and A2BAR−/− mice were treated with TcdA, with or without the selective A2BAR antagonist ATL692 or PSB1115. A murine model of CDI was used to determine the effect of A2BAR deletion or blockade with the orally available agent ATL801, on clinical outcome, histopathology and intestinal interleukin-6 (IL-6) expression from infection. TcdA and TcdB upregulated A2BAR gene expression in HCT-8 cells. ATL692 decreased TcdA-induced secretion and epithelial injury in rabbit ileum. Deletion of A2BARs reduced secretion and histopathology in TcdA-challenged mouse ileum. Deletion or blockade of A2BARs reduced histopathology, IL-6 expression, weight loss, diarrhea, and mortality in C. difficile-infected mice. A2BARs mediate C. difficile toxin-induced enteritis and disease. Inhibition of A2BAR activation may be a potential strategy to limit morbidity and mortality from CDI. PMID:23045479

  9. [Mast cells, their adenosine receptors and reactive oxygen species in chronic inflammatory pathologies of childhood].

    PubMed

    Renke, Joanna; Popadiuk, Stefan; Wozniak, Michał; Szlagatys-Sidorkiewicz, Agnieszka; Hansdorfer-Korzon, Rita

    2006-01-01

    Mast cells were described by Erhlich at the end of XIX-th century. Their role was deeply investigated in asthma and allergy. The massive degranulation of mast cells in allergy can lead to anaphylactic shock. Recently, mast cells have been recognized again as a very interesting topic for investigation, due to their possible role in chronic inflammation. Moreover, through adenosine receptors, mast cells can be activated or inactivated. That is why these cells are regarded as a potential target of new drugs. It has been reported, that mast cells generate intracellular reactive oxygen species (ROS) in response to stimulation with divergent physiologically relevant stimulants. The intensification of ROS production may be measured by the level of carbonyl groups, as a marker of protein peroxidation. However, the role of mast cells in other than asthma diseases with chronic inflammation needs further investigation. It was found out that the information about mast cell distribution in colonic mucosa may serve as help in differentiation between inflammatory bowel disease and collagenous colitis. Moreover, its accumulation in focal active gastritis was confirmed in patients with Crohn's disease. An important role in regulation of inflammatory process seems to be reserved for adenosine receptors present on mastocytes. The activation of mast cells through the adenosine receptor is connected with 11-8 release, which stimulate the migration of leukocytes and oxidation reactions. The detection of mast cells in tissues should not be limited only to the simple histologic examination. It should be completed by the detection of products of degranulation, e.g. tryptase. This is the way to find out their actual function and state of activation. PMID:17203808

  10. Activation of NTS A2a adenosine receptors differentially resets baroreflex control of renal vs. adrenal sympathetic nerve activity.

    PubMed

    Ichinose, Tomoko K; O'Leary, Donal S; Scislo, Tadeusz J

    2009-04-01

    The role of nucleus of solitary tract (NTS) A(2a) adenosine receptors in baroreflex mechanisms is controversial. Stimulation of these receptors releases glutamate within the NTS and elicits baroreflex-like decreases in mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA), whereas inhibition of these receptors attenuates HR baroreflex responses. In contrast, stimulation of NTS A(2a) adenosine receptors increases preganglionic adrenal sympathetic nerve activity (pre-ASNA), and the depressor and sympathoinhibitory responses are not markedly affected by sinoaortic denervation and blockade of NTS glutamatergic transmission. To elucidate the role of NTS A(2a) adenosine receptors in baroreflex function, we compared full baroreflex stimulus-response curves for HR, RSNA, and pre-ASNA (intravenous nitroprusside/phenylephrine) before and after bilateral NTS microinjections of selective adenosine A(2a) receptor agonist (CGS-21680; 2.0, 20 pmol/50 nl), selective A(2a) receptor antagonist (ZM-241385; 40 pmol/100 nl), and nonselective A(1) + A(2a) receptor antagonist (8-SPT; 1 nmol/100 nl) in urethane/alpha-chloralose anesthetized rats. Activation of A(2a) receptors decreased the range, upper plateau, and gain of baroreflex-response curves for RSNA, whereas these parameters all increased for pre-ASNA, consistent with direct effects of the agonist on regional sympathetic activity. However, no resetting of baroreflex-response curves along the MAP axis occurred despite the marked decreases in baseline MAP. The antagonists had no marked effects on baseline variables or baroreflex-response functions. We conclude that the activation of NTS A(2a) adenosine receptors differentially alters baroreflex control of HR, RSNA, and pre-ASNA mostly via non-baroreflex mechanism(s), and these receptors have virtually no tonic action on baroreflex control of these sympathetic outputs.

  11. 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

  12. 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.

  13. Homodimerization of adenosine A₁ receptors in brain cortex explains the biphasic effects of caffeine.

    PubMed

    Gracia, Eduard; Moreno, Estefania; Cortés, Antoni; Lluís, Carme; Mallol, Josefa; McCormick, Peter J; Canela, Enric I; Casadó, Vicent

    2013-08-01

    Using bioluminescence resonance energy transfer and proximity ligation assays, we obtained the first direct evidence that adenosine A₁ receptors (A₁Rs) form homomers not only in cell cultures but also in brain cortex. By radioligand binding experiments in the absence or in the presence of the A₁Rs allosteric modulator, adenosine deaminase, and by using the two-state dimer receptor model to fit binding data, we demonstrated that the protomer-protomer interactions in the A₁R homomers account for some of the pharmacological characteristics of agonist and antagonist binding to A₁Rs. These pharmacological properties include the appearance of cooperativity in agonist binding, the change from a biphasic saturation curve to a monophasic curve in self-competition experiments and the molecular cross-talk detected when two different specific molecules bind to the receptor. In this last case, we discovered that caffeine binding to one protomer increases the agonist affinity for the other protomer in the A₁R homomer, a pharmacological characteristic that correlates with the low caffeine concentrations-induced activation of agonist-promoted A₁R signaling. This pharmacological property can explain the biphasic effects reported at low and high concentration of caffeine on locomotor activity.

  14. Quantitative insights towards the design of potent deazaxanthine antagonists of adenosine 2B receptors.

    PubMed

    Paz, Odailson Santos; Brito, Camila Carane Bitencourt; Castilho, Marcelo Santos

    2014-08-01

    Adenosine receptors have been considered as potential targets for drug development, but one of the main obstacles to this goal is to selectively inhibit one receptor subtype over the others. This subject is particularly crucial for adenosine A2b receptor antagonists (AdoRA2B). The structure–activity relationships of xanthine derivatives which are AdoRA2B have been comprehensively investigated, but the steric and electronic requirements of deazaxanthine AdoRA2B have not been described from a quantitative standpoint of view. Herein we report our efforts to shorten this knowledge gap through 2D-QSAR (HQSAR) and 3D-QSAR (CoMFA) approaches. The good statistical quality (HQSAR--r(2) = 0.85, q(2)(LOO) = 0.77; CoMFA – r(2) = 0.86, q(2) = 0.70) and predictive ability (r(2) = (pred1) = 0.78, r(2)(pred2) = 0.78 and r(2) = (pred1) = 0.70, r(2) = (pred2) = 0.70,respectively) of the models, along with the information provided by contribution and contour maps hints their usefulness to the design of more potent 9-deazaxanthine derivatives.

  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. 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.

  17. /sup 125/I-labeled 8-phenylxanthine derivatives: antagonist radioligands for adenosine A1 receptors

    SciTech Connect

    Linden, J.; Patel, A.; Earl, C.Q.; Craig, R.H.; Daluge, S.M.

    1988-04-01

    A series of 8-phenylxanthine derivatives has been synthesized with oxyacetic acid on the para phenyl position to increase aqueous solubility and minimize nonspecific binding and iodinatable groups on the 1- or 3-position of the xanthine ring. The structure-activity relationship for binding of these compounds to A1 adenosine receptors of bovine and rat brain and A2 receptors of human platelets was examined. The addition of arylamine or photosensitive aryl azide groups to the 3-position of xanthine had little effect on A1 binding affinity with or without iodination, whereas substitutions at the 1-position caused greatly reduced A1 binding affinity. The addition of an aminobenzyl group to the 3-position of the xanthine had little effect on A2 binding affinity, but 3-aminophenethyl substitution decreased A2 binding affinity. Two acidic 3-(arylamino)-8-phenylxanthine derivatives were labeled with /sup 125/I and evaluated as A1 receptor radioligands. The new radioligands bound to A1 receptors with KD values of 1-1.25 nM. Specific binding represented over 80% of total binding. High concentrations of NaCl or other salts increased the binding affinity of acidic but not neutral antagonists, suggesting that interactions between ionized xanthines and receptors may be affected significantly by changes in ionic strength. On the basis of binding studies with these antagonists and isotope dilution with the agonist (/sup 125/I)N6-(4-amino-3-iodobenzyl)adenosine, multiple agonist affinity states of A1 receptors have been identified.

  18. A type III effector antagonizes death receptor signalling during bacterial gut infection.

    PubMed

    Pearson, Jaclyn S; Giogha, Cristina; Ong, Sze Ying; Kennedy, Catherine L; Kelly, Michelle; Robinson, Keith S; Lung, Tania Wong Fok; Mansell, Ashley; Riedmaier, Patrice; Oates, Clare V L; Zaid, Ali; Mühlen, Sabrina; Crepin, Valerie F; Marches, Olivier; Ang, Ching-Seng; Williamson, Nicholas A; O'Reilly, Lorraine A; Bankovacki, Aleksandra; Nachbur, Ueli; Infusini, Giuseppe; Webb, Andrew I; Silke, John; Strasser, Andreas; Frankel, Gad; Hartland, Elizabeth L

    2013-09-12

    Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.

  19. Antagonism of the prostaglandin E receptor EP4 inhibits metastasis and enhances NK function.

    PubMed

    Kundu, Namita; Ma, Xinrong; Holt, Dawn; Goloubeva, Olga; Ostrand-Rosenberg, Suzanne; Fulton, Amy M

    2009-09-01

    Cyclooxygenase-2 (COX-2) is associated with aggressive breast cancers. The COX-2 product prostaglandin E(2) (PGE(2)) acts through four G-protein-coupled receptors designated EP1-4. Malignant and immortalized normal mammary epithelial cell lines express all four EP. The EP4 antagonist AH23848 reduced the ability of tumor cells to colonize the lungs or to spontaneously metastasize from the mammary gland. EP4 gene silencing by shRNA also reduced the ability of mammary tumor cells to metastasize. Metastasis inhibition was lost in mice lacking either functional Natural Killer (NK) cells or interferon-gamma. EP4 antagonism inhibited MHC class I expression resulting in enhanced ability of NK cells to lyse mammary tumor target cells. These studies support the hypothesis that EP4 receptor antagonists reduce metastatic potential by facilitating NK-mediated tumor cell killing and that therapeutic targeting of EP4 may be an alternative approach to the use of COX inhibitors to limit metastatic disease.

  20. Hypocretin receptor 2 antagonism dose-dependently reduces escalated heroin self-administration in rats.

    PubMed

    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

    2015-03-13

    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.

  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. Adenosine receptors located in the NTS contribute to renal sympathoinhibition during hypotensive phase of severe hemorrhage in anesthetized rats.

    PubMed

    Scislo, Tadeusz J; O'Leary, Donal S

    2006-11-01

    Stimulation of nucleus of the solitary tract (NTS) A(2a)-adenosine receptors elicits cardiovascular responses quite similar to those observed with rapid, severe hemorrhage, including bradycardia, hypotension, and inhibition of renal but activation of preganglionic adrenal sympathetic nerve activity (RSNA and pre-ASNA, respectively). Because adenosine levels in the central nervous system increase during severe hemorrhage, we investigated to what extent these responses to hemorrhage may be due to activation of NTS adenosine receptors. In urethane- and alpha-chloralose-anesthetized male Sprague-Dawley rats, rapid hemorrhage was performed before and after bilateral nonselective or selective blockade of NTS adenosine-receptor subtypes [A(1)- and A(2a)-adenosine-receptor antagonist 8-(p-sulfophenyl)theophylline (1 nmol/100 nl) and A(2a)-receptor antagonist ZM-241385 (40 pmol/100 nl)]. The nonselective blockade reversed the response in RSNA (-21.0 +/- 9.6 Delta% vs. +7.3 +/- 5.7 Delta%) (where Delta% is averaged percent change from baseline) and attenuated the average heart rate response (change of -14.8 +/- 4.8 vs. -4.4 +/- 3.4 beats/min). The selective blockade attenuated the RSNA response (-30.4 +/- 5.2 Delta% vs. -11.1 +/- 7.7 Delta%) and tended to attenuate heart rate response (change of -27.5 +/- 5.3 vs. -15.8 +/- 8.2 beats/min). Microinjection of vehicle (100 nl) had no significant effect on the responses. The hemorrhage-induced increases in pre-ASNA remained unchanged with either adenosine-receptor antagonist. We conclude that adenosine operating in the NTS via A(2a) and possibly A(1) receptors may contribute to posthemorrhagic sympathoinhibition of RSNA but not to the sympathoactivation of pre-ASNA. The differential effects of NTS adenosine receptors on RSNA vs. pre-ASNA responses to hemorrhage supports the hypothesis that these receptors are differentially located/expressed on NTS neurons/synaptic terminals controlling different sympathetic outputs.

  3. Novel opioid cyclic tetrapeptides: Trp isomers of CJ-15,208 exhibit distinct opioid receptor agonism and short-acting κ opioid receptor antagonism

    PubMed Central

    Ross, Nicolette C; Reilley, Kate J; Murray, Thomas F; Aldrich, Jane V; McLaughlin, Jay P

    2012-01-01

    BACKGROUND AND PURPOSE The κ opioid receptor antagonists demonstrate potential for maintaining abstinence from psychostimulant abuse, but existing non-peptide κ-receptor selective antagonists show exceptionally long activity. We hypothesized that the L- and D-Trp isomers of CJ-15,208, a natural cyclic tetrapeptide reported to be a κ-receptor antagonist in vitro, would demonstrate short-acting, dose-dependent antagonism in vivo, preventing reinstatement of cocaine-seeking behaviour. EXPERIMENTAL APPROACH Affinity, selectivity and efficacy of the L-Trp and D-Trp isomers for opioid receptors were assessed in vitro in radioligand and GTPγS binding assays. Opioid receptor agonist and antagonist activities were characterized in vivo following i.c.v. administration with the 55°C warm water tail-withdrawal assay. The D-Trp isomer, which demonstrated primarily κ-receptor selective antagonist activity, was further evaluated for its prevention of stress- and drug-induced reinstatement of extinguished cocaine conditioned place preference (CPP). KEY RESULTS The two isomers showed similar affinity and selectivity for κ receptors (Ki 30–35 nM) as well as κ receptor antagonism in vitro. As expected, the D-Trp cyclic tetrapeptide exhibited minimal agonist activity and induced dose-dependent κ-receptor selective antagonism lasting less than 18 h in vivo. Pretreatment with this peptide prevented stress-, but not cocaine-induced, reinstatement of extinguished cocaine CPP. In contrast, the L-Trp cyclic tetrapeptide unexpectedly demonstrated mixed opioid agonist/antagonist activity. CONCLUSIONS AND IMPLICATIONS The L-Trp and the D-Trp isomers of CJ-15,208 demonstrate stereospecific opioid activity in vivo. The relatively brief κ opioid receptor antagonism, coupled with the prevention of stress-induced reinstatement of extinguished cocaine-seeking behaviour, suggests the D-Trp isomer could be used therapeutically to maintain abstinence from psychostimulant abuse. PMID

  4. Postsynaptic Adenosine A2A Receptors Modulate Intrinsic Excitability of Pyramidal Cells in the Rat Basolateral Amygdala

    PubMed Central

    Rau, Andrew R.; Ariwodola, Olusegun J.

    2015-01-01

    Background: The basolateral amygdala plays a critical role in the etiology of anxiety disorders and addiction. Pyramidal neurons, the primary output cells of this region, display increased firing following exposure to stressors, and it is thought that this increase in excitability contributes to stress responsivity and the expression of anxiety-like behaviors. However, much remains unknown about the underlying mechanisms that regulate the intrinsic excitability of basolateral amygdala pyramidal neurons. Methods: Ex vivo gramicidin perforated patch recordings were conducted in current clamp mode where hyper- and depolarizing current steps were applied to basolateral amygdala pyramidal neurons to assess the effects of adenosine A2A receptor modulation on intrinsic excitability. Results: Activation of adenosine A2A receptors with the selective A2A receptor agonist CGS-21680 significantly increased the firing rate of basolateral amygdala pyramidal neurons in rat amygdala brain slices, likely via inhibition of the slow afterhyperpolarization potential. Both of these A2A receptor-mediated effects were blocked by preapplication of a selective A2A receptor antagonist (ZM-241385) or by intra-pipette infusion of a protein kinase A inhibitor, suggesting a postsynaptic locus of A2A receptors on basolateral amygdala pyramidal neurons. Interestingly, bath application of the A2A receptor antagonist alone significantly attenuated basolateral amygdala pyramidal cell firing, consistent with a role for tonic adenosine in the regulation of the intrinsic excitability of these neurons. Conclusions: Collectively, these data suggest that adenosine, via activation of A2A receptors, may directly facilitate basolateral amygdala pyramidal cell output, providing a possible balance for the recently described inhibitory effects of adenosine A1 receptor activation on glutamatergic excitation of basolateral amygdala pyramidal cells. PMID:25716780

  5. 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

  6. Using caffeine and other adenosine receptor antagonists and agonists as therapeutic tools against neurodegenerative diseases: a review.

    PubMed

    Rivera-Oliver, Marla; Díaz-Ríos, Manuel

    2014-04-17

    Caffeine is the most consumed pychostimulant in the world, and it is known to affect basic and fundamental human processes such as sleep, arousal, cognition and learning and memory. It works as a nonselective blocker of adenosine receptors (A1, A2a, A2b and A3) and has been related to the regulation of heart rate, the contraction/relaxation of cardiac and smooth muscles, and the neural signaling in the central nervous system (CNS). Since the late 1990s, studies using adenosine receptor antagonists, such as Caffeine, to block the A1 and A2a adenosine receptor subtypes have shown to reduce the physical, cellular and molecular damages caused by a spinal cord injury (SCI) or a stroke (cerebral infarction) and by other neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. Interestingly, other studies using adenosine receptor agonists have also shown to provide a neuroprotective effect on various models of neurodegenerative diseases through the reduction of excitatory neurotransmitter release, apoptosis and inflammatory responses, among others. The seemingly paradoxical use of both adenosine receptor agonists and antagonists as neuroprotective agents has been attributed to differences in dosage levels, drug delivery method, extracellular concentration of excitatory neurotransmitters and stage of disease progression. We discuss and compare recent findings using both antagonists and agonists of adenosine receptors in animal models and patients that have suffered spinal cord injuries, brain strokes, and Parkinson's and Alzheimer's diseases. Additionally, we propose alternative interpretations on the seemingly paradoxical use of these drugs as potential pharmacological tools to treat these various types of neurodegenerative diseases.

  7. Regional vascular responses to ATP and ATP analogues in the rabbit kidney in vivo: roles for adenosine receptors and prostanoids

    PubMed Central

    Eppel, G A; Ventura, S; Evans, R G

    2006-01-01

    Background and purpose: Our knowledge of the effects of P2-receptor activation on renal vascular tone comes mostly from in vitro models. We aimed to characterise the pharmacology of ATP in the renal circulation in vivo. Experimental approach: In pentobarbitone anaesthetized rabbits, we examined total renal and medullary vascular responses to ATP (0.2 and 0.8 mg kg-1), β,γ-methylene ATP (β,γ-mATP, 7 and 170 μg kg-1), α,β-mATP (0.2 and 2 μg kg-1) and adenosine (2 and 6 μg kg-1) using transit-time ultrasound and laser Doppler flowmetry, respectively. We also determined whether adenosine receptors, NO or prostanoids contribute to the actions of the purinoceptor agonists. Key results: Renal arterial boluses of ATP, β,γ-mATP, and adenosine produced biphasic changes; ischaemia followed by hyperaemia, in total renal and medullary blood flow. α,β-mATP induced only ischaemia. The adenosine receptor antagonist 8-(p-sulphophenyl)theophylline reduced the responses to adenosine and the hyperaemic responses to ATP and β,γ-mATP only. NO synthase inhibition (Nω-nitro-L-arginine) did not significantly alter responses to the P2 receptor agonists. Subsequent cyclooxygenase inhibition (ibuprofen) reduced the ATP- and β,γ-mATP-induced increases in renal blood flow. All other responses remained unchanged. Conclusions and implications: In the rabbit kidney in vivo, α,β-mATP sensitive receptors mediate vasoconstriction. β,γ-mATP and ATP induce vasodilation at least partly through adenosine receptors. ATP induced renal vasodilatation is independent of NO and partly dependent on prostanoids in the bulk of the kidney, but not in the vasculature controlling medullary blood flow. PMID:16981003

  8. Using caffeine and other adenosine receptor antagonists and agonists as therapeutic tools against neurodegenerative diseases: a review.

    PubMed

    Rivera-Oliver, Marla; Díaz-Ríos, Manuel

    2014-04-17

    Caffeine is the most consumed pychostimulant in the world, and it is known to affect basic and fundamental human processes such as sleep, arousal, cognition and learning and memory. It works as a nonselective blocker of adenosine receptors (A1, A2a, A2b and A3) and has been related to the regulation of heart rate, the contraction/relaxation of cardiac and smooth muscles, and the neural signaling in the central nervous system (CNS). Since the late 1990s, studies using adenosine receptor antagonists, such as Caffeine, to block the A1 and A2a adenosine receptor subtypes have shown to reduce the physical, cellular and molecular damages caused by a spinal cord injury (SCI) or a stroke (cerebral infarction) and by other neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. Interestingly, other studies using adenosine receptor agonists have also shown to provide a neuroprotective effect on various models of neurodegenerative diseases through the reduction of excitatory neurotransmitter release, apoptosis and inflammatory responses, among others. The seemingly paradoxical use of both adenosine receptor agonists and antagonists as neuroprotective agents has been attributed to differences in dosage levels, drug delivery method, extracellular concentration of excitatory neurotransmitters and stage of disease progression. We discuss and compare recent findings using both antagonists and agonists of adenosine receptors in animal models and patients that have suffered spinal cord injuries, brain strokes, and Parkinson's and Alzheimer's diseases. Additionally, we propose alternative interpretations on the seemingly paradoxical use of these drugs as potential pharmacological tools to treat these various types of neurodegenerative diseases. PMID:24530739

  9. Structure Based Prediction of Subtype-Selectivity for Adenosine Receptor Antagonists

    PubMed Central

    Katritch, Vsevolod; Kufareva, Irina; Abagyan, Ruben

    2010-01-01

    One of the major hurdles in the development of safe and effective drugs targeting G-protein coupled receptors (GPCRs) is finding ligands that are highly selective for a specific receptor subtype. Structural understanding of subtype-specific binding pocket variations and ligand-receptor interactions may greatly facilitate design of selective ligands. To gain insights into the structural basis of ligand subtype selectivity within the family of adenosine receptors (AR: A1, A2A, A2B, and A3) we generated 3D models of all four subtypes using the recently determined crystal structure of the AA2AR as a template, and employing the methodology of ligand-guided receptor optimization for refinement. This approach produced 3D conformational models of AR subtypes that effectively explain binding modes and subtype selectivity for a diverse set of known AR antagonists. Analysis of the subtype-specific ligand-receptor interactions allowed identification of the major determinants of ligand selectivity, which may facilitate discovery of more efficient drug candidates. PMID:20637786

  10. 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

  11. Adenosine A2a receptors form distinct oligomers in protein detergent complexes.

    PubMed

    Schonenbach, Nicole S; Rieth, Monica D; Han, Songi; O'Malley, Michelle A

    2016-09-01

    The human adenosine A2a receptor (A2aR) tunes its function by forming homo-oligomers and hetero-oligomers with other G protein-coupled receptors, but the biophysical characterization of these oligomeric species is limited. Here, we show that upon reconstitution into an optimized mixed micelle system, and purification via an antagonist affinity column, full-length A2aR exists as a distribution of oligomers. We isolated the dimer population from the other oligomers via size exclusion chromatography and showed that it is stable upon dilution, thus supporting the hypotheses that the A2aR dimer has a defined structure and function. This study presents a crucial enabling step to a detailed biophysical characterization of A2aR homodimers. PMID:27543907

  12. The imbalanced expression of adenosine receptors in an epilepsy model corrected using targeted mesenchymal stem cell transplantation.

    PubMed

    Huicong, Kang; Zheng, Xue; Furong, Wang; Zhouping, Tang; Feng, Xu; Qi, Hu; Xiaoyan, Liu; Xiaojiang, Huang; Na, Zhang; Ke, Xu; Zheng, Zeng; Suiqiang, Zhu

    2013-12-01

    Adenosine inhibits epileptic episodes by interacting with G-protein-coupled receptors. This study examined the mechanism by which the inhibitory effect of adenosine becomes impaired during epileptogenesis. Dynamic changes in adenosine A1 receptors (A1Rs) and A2a receptors (A2aRs) were investigated in a kindling model of epilepsy. RT-PCR, Western blotting, and immunofluorescence results indicated that expression of A1Rs was increased in the hippocampus 24 h after kindling, but progressively decreased 1 and 6 months after kindling. Opposite changes were seen in the expression of A2aRs. This bidirectional change resulted in an imbalance between A1Rs and A2aRs and dysregulation of the adenosine system. Autologous mesenchymal stem cell (MSC) transplantation was used to correct this disorder and avoid side effects of systematic adenosine therapy. Paramagnetic iron oxide particles were used to mark and track the MSCs in vivo using MRI. The results indicated that the transplanted cells migrated along the callosum and settled at the ependymal layer. The MSCs displayed a relatively long survival time, at least 3 months. The improved AR expression and EEG findings suggested that MSC transplantation was a potentially effective means of treating refractory epilepsy.

  13. The stimulant effects of caffeine on locomotor behaviour in mice are mediated through its blockade of adenosine A2A receptors

    PubMed Central

    Yacoubi, Malika El; Ledent, Catherine; Ménard, Jean-François; Parmentier, Marc; Costentin, Jean; Vaugeois, Jean-Marie

    2000-01-01

    The locomotor stimulatory effects induced by caffeine (1,3,7-trimethylxanthine) in rodents have been attributed to antagonism of adenosine A1 and A2A receptors. Little is known about its locomotor depressant effects seen when acutely administered at high doses. The roles of adenosine A1 and A2A receptors in these activities were investigated using a Digiscan actimeter in experiments carried out in mice. Besides caffeine, the A2A antagonist SCH 58261 (5-amino-7-(β-phenylethyl)-2-(8-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine), the A1 antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine), the A1 agonist CPA (N6-cyclopentyladenosine) and A2A receptor knockout mice were used.Caffeine had a biphasic effect on locomotion of wild-type mice not habituated to the open field, stimulating locomotion at 6.25–25 mg kg−1 i.p. doses, while depressing it at 100 mg kg−1. In sharp contrast, caffeine dose-dependently decreased locomotion in A2A receptor knockout mice over the whole range of tested doses.The depressant effects induced by high doses of caffeine were lost in control CD1 mice habituated to the open field.The A1 agonist CPA depressed locomotion at 0.3–1 mg kg−1 i.p. doses.The A1 antagonist DPCPX decreased locomotion of A2A receptor knockouts and CD1 mice at 5 mg kg−1 i.p. and 25 mg kg−1 i.p. respectively.DPCPX (0.2–1 mg kg−1 i.p.) left unaltered or even reduced the stimulant effect of SCH 58261 (1–3 mg kg−1 i.p.) on CD1 mice.These results suggest therefore that the stimulant effect of low doses of caffeine is mediated by A2A receptor blockade while the depressant effect seen at higher doses under some conditions is explained by A1 receptor blockade. PMID:10742303

  14. Mechanisms mediating regional sympathoactivatory responses to stimulation of NTS A(1) adenosine receptors.

    PubMed

    Scislo, Tadeusz J; O'Leary, Donal S

    2002-10-01

    Selective activation of adenosine A(1) and A(2a) receptors in the subpostremal nucleus tractus solitarius (NTS) increases and decreases mean arterial pressure (MAP), respectively, and decreases heart rate (HR). We have previously shown that the decreases in MAP evoked by NTS A(2a) receptor stimulation were accompanied with differential sympathetic responses in renal (RSNA), lumbar (LSNA), and preganglionic adrenal sympathetic nerve activity (pre-ASNA). Therefore, now we investigated whether stimulation of NTS A(1) receptors via unilateral microinjection of N(6)-cyclopentyladenosine (CPA) elicits differential activation of the same sympathetic outputs in alpha-chloralose-urethane-anesthetized male Sprague-Dawley rats. CPA (0.33-330.0 pmol in 50 nl) evoked dose-dependent increases in MAP, variable decreases in HR, and differential increases in all recorded sympathetic outputs: upward arrow pre-ASNA > upward arrow RSNA > or = upward arrow LSNA. Sinoaortic denervation + vagotomy abolished the MAP and LSNA responses, reversed the normal increases in RSNA into decreases, and significantly attenuated increases in pre-ASNA. NTS ionotropic glutamatergic receptor blockade with kynurenate sodium (4.4 nmol/100 nl) reversed the responses in MAP, LSNA, and RSNA and attenuated the responses in pre-ASNA. We conclude that afferent inputs and intact glutamatergic transmission in the NTS are necessary to mediate the pressor and differential sympathoactivatory responses to stimulation of NTS A(1) receptors.

  15. Functional expression of adenosine A2A and A3 receptors in the mouse dendritic cell line XS-106.

    PubMed

    Dickenson, John M; Reeder, Steve; Rees, Bob; Alexander, Steve; Kendall, Dave

    2003-08-01

    There is increasing evidence to suggest that adenosine receptors can modulate the function of cells involved in the immune system. For example, human dendritic cells derived from blood monocytes have recently been described to express functional adenosine A1, A2A and A3 receptors. Therefore, in the present study, we have investigated whether the recently established murine dendritic cell line XS-106 expresses functional adenosine receptors. The selective adenosine A3 receptor agonist 1-[2-chloro-6[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-beta-D-ribofuranuronamide (2-Cl-IB-MECA) inhibited forskolin-mediated [3H]cyclic AMP accumulation and stimulated concentration-dependent increases in p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation. The selective adenosine A2A receptor agonist 4-[2-[[-6-amino-9-(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene-propanoic acid (CGS 21680) stimulated a robust increase in [3H]cyclic AMP accumulation and p42/p44 MAPK phosphorylation. In contrast, the selective adenosine A1 receptor agonist CPA (N6-cyclopentyladenosine) did not inhibit forskolin-mediated [3H]cyclic AMP accumulation or stimulate increases in p42/p44 MAPK phosphorylation. These observations suggest that XS-106 cells express functional adenosine A2A and A3 receptors. The non-selective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) inhibited lipopolysaccharide-induced tumour necrosis factor-alpha (TNF-alpha) release from XS-106 cells in a concentration-dependent fashion. Furthermore, treatment with Cl-IB-MECA (1 microM) or CGS 21680 (1 microM) alone produced a partial inhibition of lipopolysaccharide-induced TNF-alpha release (when compared to NECA), whereas a combination of both agonists resulted in the inhibition of TNF-alpha release comparable to that observed with NECA alone. Treatment of cells with the adenosine A2A receptor selective antagonists 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a

  16. The role of adenosine A2A and A3 receptors on the differential modulation of norepinephrine and neuropeptide Y release from peripheral sympathetic nerve terminals.

    PubMed

    Donoso, M Verónica; Aedo, Felipe; Huidobro-Toro, J Pablo

    2006-03-01

    The pre-synaptic sympathetic modulator role of adenosine was assessed by studying transmitter release following electrical depolarization of nerve endings from the rat mesenteric artery. Mesentery perfusion with exogenous adenosine exclusively inhibited the release of norepinephrine (NA) but did not affect the overflow of neuropeptide Y (NPY), establishing the basis for a differential pre-synaptic modulator mechanism. Several adenosine structural analogs mimicked adenosine's effect on NA release and their relative order of potency was: 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride = 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-beta-d-ribofuranuronamide = 5'-(N-ethylcarboxamido)adenosine > adenosine > N(6)-cyclopentyladenosine. The use of selective receptor subtype antagonists confirmed the involvement of A(2A) and A(3) adenosine receptors. The modulator role of adenosine is probably due to the activation of both receptors; co-application of 1 nM 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride plus 1 nM 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-beta-D-ribofuranuronamide caused additive reductions in NA released. Furthermore, while 1 nM of an A(2A) or A(3) receptor antagonist only partially reduced the inhibitory action of adenosine, the combined co-application of the two antagonists fully blocked the adenosine-induced inhibition. Only the simultaneous blockade of the adenosine A(2A) plus A(3) receptors with selective antagonists elicited a significant increase in NA overflow. H 89 reduced the release of both NA and NPY. We conclude that pre-synaptic A(2A) and A(3) adenosine receptor activation modulates sympathetic co-transmission by exclusively inhibiting the release of NA without affecting immunoreactive (ir)-NPY and we suggest separate mechanisms for vesicular release modulation.

  17. Aldosterone receptor antagonism normalizes vascular function in liquorice-induced hypertension.

    PubMed

    Quaschning, T; Ruschitzka, F; Shaw, S; Lüscher, T F

    2001-02-01

    aldosterone receptor antagonism normalizes blood pressure, prevents upregulation of vascular ET-1, restores NO-mediated endothelial dysfunction, and thus, may advance as a novel and specific therapeutic approach in 11beta-HSD2-deficient hypertension.

  18. 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

  19. 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.

  20. 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.

  1. Probing biased/partial agonism at the G protein-coupled A(2B) adenosine receptor.

    PubMed

    Gao, Zhan-Guo; Balasubramanian, Ramachandran; Kiselev, Evgeny; Wei, Qiang; Jacobson, Kenneth A

    2014-08-01

    G protein-coupled A(2B) adenosine receptor (AR) regulates numerous important physiological functions, but its activation by diverse A(2B)AR agonists is poorly profiled. We probed potential partial and/or biased agonism in cell lines expressing variable levels of endogenous or recombinant A(2B)AR. In cAMP accumulation assays, both 5'-substituted NECA and C2-substituted MRS3997 are full agonists. However, only 5'-substituted adenosine analogs are full agonists in calcium mobilization, ERK1/2 phosphorylation and β-arrestin translocation. A(2B)AR overexpression in HEK293 cells markedly increased the agonist potency and maximum effect in cAMP accumulation, but less in calcium and ERK1/2. A(2B)AR siRNA silencing was more effective in reducing the maximum cAMP effect of non-nucleoside agonist BAY60-6583 than NECA's. A quantitative 'operational model' characterized C2-substituted MRS3997 as either balanced (cAMP accumulation, ERK1/2) or strongly biased agonist (against calcium, β-arrestin). N⁶-substitution biased against ERK1/2 (weakly) and calcium and β-arrestin (strongly) pathways. BAY60-6583 is ERK1/2-biased, suggesting a mechanism distinct from adenosine derivatives. BAY60-6583, as A(2B)AR antagonist in MIN-6 mouse pancreatic β cells expressing low A(2B)AR levels, induced insulin release. This is the first relatively systematic study of structure-efficacy relationships of this emerging drug target.

  2. 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

  3. 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

  4. 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

  5. Trace amines inhibit insect odorant receptor function through antagonism of the co-receptor subunit

    PubMed Central

    Chen, Sisi; Luetje, Charles W.

    2014-01-01

    Many insect behaviors are driven by olfaction, making insect olfactory receptors (ORs) appealing targets for insect control.  Insect ORs are odorant-gated ion channels, with each receptor thought to be composed of a representative from a large, variable family of odorant binding subunits and a highly conserved co-receptor subunit (Orco), assembled in an unknown stoichiometry.  Synthetic Orco directed agonists and antagonists have recently been identified.  Several Orco antagonists have been shown to act via an allosteric mechanism to inhibit OR activation by odorants.  The high degree of conservation of Orco across insect species results in Orco antagonists having broad activity at ORs from a variety of insect species and suggests that the binding site for Orco ligands may serve as a modulatory site for compounds endogenous to insects or may be a target of exogenous compounds, such as those produced by plants.  To test this idea, we screened a series of biogenic and trace amines, identifying several as Orco antagonists.  Of particular interest were tryptamine, a plant-produced amine, and tyramine, an amine endogenous to the insect nervous system.  Tryptamine was found to be a potent antagonist of Orco, able to block Orco activation by an Orco agonist and to allosterically inhibit activation of ORs by odorants.  Tyramine had effects similar to those of tryptamine, but was less potent.  Importantly, both tryptamine and tyramine displayed broad activity, inhibiting odorant activation of ORs of species from three different insect orders (Diptera, Lepidoptera and Coleoptera), as well as odorant activation of six diverse ORs from a single species (the human malaria vector mosquito, Anopheles gambiae).  Our results suggest that endogenous and exogenous natural compounds serve as Orco ligands modulating insect olfaction and that Orco can be an important target for the development of novel insect repellants. PMID:25075297

  6. Effects of adenosine receptor agonists on efferent renal nerve activity in anesthetized rats.

    PubMed

    Genovesi, S; Pieruzzi, F; Camisasca, P; Ragonesi, G; Protasoni, G; Golin, R; Zanchetti, A; Stella, A

    2000-02-01

    The aim of this study was to investigate the effects of A1 and A2 adenosine-receptor activation on the sympathetic nervous system. The effects on efferent renal nerve activity of selective A1 (CCPA; 2-chloro-N-6-cyclopentyladenosine) and A2 (2HE-NECA; 2-hexynyl-5'-N-ethylcarboxamidoadenosine) adenosine-receptor agonists were studied in anesthetized rats either with intact baroreflexes (intact rats) or with bilateral sinoaortic denervation and vagotomy (denervated rats). After a control period of 5 min, A1 or A2 agonist or vehicle were intravenously infused for 8 min in separate groups of intact or denervated rats, in which arterial pressure and heart rate were continuously recorded. CCPA (5.0 microg/kg/min) and 2HE-NECA (0.7 microg/kg/min) were selected to obtain comparable blood pressure changes over the period of observation. Arterial pressure significantly and equally decreased during the A1 (-41 +/- 8%), and A2 (-35 +/- 5%) agonist administration. Heart rate significantly decreased during A1 agonist infusion, but it did not change during A2 agonist administration. Bilateral sinoaortic denervation and vagotomy did not modify the hemodynamic responses to both drugs. The A1 and A2 administration caused a large and significant increase in efferent renal nerve activity (+66 +/- 22% and +76 +/- 15%, respectively), and this effect was entirely abolished in denervated rats. A linear relation with a significant negative slope between changes in arterial pressure and changes in neural discharge was observed for each treatment. The comparison of the regression slopes showed that the reflex increase of efferent sympathetic activity caused by the administration of both agonists was significantly smaller than the increment induced by equipotent hypotensive dose of sodium nitroprusside (10 microg/kg). These data show that the selective activation of A1 and A2 receptors elicits a reflex increase in efferent renal nerve activity. This neural activation is smaller as compared

  7. 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

  8. Differential effects of presynaptic versus postsynaptic adenosine A2A receptor blockade on Δ9-tetrahydrocannabinol (THC) self-administration in squirrel monkeys.

    PubMed

    Justinová, Zuzana; Redhi, Godfrey H; Goldberg, Steven R; Ferré, Sergi

    2014-05-01

    Different doses of an adenosine A2A receptor antagonist MSX-3 [3,7-dihydro-8-[(1E)-2-(3-ethoxyphenyl)ethenyl]-7 methyl-3-[3-(phosphooxy)propyl-1-(2 propynil)-1H-purine-2,6-dione] were found previously to either decrease or increase self-administration of cannabinoids delta-9-tetrahydrocannabinol (THC) or anandamide in squirrel monkeys. It was hypothesized that the decrease observed with a relatively low dose of MSX-3 was related to blockade of striatal presynaptic A2A receptors that modulate glutamatergic neurotransmission, whereas the increase observed with a higher dose was related to blockade of postsynaptic A2A receptors localized in striatopallidal neurons. This hypothesis was confirmed in the present study by testing the effects of the preferential presynaptic and postsynaptic A2A receptor antagonists SCH-442416 [2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] and KW-6002 [(E)-1, 3-diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione], respectively, in squirrel monkeys trained to intravenously self-administer THC. SCH-442416 produced a significant shift to the right of the THC self-administration dose-response curves, consistent with antagonism of the reinforcing effects of THC. Conversely, KW-6002 produced a significant shift to the left, consistent with potentiation of the reinforcing effects of THC. These results show that selectively blocking presynaptic A2A receptors could provide a new pharmacological approach to the treatment of marijuana dependence and underscore corticostriatal glutamatergic neurotransmission as a possible main mechanism involved in the rewarding effects of THC.

  9. Communication over the Network of Binary Switches Regulates the Activation of A2A Adenosine Receptor

    PubMed Central

    Lee, Yoonji; Choi, Sun; Hyeon, Changbong

    2015-01-01

    Dynamics and functions of G-protein coupled receptors (GPCRs) are accurately regulated by the type of ligands that bind to the orthosteric or allosteric binding sites. To glean the structural and dynamical origin of ligand-dependent modulation of GPCR activity, we performed total ~ 5 μsec molecular dynamics simulations of A2A adenosine receptor (A2AAR) in its apo, antagonist-bound, and agonist-bound forms in an explicit water and membrane environment, and examined the corresponding dynamics and correlation between the 10 key structural motifs that serve as the allosteric hotspots in intramolecular signaling network. We dubbed these 10 structural motifs “binary switches” as they display molecular interactions that switch between two distinct states. By projecting the receptor dynamics on these binary switches that yield 210 microstates, we show that (i) the receptors in apo, antagonist-bound, and agonist-bound states explore vastly different conformational space; (ii) among the three receptor states the apo state explores the broadest range of microstates; (iii) in the presence of the agonist, the active conformation is maintained through coherent couplings among the binary switches; and (iv) to be most specific, our analysis shows that W246, located deep inside the binding cleft, can serve as both an agonist sensor and actuator of ensuing intramolecular signaling for the receptor activation. Finally, our analysis of multiple trajectories generated by inserting an agonist to the apo state underscores that the transition of the receptor from inactive to active form requires the disruption of ionic-lock in the DRY motif. PMID:25664580

  10. Ultraslow Water-Mediated Transmembrane Interactions Regulate the Activation of A2A Adenosine Receptor.

    PubMed

    Lee, Yoonji; Kim, Songmi; Choi, Sun; Hyeon, Changbong

    2016-09-20

    Water molecules inside a G-protein coupled receptor (GPCR) have recently been spotlighted in a series of crystal structures. To decipher the dynamics and functional roles of internal water molecules in GPCR activity, we studied the A2A adenosine receptor using microsecond molecular-dynamics simulations. Our study finds that the amount of water flux across the transmembrane (TM) domain varies depending on the receptor state, and that the water molecules of the TM channel in the active state flow three times more slowly than those in the inactive state. Depending on the location in solvent-protein interface as well as the receptor state, the average residence time of water in each residue varies from ∼O(10(2)) ps to ∼O(10(2)) ns. Especially, water molecules, exhibiting ultraslow relaxation (∼O(10(2)) ns) in the active state, are found around the microswitch residues that are considered activity hotspots for GPCR function. A continuous allosteric network spanning the TM domain, arising from water-mediated contacts, is unique in the active state, underscoring the importance of slow water molecules in the activation of GPCRs. PMID:27653477

  11. [60]Fullerene derivative modulates adenosine and metabotropic glutamate receptors gene expression: a possible protective effect against hypoxia

    PubMed Central

    2014-01-01

    Background Glutamate, the main excitatory neurotransmitter, is involved in learning and memory processes but at higher concentration results excitotoxic causing degeneration and neuronal death. Adenosine is a nucleoside that exhibit neuroprotective effects by modulating of glutamate release. Hypoxic and related oxidative conditions, in which adenosine and metabotropic glutamate receptors are involved, have been demonstrated to contribute to neurodegenerative processes occurring in certain human pathologies. Results Human neuroblastoma cells (SH-SY5Y) were used to evaluate the long time (24, 48 and 72 hours) effects of a [60]fullerene hydrosoluble derivative (t3ss) as potential inhibitor of hypoxic insult. Low oxygen concentration (5% O2) caused cell death, which was avoided by t3ss exposure in a concentration dependent manner. In addition, gene expression analysis by real time PCR of adenosine A1, A2A and A2B and metabotropic glutamate 1 and 5 receptors revealed that t3ss significantly increased A1 and mGlu1 expression in hypoxic conditions. Moreover, t3ss prevented the hypoxia-induced increase in A2A mRNA expression. Conclusions As t3ss causes overexpression of adenosine A1 and metabotropic glutamate receptors which have been shown to be neuroprotective, our results point to a radical scavenger protective effect of t3ss through the enhancement of these neuroprotective receptors expression. Therefore, the utility of these nanoparticles as therapeutic target to avoid degeneration and cell death of neurodegenerative diseases is suggested. PMID:25123848

  12. 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.

  13. Exploring human adenosine A3 receptor complementarity and activity for adenosine analogues modified in the ribose and purine moiety

    PubMed Central

    Van Rompaey, Philippe; Jacobson, Kenneth A.; Gross, Ariel S.; Gao, Zhan-Guo; Van Calenbergh, Serge

    2012-01-01

    In this paper we investigated the influence on affinity, selectivity and intrinsic activity upon modification of the adenosine agonist scaffold at the 3′- and 5′-positions of the ribofuranosyl moiety and the 2- and N6-positions of the purine base. This resulted in the synthesis of various analogues, that is, 3–12 and 24–33, with good hA3AR selectivity and moderate-to-high affinities (as in 32, Ki = 27 nM). Interesting was the ability to tune the intrinsic activity depending on the substituent introduced at the 3′-position. PMID:15670905

  14. Adenosine A2A Receptor Antagonists and Parkinson’s Disease

    PubMed Central

    2011-01-01

    This Review summarizes and updates the work on adenosine A2A receptor antagonists for Parkinson’s disease from 2006 to the present. There have been numerous publications, patent applications, and press releases within this time frame that highlight new medicinal chemistry approaches to this attractive and promising target to treat Parkinson’s disease. The Review is broken down by scaffold type and will discuss the efforts to optimize particular scaffolds for activity, pharmacokinetics, and other drug discovery parameters. The majority of approaches focus on preparing selective A2A antagonists, but a few approaches to dual A2A/A1 antagonists will also be highlighted. The in vivo profiles of compounds will be highlighted and discussed to compare activities across different chemical series. A clinical report and update will be given on compounds that have entered clinical trials. PMID:22860156

  15. Structure-kinetics relationships of Capadenoson derivatives as adenosine A1 receptor agonists.

    PubMed

    Louvel, Julien; Guo, Dong; Soethoudt, Marjolein; Mocking, Tamara A M; Lenselink, Eelke B; Mulder-Krieger, Thea; Heitman, Laura H; IJzerman, Adriaan P

    2015-08-28

    We report the synthesis and biological evaluation of new derivatives of Capadenoson, a former drug candidate that was previously advanced to phase IIa clinical trials. 19 of the 20 ligands show an affinity below 100 nM at the human adenosine A1 receptor (hA1AR) and display a wide range of residence times at this target (from approx. 5 min (compound 10) up to 132 min (compound 5)). Structure-affinity and structure-kinetics relationships were established, and computational studies of a homology model of the hA1AR revealed crucial interactions for both the affinity and dissociation kinetics of this family of ligands. These results were also combined with global metrics (Ligand Efficiency, cLogP), showing the importance of binding kinetics as an additional way to better select a drug candidate amongst seemingly similar leads.

  16. 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

  17. Agonism, Antagonism, and Inverse Agonism Bias at the Ghrelin Receptor Signaling.

    PubMed

    M'Kadmi, Céline; Leyris, Jean-Philippe; Onfroy, Lauriane; Galés, Céline; Saulière, Aude; Gagne, Didier; Damian, Marjorie; Mary, Sophie; Maingot, Mathieu; Denoyelle, Séverine; Verdié, Pascal; Fehrentz, Jean-Alain; Martinez, Jean; Banères, Jean-Louis; Marie, Jacky

    2015-11-01

    The G protein-coupled receptor GHS-R1a mediates ghrelin-induced growth hormone secretion, food intake, and reward-seeking behaviors. GHS-R1a signals through Gq, Gi/o, G13, and arrestin. Biasing GHS-R1a signaling with specific ligands may lead to the development of more selective drugs to treat obesity or addiction with minimal side effects. To delineate ligand selectivity at GHS-R1a signaling, we analyzed in detail the efficacy of a panel of synthetic ligands activating the different pathways associated with GHS-R1a in HEK293T cells. Besides β-arrestin2 recruitment and ERK1/2 phosphorylation, we monitored activation of a large panel of G protein subtypes using a bioluminescence resonance energy transfer-based assay with G protein-activation biosensors. We first found that unlike full agonists, Gq partial agonists were unable to trigger β-arrestin2 recruitment and ERK1/2 phosphorylation. Using G protein-activation biosensors, we then demonstrated that ghrelin promoted activation of Gq, Gi1, Gi2, Gi3, Goa, Gob, and G13 but not Gs and G12. Besides, we identified some GHS-R1a ligands that preferentially activated Gq and antagonized ghrelin-mediated Gi/Go activation. Finally, we unambiguously demonstrated that in addition to Gq, GHS-R1a also promoted constitutive activation of G13. Importantly, we identified some ligands that were selective inverse agonists toward Gq but not of G13. This demonstrates that bias at GHS-R1a signaling can occur not only with regard to agonism but also to inverse agonism. Our data, combined with other in vivo studies, may facilitate the design of drugs selectively targeting individual signaling pathways to treat only the therapeutically relevant function.

  18. Farnesoid X Receptor Antagonizes JNK Signaling Pathway in Liver Carcinogenesis by Activating SOD3

    PubMed Central

    Li, Cunbao; Guo, Cong; Li, Yanyan; Qi, Hui; Shen, Hailing; Kong, Jing; Long, Xuecheng; Yuan, Frank; Wang, Xichun

    2015-01-01

    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 H2O2 or 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 H2O2 in 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 H2O2 in 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

  19. 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.

  20. Discovery and optimization of potent and selective functional antagonists of the human adenosine A2B receptor.

    PubMed

    Bedford, Simon T; Benwell, Karen R; Brooks, Teresa; Chen, Ijen; Comer, Mike; Dugdale, Sarah; Haymes, Tim; Jordan, Allan M; Kennett, Guy A; Knight, Anthony R; Klenke, Burkhard; LeStrat, Loic; Merrett, Angela; Misra, Anil; Lightowler, Sean; Padfield, Anthony; Poullennec, Karine; Reece, Mark; Simmonite, Heather; Wong, Melanie; Yule, Ian A

    2009-10-15

    We herein report the discovery of a novel class of antagonists of the human adenosine A2B receptor. This low molecular weight scaffold has been optimized to offer derivatives with potential utility for the alleviation of conditions associated with this receptor subtype, such as nociception, diabetes, asthma and COPD. Furthermore, preliminary pharmacokinetic analysis has revealed compounds with profiles suitable for either inhaled or systemic routes of administration.

  1. Protective Effects of Adenosine Receptor Agonist in a Cirrhotic Liver Resection Model

    PubMed Central

    Iskandarov, Emil; Kadaba Srinivasan, Pramod; Xin, Wang; Bleilevens, Christian; Afify, Mamdouh; Hamza, Astrit; Wei, Lai; Hata, Koichiro; Agayev, Boyukkishi; Tolba, Rene

    2016-01-01

    Objectives To investigate the role of CGS21680, a selective adenosine A2A receptor agonist, on a bile-duct-ligated cirrhotic liver resection model in rats. Methods Male Wistar rats were allotted into 3 groups (n = 7 per time-point): the control group, the bile duct ligation + CGS21680 group (BDL + CGS), and the bile duct ligation group (BDL). Biliary cirrhosis had been previously induced by ligature of the common bile duct in the BDL + CGS and BDL groups. After 2 weeks, the animals underwent partial hepatectomy (50%). The BDL + CGS group received a single dose of CGS21680 15 minutes prior to hepatectomy. Blood samples were collected and analyzed. Results Aspartate transaminase levels were found to be lower in the control vs BDL groups (1, 3, and 24 h) (P < 0.01) and the BDL + CGS (1 and 3 hours) (P < 0.01) and BDL + CGS vs BDL (24 hours) (P < 0.05) groups. Hepatic flow was measured and BDL showed significantly lower values at the 3, 24, and 168 h time-points compared to the control (P < 0.01) and BDL + CGS groups (P < 0.05 at 3 and 168 hours; P < 0.01 at 24 h). O2C velocity was reduced in the BDL compared to the control group (P < 0.001 at 3 hours; P < 0.01 at 24 and 168 hours) and the BDL + CGS group (P < 0.01 at 24 hours). Interleukin-6 levels were abrogated in the BDL + CGS (P < 0.05) and control (P < 0.01) groups versus BDL. Histone-bound low-molecular-weight DNA fragments in the BDL + CGS (P < 0.01) and control (P < 0.05) groups were low compared to the BDL group. Conclusions Administration of CGS21680, an adenosine receptor agonist, after the resection of bile-duct-ligated cirrhotic livers led to improved liver function, regeneration, and microcirculation. PMID:27799962

  2. Adenosine A1 receptors contribute to immune regulation after neonatal hypoxic ischemic brain injury.

    PubMed

    Winerdal, Max; Winerdal, Malin E; Wang, Ying-Qing; Fredholm, Bertil B; Winqvist, Ola; Ådén, Ulrika

    2016-03-01

    Neonatal brain hypoxic ischemia (HI) often results in long-term motor and cognitive impairments. Post-ischemic inflammation greatly effects outcome and adenosine receptor signaling modulates both HI and immune cell function. Here, we investigated the influence of adenosine A1 receptor deficiency (A1R(-/-)) on key immune cell populations in a neonatal brain HI model. Ten-day-old mice were subjected to HI. Functional outcome was assessed by open locomotion and beam walking test and infarction size evaluated. Flow cytometry was performed on brain-infiltrating cells, and semi-automated analysis of flow cytometric data was applied. A1R(-/-) mice displayed larger infarctions (+33%, p < 0.05) and performed worse in beam walking tests (44% more mistakes, p < 0.05) than wild-type (WT) mice. Myeloid cell activation after injury was enhanced in A1R(-/-) versus WT brains. Activated B lymphocytes expressing IL-10 infiltrated the brain after HI in WT, but were less activated and did not increase in relative frequency in A1R(-/-). Also, A1R(-/-) B lymphocytes expressed less IL-10 than their WT counterparts, the A1R antagonist DPCPX decreased IL-10 expression whereas the A1R agonist CPA increased it. CD4(+) T lymphocytes including FoxP3(+) T regulatory cells, were unaffected by genotype, whereas CD8(+) T lymphocyte responses were smaller in A1R(-/-) mice. Using PCA to characterize the immune profile, we could discriminate the A1R(-/-) and WT genotypes as well as sham operated from HI-subjected animals. We conclude that A1R signaling modulates IL-10 expression by immune cells, influences the activation of these cells in vivo, and affects outcome after HI. PMID:26608888

  3. Activation of adenosine receptor potentiates the anticonvulsant effect of phenytoin against amygdala kindled seizures.

    PubMed

    Sun, Zhen; Zhong, Xiao-Ling; Zong, Yu; Wu, Zhong-Chen; Zhang, Qun; Yu, Jin-Tai; Tan, Lan

    2015-01-01

    Drug resistance in epilepsy is considered as a complicated and multifactorial problem. Poor penetration of antiepileptic drugs (AEDs) across blood-brain barrier (BBB) into the brain, which results in insufficient level of the drugs at the targeted brain region, has been discussed as one mechanism contributing to pharmacoresistance of epilepsies. Therefore, modulating permeability of BBB is the effective treatment strategy since it facilitates the entry of AEDs into the central nervous system (CNS). Recently, signaling through receptors for the adenosine has been identified as a potent modulator of BBB permeability. This paper aimed to investigate the effects of auxiliary application of adenosine receptor (AR) agonist on amygdala-kindled seizures in adult male Wistar rats. When fully kindled seizures were achieved by daily electrical stimulation of the amygdala, rats were randomly divided into three groups: control, phenytoin, and phenytoin (PHT)+5'-N-ethylcarboxamidoadenosine (NECA) groups. NECA (0.08 mg/kg, i.v.) was applied to the PHT+NECA group after the administration of PHT (75 mg/kg, i.p. on the first day; 50mg/kg, i.p. on the following 9 days). Intravenous infusion of NECA resulted in a significant increase in brain PHT levels as compared with the PHT treatment alone. On the other hand, the auxiliary application of NECA dramatically decreased the frequency of generalized seizures and seizure stage, shortened duration of afterdischarge and generalized seizures, as well as the elevated the afterdischarge threshold and generalized seizures threshold. Our study demonstrated that auxiliary application of AR agonist enhanced brain antiepileptic drug levels and strengthened the anticonvulsant properties of PHT against amygdala kindled seizures.

  4. The effects of saponin on the binding and functional properties of the human adenosine A1 receptor.

    PubMed Central

    Cohen, F. R.; Lazareno, S.; Birdsall, N. J.

    1996-01-01

    1. Experiments with adenosine deaminase suggest that adenosine is present in membrane preparations from CHO cells bearing adenosine A1 receptors. 2. Pretreatment of the membranes (ca 0.6 mg protein ml-1) with the permeabilizing agent saponin (100 micrograms ml-1) or addition of saponin (10 micrograms ml-1) to the membranes (0.02-0.08 mg protein ml-1) in the assay, generates homogeneous low affinity agonist binding curves in the presence of GTP and an increased function, assessed by agonist stimulation of [35S]-GTP gamma S binding. The affinity constants for the binding of an agonist and an antagonist are not affected by this saponin treatment. Saponin facilitates the interaction of guanine nucleotides with receptor G-protein complexes, possibly by removing a permeability barrier to access of G-proteins by GTP. However, adenosine is still present in the binding assays after saponin treatment. 3. The agonist binding properties of the human A1 receptor have been characterized. In saponin pretreated membranes, 80-90% of the A1 receptors are capable of forming agonist-receptor-G protein complexes in the absence of GTP. These complexes have a 300-600 fold higher affinity than uncoupled receptors for N6-cyclohexyladenosine. 4. A very slow component is observed in the association and dissociation kinetics of the agonist [3H]-N6-cyclohexyladenosine ([3H]-CHA) and in the association but not dissociation kinetics of the antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). The slow association component of [3H]-DPCPX is essentially absent when incubations are carried out in the presence of GTP. The slow dissociation component of [3H]-CHA binding is rapidly disrupted by GTP. 5. It is hypothesized that long-lasting adenosine-receptor-G protein complexes are present in the CHO membrane preparations. The existence of these complexes, resistant to the action of adenosine deaminase but sensitive to GTP, may rationalize the observed kinetics and the increase in 3H

  5. 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.

  6. Muscarinic M(3) facilitation of acetylcholine release from rat myenteric neurons depends on adenosine outflow leading to activation of excitatory A(2A) receptors.

    PubMed

    Vieira, C; Duarte-Araújo, M; Adães, S; Magalhães-Cardoso, T; Correia-de-Sá, P

    2009-10-01

    Acetylcholine (ACh) is a major excitatory neurotransmitter in the myenteric plexus, and it regulates its own release acting via muscarinic autoreceptors. Adenosine released from stimulated myenteric neurons modulates ACh release preferentially via facilitatory A(2A) receptors. In this study, we investigated how muscarinic and adenosine receptors interplay to regulate ACh from the longitudinal muscle-myenteric plexus of the rat ileum. Blockade of the muscarinic M(2) receptor with 11-[[2-1[(diethylamino) methyl-1-piperidinyl]- acetyl

  7. 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.

  8. 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.

  9. Stimulation of NTS A1 adenosine receptors evokes counteracting effects on hindlimb vasculature.

    PubMed

    McClure, Joseph M; O'Leary, Donal S; Scislo, Tadeusz J

    2005-12-01

    Our previous studies concluded that stimulation of the nucleus of the solitary tract (NTS) A2a receptors evokes preferential hindlimb vasodilation mainly via inducing increases in preganglionic sympathetic nerve activity (pre-ASNA) directed to the adrenal medulla. This increase in pre-ASNA causes the release of epinephrine and subsequent activation of beta-adrenergic receptors that are preferentially located in the skeletal muscle vasculature. Selective activation of NTS A1 adenosine receptors evokes variable, mostly pressor effects and increases pre-ASNA, as well as lumbar sympathetic activity, which is directed to the hindlimb. These counteracting factors may have opposite effects on the hindlimb vasculature resulting in mixed vascular responses. Therefore, in chloralose-urethane-anesthetized rats, we evaluated the contribution of vasodilator versus vasoconstrictor effects of stimulation of NTS A1 receptors on the hindlimb vasculature. We compared the changes in iliac vascular conductance evoked by microinejctions into the NTS of the selective A1 receptor agonist N6-cyclopentyladenosine (330 pmol in 50 nl volume) in intact animals with the responses evoked after beta-adrenergic blockade, bilateral adrenalectomy, bilateral lumbar sympathectomy, and combined adrenalectomy + lumbar sympathectomy. In intact animals, stimulation of NTS A1 receptors evoked variable effects: increases and decreases in mean arterial pressure and iliac conductance with prevailing pressor and vasoconstrictor effects. Peripheral beta-adrenergic receptor blockade and bilateral adrenalectomy eliminated the depressor component of the responses, markedly potentiated iliac vasoconstriction, and tended to increase the pressor responses. Lumbar sympathectomy tended to decrease the pressor and vasoconstrictor responses. After bilateral adrenalectomy plus lumbar sympathectomy, a marked vasoconstriction in iliac vascular bed still persisted, suggesting that the vasoconstrictor component of the

  10. 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.

  11. Evaluation of structural effects on 5-HT2A receptor antagonism by aporphines: identification of a new aporphine with 5-HT2A antagonist activity

    PubMed Central

    Ponnala, Shashikanth; Gonzales, Junior; Kapadia, Nirav; Navarro, Hernan A.; Harding, Wayne W.

    2014-01-01

    A set of aporphine analogs related to nantenine was evaluated for antagonist activity at 5-HT2A and α1A adrenergic receptors. With regards to 5-HT2A receptor antagonism, a C2 allyl group is detrimental to activity. The chiral center of nantenine is not important for 5-HT2A antagonist activity, however the N6 nitrogen atom is a critical feature for 5-HT2A antagonism. Compound 12b was the most potent 5-HT2A aporphine antagonist identified in this study and has similar potency to previously identified aporphine antagonists 2 and 3. The ring A and N6 modifications examined were detrimental to α1A antagonism. A slight eutomeric preference for the R enantiomer of nantenine was observed in relation to α1A antagonism. PMID:24630561

  12. Blockade of orexin-1 receptors attenuates orexin-2 receptor antagonism-induced sleep promotion in the rat.

    PubMed

    Dugovic, Christine; Shelton, Jonathan E; Aluisio, Leah E; Fraser, Ian C; Jiang, Xiaohui; Sutton, Steven W; Bonaventure, Pascal; Yun, Sujin; Li, Xiaorong; Lord, Brian; Dvorak, Curt A; Carruthers, Nicholas I; Lovenberg, Timothy W

    2009-07-01

    Orexins are peptides produced by lateral hypothalamic neurons that exert a prominent role in the maintenance of wakefulness by activating orexin-1 (OX1R) and orexin-2 (OX2R) receptor located in wake-active structures. Pharmacological blockade of both receptors by the dual OX1/2R antagonist (2R)-2-[(1S)-6,7-dimethoxy-1-{2-[4-(trifluoromethyl)phenyl]ethyl}-3,4-dihydroisoquinolin-2(1H)-yl]-N-methyl-2-phenylethanamide (almorexant) has been shown to promote sleep in animals and humans during their active period. However, the selective distribution of OX1R and OX2R in distinct neuronal circuits may result in a differential impact of these receptors in sleep-wake modulation. The respective role of OX1R and OX2R on sleep in correlation with monoamine release was evaluated in rats treated with selective antagonists alone or in combination. When administered in either phase of the light/dark cycle, the OX2R antagonist 1-(2,4-dibromophenyl)-3-[(4S,5S)-2,2-dimethyl-4-phenyl-1,3-dioxan-5-yl]urea (JNJ-10397049) decreased the latency for persistent sleep and increased nonrapid eye movement and rapid eye movement sleep time. Almorexant produced less hypnotic activity, whereas the OX1R antagonist 1-(6,8-difluoro-2-methylquinolin-4-yl)-3-[4-(dimethylamino)phenyl]urea (SB-408124) had no effect. Microdialysis studies showed that either OX2R or OX1/2R antagonism decreased extracellular histamine concentration in the lateral hypothalamus, whereas both OX1R and OX1/2R antagonists increased dopamine release in the prefrontal cortex. Finally, coadministration of the OX1R with the OX2R antagonist greatly attenuated the sleep-promoting effects of the OX2R antagonist. These results indicate that blockade of OX2R is sufficient to initiate and prolong sleep, consistent with the hypothesis of a deactivation of the histaminergic system. In addition, it is suggested that simultaneous inhibition of OX1R attenuates the sleep-promoting effects mediated by selective OX2R blockade, possibly correlated

  13. Effects of tianeptine on onset time of pentylenetetrazole-induced seizures in mice: possible role of adenosine A1 receptors.

    PubMed

    Uzbay, Tayfun I; Kayir, Hakan; Ceyhan, Mert

    2007-02-01

    Depression is a common psychiatric problem in epileptic patients. Thus, it is important that an antidepressant agent has anticonvulsant activity. This study was organized to investigate the effects of tianeptine, an atypical antidepressant, on pentylenetetrazole (PTZ)-induced seizure in mice. A possible contribution of adenosine receptors was also evaluated. Adult male Swiss-Webster mice (25-35 g) were subjects. PTZ (80 mg/kg, i.p.) was injected to mice 30 min after tianeptine (2.5-80 mg/kg, i.p.) or saline administration. The onset times of 'first myoclonic jerk' (FMJ) and 'generalized clonic seizures' (GCS) were recorded. Duration of 600 s was taken as a cutoff time in calculation of the onset time of the seizures. To evaluate the contribution of adenosine receptors in the effect of tianeptine, a nonspecific adenosine receptor antagonist caffeine, a specific A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a specific A2A receptor antagonist 8-(3-chlorostyryl) caffeine (CSC) or their vehicles were administered to the mice 15 min before tianeptine (80 mg/kg) or saline treatments. Tianeptine (40 and 80 mg/kg) pretreatment significantly delayed the onset time of FMJ and GCS. Caffeine (10-60 mg/kg, i.p.) dose-dependently blocked the retarding effect of tianeptine (80 mg/kg) on the onset times of FMJ and GCS. DPCPX (20 mg/kg) but not CSC (1-8 mg/kg) blocked the effect of tianeptine (80 mg/kg) on FMJ. Our results suggest that tianeptine delayed the onset time of PTZ-induced seizures via adenosine A1 receptors in mice. Thus, this drug may be a useful choice for epileptic patients with depression.

  14. 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

  15. 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

  16. 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

  17. Striatal pre- and postsynaptic profile of adenosine A(2A) receptor antagonists.

    PubMed

    Orru, Marco; Bakešová, Jana; Brugarolas, Marc; Quiroz, César; Beaumont, Vahri; Goldberg, Steven R; Lluís, Carme; Cortés, Antoni; Franco, Rafael; Casadó, Vicent; Canela, Enric I; Ferré, Sergi

    2011-01-11

    Striatal adenosine A(2A) receptors (A(2A)Rs) are highly expressed in medium spiny neurons (MSNs) of the indirect efferent pathway, where they heteromerize with dopamine D(2) receptors (D(2)Rs). A(2A)Rs are also localized presynaptically in cortico-striatal glutamatergic terminals contacting MSNs of the direct efferent pathway, where they heteromerize with adenosine A(1) receptors (A(1)Rs). It has been hypothesized that postsynaptic A(2A)R antagonists should be useful in Parkinson's disease, while presynaptic A(2A)R 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 A(2A)R 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 A(2A)R-D(2)R and A(1)R-A(2A)R heteromers to determine possible differences in the affinity of these compounds for different A(2A)R heteromers. Heteromerization played a key role in the presynaptic profile of SCH-442416, since it bound with much less affinity to A(2A)R when co-expressed with D(2)R than with A(1)R. KW-6002 showed the best relative affinity for A(2A)R co-expressed with D(2)R than co-expressed with A(1)R, 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

  18. 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

  19. 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

  20. Adenosine A2B receptor stimulates angiogenesis by inducing VEGF and eNOS in human microvascular endothelial cells

    PubMed Central

    Du, Xiaolong; Ou, Xuehai; Song, Tao; Zhang, Wentao; Cong, Fei; Zhang, Shihui

    2015-01-01

    Angiogenesis is critical to wound repair due to its role in providing oxygen and nutrients that are required to support the growth and function of reparative cells in damaged tissues. Adenosine receptors are claimed to be of paramount importance in driving wound angiogenesis by inducing VEGF. However, the underlying mechanisms for the regulation of adenosine receptors in VEGF as well as eNOS remain poorly understood. In the present study, we found that adenosine and the non-selective adenosine receptor agonists (NECA) induced tube formation in HMEC-1 in a dose-dependent manner. Adenosine or NECA (10 µmol/L) significantly augmented the number and length of the segments in comparison with the control. Simultaneously, VEGF and eNOS were significantly upregulated following the administration of 10 µmol/L NECA, while they were suppressed after A2B AR genetic silencing and pharmacological inhibition by MRS1754. In addition, VEGF expression and eNOS bioavailability elimination significantly reduced the formation of capillary-like structures. Furthermore, the activation of A2B AR by NECA significantly increased the intracellular cAMP levels and concomitant CREB phosphorylation, eventually leading to the production of VEGF in HMEC-1. However, the activated PKA-CREB pathway seemed to be invalidated in the induction of eNOS. Moreover, we found that the elicited PI3K/AKT signaling in response to the induction of NECA assisted in regulating eNOS but failed to impact on VEGF generation. In conclusion, the A2B AR activation-driven angiogenesis via cAMP-PKA-CREB mediated VEGF production and PI3K/AKT-dependent upregulation of eNOS in HMEC-1. PMID:25966978

  1. 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.

  2. 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.

  3. Palmitoylation of the recombinant human A1 adenosine receptor: enhanced proteolysis of palmitoylation-deficient mutant receptors.

    PubMed Central

    Gao, Z; Ni, Y; Szabo, G; Linden, J

    1999-01-01

    Palmitoylation of the recombinant human A(1) adenosine receptor (A(1)AR) expressed in HEK-293 cells is demonstrated by showing that hexahistidine (His(6))/Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (FLAG) (H/F) A(1)ARs, purified to homogeneity from cells metabolically labelled with [(3)H]palmitate, incorporate tritium into a 38-42 kDa receptor glycoprotein. The amount of palmitoylation is not affected by incubation of cells with the A(1)AR-selective agonist N(6)-cyclopentyladenosine (CPA). A(1)AR palmitoylation is abolished by treatment with neutral hydroxylamine or by mutation of Cys-309 to Ala (C(309)-->A). Based on Western blotting and pulse-chase experiments with [(35)S]methionine, at least 90% of wild-type receptors are palmitoylated and turn over with a t1/2 of 6.4 h. Of the C(309)-->A mutated receptors, 40% appear to turn over like wild-type receptors, with a t1/2 of 7.1 h, and 60% appear to be rapidly cleaved to form a 25 kDa receptor fragment that turns over with a t1/2 of 0.8 h. In HEK-293 cell lines expressing similar numbers of wild-type or C(309)-->A mutant A(1)Rs, there is little difference in the kinetics of CPA-induced receptor internalization (1 h), down-regulation (24 h), inhibition of forskolin-stimulated cAMP accumulation, or activation of co-transfected G-protein-activated inward rectifier K(+)/cardiac inward rectifying K(+) (GIRK1/CIR K(+)) channels. Also unaffected by palmitoylation is guanosine 5'-[gamma-thio]-triphosphate ([S]GTP)-sensitive binding to membranes by the agonist (125)I-labelled aminobenzyladenosine. The results suggest that palmitoylation has little effect on receptor-effector coupling, agonist-induced internalization or down-regulation. We speculate that palmitoylation may divert newly synthesized A(1)ARs from a pathway leading to rapid degradation. PMID:10455026

  4. Key modulatory role of presynaptic adenosine A2A receptors in cortical neurotransmission to the striatal direct pathway.

    PubMed

    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

    2009-11-18

    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 antiparkinsonian 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 corticostriatal neurotransmission. Presynaptic striatal A2A receptors could provide a new target for the treatment of neuropsychiatric disorders.

  5. 2-Alkynyl derivatives of adenosine-5'-N-ethyluronamide: selective A2 adenosine receptor agonists with potent inhibitory activity on platelet aggregation.

    PubMed

    Cristalli, G; Volpini, R; Vittori, S; Camaioni, E; Monopoli, A; Conti, A; Dionisotti, S; Zocchi, C; Ongini, E

    1994-05-27

    A series of new 2-alkynyl and 2-cycloalkynyl derivatives of adenosine-5'-N-ethyluronamide (NECA) and of N-ethyl-1'-deoxy-1'-(6-amino-2-hexynyl-9H-purin-9-yl)-beta-D- ribofuranuronamide (1, HE-NECA), bearing hydroxy, amino, chloro, and cyano groups in the side chain, were synthesized. The compounds were studied in binding and functional assays to assess their potency for the A2 compared to A1 adenosine receptor. The presence of an alpha-hydroxyl group in the alkynyl chain of NECA derivatives accounts for the A2 agonist potency, leading to compounds endowed with sub-nanomolar affinity in binding studies. However, these analogues also possess good A1 receptor affinity resulting in low A2 selectivity. From functional experiments the 4-hydroxy-1-butynyl (6) and the 4-(2-tetrahydro-2H-pyranyloxy)-1-butynyl (16) derivatives appear to be very potent in inducing vasorelaxation without appreciable effect on heart rate. The new compounds were also tested as inhibitors of platelet aggregation induced by ADP. Introduction of an alpha-hydroxyl group in the alkynyl side chain caused a greater increase in antiaggregatory activity than either NECA or HE-NECA, resulting in the most potent inhibitors of platelet aggregation so far known in the nucleoside series. The presence of an alpha-quaternary carbon such as the 3-hydroxy-3,5-dimethyl-1-hexynyl (12) and the 3-hydroxy-3-phenyl-1-butynyl (15) derivatives markedly reduced the antiaggregatory potency without affecting the A2 affinity. The hydrophobicity index (k') of the new nucleosides barely correlated with the binding data, whereas high k' values were associated with increased A2 vs A1 selectivity but with reduced activity in all functional assays. Some of the compounds synthesized possess interesting pharmacological properties. Compounds having an appropriate balance between vasorelaxation and antiplatelet activity, if confirmed in vivo, deserve further development for the treatments of cardiovascular disorders.

  6. Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site.

    PubMed Central

    Vandenberg, R J; French, C R; Barry, P H; Shine, J; Schofield, P R

    1992-01-01

    The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily. PMID:1311851

  7. Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator.

    PubMed

    Montesinos, M Carmen; Desai-Merchant, Avani; Cronstein, Bruce N

    2015-12-01

    Impaired wound healing, as it occurs in diabetes mellitus or long-term corticoid treatment, is commonly associated with disability, diminished quality of life, and high economic costs. Selective agonists of the A2A receptor subtype of adenosine, an endogenous regulator of inflammation, promote tissue repair in animal models, both healthy and with impaired healing. Plasmin-mediated proteolysis of fibrin and other matrix proteins is essential for cell migration at sites of injury. Since adenosine A2A receptor activation increases plasminogen activator release from macrophages and mast cells, we studied the effect of a selective agonist, CGS-21680, on full-thickness excisional wound closure in wild-type, urokinase plasminogen activator (uPA)-deficient, and tissue plasminogen activator (tPA)-deficient mice. Wound closure was impaired in tPA- and uPA-deficient mice as compared with wild-type mice, and topical application of CGS-21680 significantly increased the rate at which wounds closed in wild-type mice and uPA-deficient mice, but not in tPA-deficient mice. Immunostaining of tissue sections showed that tPA was present in endothelial cells and histiocytes by day 3 post-wound and also by day 6. In contrast, uPA was more prominent in these cell types only by day 6 post-wound. Our results confirm that plasminogen activation contributes to wound repair and are consistent with the hypothesis that adenosine A2A receptor activation promotes wound closure by a mechanism that depends upon tPA, but not uPA. Moreover, our results suggest that topical adenosine A2A receptor agonists may be useful in promotion of wound closure in patients with impaired wound healing.

  8. Doxazosin inhibits proliferation and migration of human vascular smooth-muscle cells independent of alpha1-adrenergic receptor antagonism.

    PubMed

    Hu, Z W; Shi, X Y; Hoffman, B B

    1998-06-01

    Proliferation and migration of vascular smooth-muscle cells (VSMCs), stimulated by a variety of growth factors, play a critical role in the pathogenesis of vascular diseases. We found unexpectedly that doxazosin, an alpha1-adrenergic-receptor antagonist, inhibits serum-stimulated proliferation of cultured human VSMCs. Subsequent experiments systematically investigated inhibitory effects of doxazosin on mitogenesis stimulated in VSMCs by platelet-derived growth factor (PDGF), epidermal growth factor, and G protein-coupled receptor agonists thrombin and angiotensin II. Doxazosin attenuated the stimulation of DNA synthesis for each of these ligands with median inhibitory concentrations (IC50s) from 0.3 to 1 microM. PDGF-AB (1 nM) increased cell number; doxazosin inhibited this response by 70-80%. Prazosin, a related alpha1-receptor antagonist, had similar but less potent effects on inhibiting mitogenesis in these cells. Doxazosin and prazosin inhibited PDGF-AB-stimulated and insulin-like growth factor (IGF-I)-stimulated migration of VSMCs by approximately 40-50%. These effects of doxazosin were likely unrelated to alpha1-receptor blockade because pretreatment of cells with phenoxybenzamine, an irreversible alpha1 antagonist, did not change the capacity of doxazosin to inhibit of PDGF-stimulated mitogenesis. Also, doxazosin inhibited PDGF-stimulated DNA synthesis in NIH 3T3 cells, which do not express alpha1 receptors. These results suggest that doxazosin is a potent inhibitor of VSMC proliferation and migration through a mechanism unrelated to alpha1-receptor antagonism.

  9. Effect of a toggle switch mutation in TM6 of the human adenosine A3 receptor on Gi protein-dependent signalling and Gi-independent receptor internalization

    PubMed Central

    Stoddart, Leigh A; Kellam, Barrie; Briddon, Stephen J; Hill, Stephen J

    2014-01-01

    BACKGROUND AND PURPOSE The highly conserved tryptophan (W6.48) in transmembrane domain 6 of GPCRs has been shown to play a central role in forming an active conformation in response to agonist binding. We set out to characterize the effect of this mutation on the efficacy of two agonists at multiple signalling pathways downstream of the adenosine A3 receptor. EXPERIMENTAL APPROACH Residue W6.48 in the human adenosine A3 receptor fused to yellow fluorescent protein was mutated to phenylalanine and expressed in CHO-K1 cells containing a cAMP response element reporter gene. The effects on agonist-mediated receptor internalization were monitored by automated confocal microscopy and image analysis. Further experiments were carried out to investigate agonist-mediated ERK1/2 phosphorylation, inhibition of [3H]-cAMP accumulation and β-arrestin2 binding. KEY RESULTS NECA was able to stimulate agonist-mediated internalization of the W6.48F mutant receptor, while the agonist HEMADO was inactive. Investigation of other downstream signalling pathways indicated that G-protein coupling was impaired for both agonists tested. Mutation of W6.48F therefore resulted in differential effects on agonist efficacy, and introduced signalling pathway bias for HEMADO at the adenosine A3 receptor. CONCLUSIONS AND IMPLICATIONS Investigation of the pharmacology of the W6.48F mutant of the adenosine A3 receptor confirms that this region is important in forming the active conformation of the receptor for stimulating a number of different signalling pathways and that mutations in this residue can lead to changes in agonist efficacy and signalling bias. PMID:24750014

  10. 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

  11. Pharmacological and biochemical characterization of purified A2a adenosine receptors in human platelet membranes by [3H]-CGS 21680 binding.

    PubMed Central

    Varani, K.; Gessi, S.; Dalpiaz, A.; Borea, P. A.

    1996-01-01

    1. The binding properties of human platelet A2a adenosine receptors, assayed with the A2a-selective agonist, [3H]-2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoad enosine ([3H]-CGS 21680), are masked by a non-receptorial component, the adenotin site. In order to separate A2a receptors from adenotin sites, human platelet membranes were solubilized with 1% 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulphonate (CHAPS). The soluble platelet extract was precipitated with polyethylene glycol (PEG) and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. 2. The present paper describes the binding characteristics of the selective A2a agonist, [3H]-CGS 21680, to this purified platelet membrane preparation. In addition, receptor affinity and potency of several adenosine agonists and antagonists were determined in binding and adenylyl cyclase studies. 3. Saturation experiments revealed a single class of binding site with Kd and Bmax values of 285 nM and 2.07 pmol mg-1 of protein respectively. Adenosine receptor ligands competed for the binding of 50 nM [3H]-CGS 21680 to purified protein, showing a rank order of potency consistent with that typically found for interactions with the A2a adenosine receptors. In the adenylyl cyclase assay the compounds examined exhibited a rank order of potency very close to that observed in binding experiments. 4. Thermodynamic data indicated that [3H]-CGS 21680 binding to the purified receptor is totally entropy-driven in agreement with results obtained in rat striatal A2a adenosine receptors. 5. It is concluded that in the purified platelet membranes there is a CGS 21680 binding site showing the characteristic properties of the A2a receptor. This makes it possible to use this compound for reliable radioligand binding studies on the A2a adenosine receptor of human platelets. PMID:8732278

  12. The Reno-Vascular A2B Adenosine Receptor Protects the Kidney from Ischemia

    PubMed Central

    Grenz, Almut; Osswald, Hartmut; Eckle, Tobias; Yang, Dan; Zhang, Hua; Tran, Zung Vu; Klingel, Karin; Ravid, Katya; Eltzschig, Holger K

    2008-01-01

    Background Acute renal failure from ischemia significantly contributes to morbidity and mortality in clinical settings, and strategies to improve renal resistance to ischemia are urgently needed. Here, we identified a novel pathway of renal protection from ischemia using ischemic preconditioning (IP). Methods and Findings For this purpose, we utilized a recently developed model of renal ischemia and IP via a hanging weight system that allows repeated and atraumatic occlusion of the renal artery in mice, followed by measurements of specific parameters or renal functions. Studies in gene-targeted mice for each individual adenosine receptor (AR) confirmed renal protection by IP in A1−/−, A2A−/−, or A3AR−/− mice. In contrast, protection from ischemia was abolished in A2BAR−/− mice. This protection was associated with corresponding changes in tissue inflammation and nitric oxide production. In accordance, the A2BAR-antagonist PSB1115 blocked renal protection by IP, while treatment with the selective A2BAR-agonist BAY 60–6583 dramatically improved renal function and histology following ischemia alone. Using an A2BAR-reporter model, we found exclusive expression of A2BARs within the reno-vasculature. Studies using A2BAR bone-marrow chimera conferred kidney protection selectively to renal A2BARs. Conclusions These results identify the A2BAR as a novel therapeutic target for providing potent protection from renal ischemia. PMID:18578565

  13. 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

  14. 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

  15. Macrophage A2A Adenosine Receptors Are Essential to Protect from Progressive Kidney Injury.

    PubMed

    Truong, Luan D; Trostel, Jessica; McMahan, Rachel; Chen, Jiang-Fan; Garcia, Gabriela E

    2016-10-01

    A2A adenosine receptors (A2ARs) are endogenous inhibitor of inflammation. Macrophages that are key effectors of kidney disease progression express A2ARs. We investigated the role of A2ARs in kidney inflammation in a macrophage-mediated anti-glomerular basement membrane reactive serum-induced immune nephritis in A2AR-deficient mice. Sub-threshold doses of glomerular basement membrane-reactive serum induced more severe and prolonged kidney damage with higher levels of proinflammatory cytokines and greater accumulation of inflammatory cells in A2AR(-/-) mice than wild-type (WT) mice. To investigate the role of macrophage A2AR in progressive kidney injury, glomerulonephritis was induced in CD11b-DTR transgenic mice. Macrophages were selectively depleted in the established phase of the disease and reconstituted with macrophages from WT or A2AR-deficient mice and then treated with an A2AR agonist. In mice receiving WT macrophages and treated with an A2AR agonist, the glomerular cellularity, crescent formation, sclerotic glomeruli, and tubulointerstitial injury were significantly reduced compared with the control group. In contrast, in mice reconstituted with A2AR-deficient macrophages and treated with an A2AR agonist, the kidney injury was more severe with increased deposition of collagen I, III, and IV. These findings suggest that disruption of the protective A2AR amplifies inflammation to accelerate glomerular damage and endogenous macrophage A2ARs are essential to protect from progressive kidney fibrosis.

  16. Control and Function of the Homeostatic Sleep Response by Adenosine A1 Receptors

    PubMed Central

    Bjorness, Theresa E.; Kelly, Christine L.; Gao, Tianshu; Poffenberger, Virginia; Greene, Robert W.

    2009-01-01

    During sleep, the mammalian CNS undergoes widespread, synchronized slow wave activity (SWA) that directly varies with prior waking duration (Borbely, 1982;Dijk et al., 1990a). When sleep is restricted, an enhanced SWA response follows in the next sleep period. The enhancement of SWA is associated with improved cognitive performance (Huber et al., 2004c), but it is unclear either how the SWA is enhanced or whether SWA is needed to maintain normal cognitive performance. A conditional, CNS knockout of the adenosine receptor, AdoA1R gene, shows selective attenuation of the SWA rebound response to restricted sleep, but sleep duration is not affected. During sleep restriction, wild phenotype animals, express a rebound SWA response and maintain cognitive performance in a working memory task. However, the knockout animals not only show a reduced rebound SWA response but they also fail to maintain normal cognitive function, although this function is normal when sleep is not restricted. Thus, AdoA1R activation is needed for normal rebound SWA, and when the SWA rebound is reduced, there is a failure to maintain working memory function suggesting a functional role for SWA homeostasis. PMID:19193874

  17. Effects of a selective adenosine A1 receptor antagonist on the development of cyclosporin nephrotoxicity.

    PubMed Central

    Balakrishnan, V. S.; von Ruhland, C. J.; Griffiths, D. F.; Coles, G. A.; Williams, J. D.

    1996-01-01

    1. The clinical application of cyclosporin as an immunosuppressive agent is limited by its nephrotoxicity. 2. The effect of FK453, a selective A1-receptor antagonist, administered twice daily to rats at a dose of 100 mg kg-1 was assessed on the development of nephrotoxicity induced by cyclosporin (10 mg kg-1 i.p. daily) administered for 14 days. The effects of nifedipine administered twice daily (0.3 mg kg-1 s.c.) for 14 days, on cyclosporin nephrotoxicity were also studied. 3. Cyclosporin induced a 46.58% and 35.78% decline in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) respectively and a reduction of 16.69% in filtration fraction (FF). Co-administration of FK453 resulted in falls of 30.5%, 18.59% and 14.7% in GFR, ERPF and FF respectively, the former two significantly less than the falls seen with cyclosporin (CyA) alone (P < 0.05 vs CyA, ANOVA). 4. Nifedipine appeared to have a more pronounced protective effect resulting in a decline of only 20.91% in GFR, with no significant change in ERPF (increase of 0.93%) when co-administered with CyA. 5. These observations indicate adenosine plays a minor role in the pathophysiology of CyA nephrotoxicity. PMID:8851505

  18. 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.

  19. Adenosine (ADO) receptors in the PC12 phenochromocytoma cell line: characterization by radioligand binding

    SciTech Connect

    Williams, M.; Abreu, M.; Noronha-Blob, L.

    1986-03-01

    PC12 cells contain an ado-sensitive adenylate cyclase which is exclusively A-2 in nature. Binding of A-1 selective radioligands (Cyclohexylado; CHA and Cyclopentylado; CPA) to PC12 membranes is minimal and irreproducible. In contrast, the non-selective A-1/A-2 agonist NECA (5'-N-ethylcarboxamidoado) binds with high affinity to two sites in adenosine-deaminase pretreated PC12 membranes. Using computerized curve fitting, the first site had a Kd value of 5.2 +/- 1.5 nM (mean +/- s.d.; n = 8) and an apparent Bmax of 205 fmoles/mg prot. Due to large increases in non-specific binding, the second site could not be resolved but had a Kd in the range of 1 ..mu..M. Pharmacological analysis of specific NECA binding (5 nM) gave the rank order activity profile: NECA < MECA = 2-CADO > CHA > R-PIA > CPA S-PIA > AMP = ADP = ATP. Binding was also xanthine sensitive with PACPX > 8-PT > 8-PST. The ratio of activity for the R and S diastereomers of PIA was 10, consistent with the labeling of A-2-type ado receptors in the PC12 cell line.

  20. 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

  1. Characterization by flow cytometry of fluorescent, selective agonist probes of the A(3) adenosine receptor.

    PubMed

    Kozma, Eszter; Gizewski, Elizabeth T; Tosh, Dilip K; Squarcialupi, Lucia; Auchampach, John A; Jacobson, Kenneth A

    2013-04-15

    Various fluorescent nucleoside agonists of the A3 adenosine receptor (AR) were compared as high affinity probes using radioligands and flow cytometry (FCM). They contained a fluorophore linked through the C2 or N(6) position and rigid A3AR-enhancing (N)-methanocarba modification. A hydrophobic C2-(1-pyrenyl) derivative MRS5704 bound nonselectively. C2-Tethered cyanine5-dye labeled MRS5218 bound selectively to hA3AR expressed in whole CHO cells and membranes. By FCM, binding was A3AR-mediated (blocked by A3AR antagonist, at least half through internalization), with t1/2 for association 38min in mA3AR-HEK293 cells; 26.4min in sucrose-treated hA3AR-CHO cells (Kd 31nM). Membrane binding indicated moderate mA3AR affinity, but not selectivity. Specific accumulation of fluorescence (50nM MRS5218) occurred in cells expressing mA3AR, but not other mouse ARs. Evidence was provided suggesting that MRS5218 detects endogenous expression of the A3AR in the human promyelocytic leukemic HL-60 cell line. Therefore, MRS5218 promises to be a useful tool for characterizing the A3AR.

  2. Pharmacological Preconditioning by Adenosine A2a Receptor Stimulation: Features of the Protected Liver Cell Phenotype

    PubMed Central

    Alchera, Elisa; Imarisio, Chiara; Mandili, Giorgia; Merlin, Simone; Chandrashekar, Bangalore R.; Novelli, Francesco; Follenzi, Antonia; Carini, Rita

    2015-01-01

    Ischemic preconditioning (IP) of the liver by a brief interruption of the blood flow protects the damage induced by a subsequent ischemia/reperfusion (I/R) preventing parenchymal and nonparenchymal liver cell damage. The discovery of IP has shown the existence of intrinsic systems of cytoprotection whose activation can stave off the progression of irreversible tissue damage. Deciphering the molecular mediators that underlie the cytoprotective effects of preconditioning can pave the way to important therapeutic possibilities. Pharmacological activation of critical mediators of IP would be expected to emulate or even to intensify its salubrious effects. In vitro and in vivo studies have demonstrated the role of the adenosine A2a receptor (A2aR) as a trigger of liver IP. This review will provide insight into the phenotypic changes that underline the resistance to death of liver cells preconditioned by pharmacological activation of A2aR and their implications to develop innovative strategies against liver IR damage. PMID:26539478

  3. Adenosine A2A receptors modulate glutamate uptake in cultured astrocytes and gliosomes.

    PubMed

    Matos, Marco; Augusto, Elisabete; Santos-Rodrigues, Alexandre Dos; Schwarzschild, Michael A; Chen, Jiang-Fan; Cunha, Rodrigo A; Agostinho, Paula

    2012-05-01

    Glutamate is the primary excitatory neurotransmitter in the central nervous system, where its toxic build-up leads to synaptic dysfunction and excitotoxic cell death that underlies many neurodegenerative diseases. Therefore, efforts have been made to understand the regulation of glutamate transporters, which are responsible for the clearance of extracellular glutamate. We now report that adenosine A(2A) receptors (A(2A) R) control the uptake of D-aspartate in primary cultured astrocytes as well as in an ex vivo preparation enriched in glial plasmalemmal vesicles (gliosomes) from adult rats, whereas A(1) R and A(3) R were devoid of effects. Thus, the acute exposure to the A(2A) R agonist, CGS 21680, inhibited glutamate uptake, an effect prevented by the A(2A) R antagonist, SCH 58261, and abbrogated in cultured astrocytes from A(2A) R knockout mice. Furthermore, the prolonged activation of A(2A) R lead to a cAMP/protein kinase A-dependent reduction of GLT-I and GLAST mRNA and protein levels, which leads to a sustained decrease of glutamate uptake. This dual mechanism of inhibition of glutamate transporters by astrocytic A(2A) R provides a novel candidate mechanism to understand the ability of A(2) (A) R to control synaptic plasticity and neurodegeneration, two conditions tightly associated with the control of extracellular glutamate levels by glutamate transporters.

  4. Unsurmountable antagonism of brain 5-hydroxytryptamine2 receptors by (+)-lysergic acid diethylamide and bromo-lysergic acid diethylamide.

    PubMed

    Burris, K D; Sanders-Bush, E

    1992-11-01

    Lysergic acid diethylamide (LSD) and its structural analogue 2-bromo-lysergic acid diethylamide (BOL) act as unsurmountable antagonists of serotonin-elicited contractions in smooth muscle preparations. Two different models, allosteric and kinetic, have been invoked to explain these findings. The present studies investigate the mechanism of antagonism of brain 5-hydroxytryptamine (5HT)2 receptors, utilizing cells transfected with 5HT2 receptor cDNA cloned from rat brain. A proximal cellular response, phosphoinositide hydrolysis, was examined in order to minimize possible postreceptor effects. Even though LSD behaved as a partial agonist and BOL as a pure antagonist, both drugs blocked the effect of serotonin in an unsurmountable manner, i.e., increasing concentrations of serotonin could not overcome the blocking effect of LSD or BOL. Radioligand binding studies showed that preincubation of membranes with either LSD or BOL reduced the density of [3H]ketanserin binding sites, suggesting that the drugs bind tightly to the 5HT2 receptor and are not displaced during the binding assay. Two additional experiments supported this hypothesis. First, the off-rate of [3H] LSD was slow (20 min), relative to that of [3H]ketanserin (approximately 4 min). Second, when the length of incubation with [3H]ketanserin was increased to 60 min, the LSD-induced decrease in Bmax was essentially eliminated. The possibility that LSD and BOL decrease [3H]ketanserin binding by interacting with an allosteric site was rejected, because neither drug altered the rate of dissociation of [3H]ketanserin. The most parsimonious interpretation of these results is that unsurmountable antagonism reflects prolonged occupancy of the receptor by slowly reversible antagonists.

  5. 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

  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. Adenosine A2A receptor stimulation decreases GAT-1-mediated GABA uptake in the globus pallidus of the rat.

    PubMed

    Gonzalez, Brenda; Paz, Francisco; Florán, Leonor; Aceves, Jorge; Erlij, David; Florán, Benjamín

    2006-07-01

    We examined modulation of [(3)H]GABA uptake in slices of the rat globus pallidus because stimulation of adenosine A(2A) receptors increases extracellular GABA in this structure. Pharmacological analysis showed that GAT-1 is the main transporter present in these slices. Both adenosine and the A(2A) agonist CGS 21680 reduced GABA uptake. Antagonist ZM 241385 prevented these effects. Agents that increase protein kinase A activity like forskolin and 8-bromo-cAMP also inhibited GABA uptake. The inhibition of uptake produced by these substances and by CGS 21680 was prevented by the protein kinase A blocker H-89. The protein phosphatase blocker okadaic acid reduced uptake; this effect and the response to CGS 21680 were not additive. The effective concentrations of adenosine (EC(50)=15.2microM) are within the range measured in the interstitial fluid under some physiological conditions. Thus, inhibition of uptake may be important in increasing interstitial GABA during endogenous adenosine release.

  8. 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

  9. 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

  10. Colocalization and regulated physical association of presynaptic serotonin transporters with A₃ adenosine receptors.

    PubMed

    Zhu, Chong-Bin; Lindler, Kathryn M; Campbell, Nicholas G; Sutcliffe, James S; Hewlett, William A; Blakely, Randy D

    2011-09-01

    Activation of A₃ adenosine receptors (A₃ARs) rapidly enhances the activity of antidepressant-sensitive serotonin (5-HT) transporters (SERTs) in vitro, ex vivo, and in vivo. A₃AR agonist stimulation of SERT activity is lost in A₃AR knockout mice. A₃AR-stimulated SERT activity is mediated by protein kinase G1 (PKGI)- and p38 mitogen-activated protein kinase (MAPK)-linked pathways that support, respectively, enhanced SERT surface expression and catalytic activation. The mechanisms by which A₃ARs target SERTs among other potential effectors is unknown. Here we present evidence that A₃ARs are coexpressed with SERT in midbrain serotonergic neurons and form a physical complex in A₃AR/hSERT cotransfected cells. Treatment of A₃AR/SERT-cotransfected Chinese hamster ovary cells with the A₃AR agonist N⁶-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (1 μM, 10 min), conditions previously reported to increase SERT surface expression and 5-HT uptake activity, enhanced the abundance of A₃AR/SERT complexes in a PKGI-dependent manner. Cotransfection of SERT with L90V-A₃AR, a hyperfunctional coding variant identified in subjects with autism spectrum disorder, resulted in a prolonged recovery of receptor/transporter complexes after A₃AR activation. Because PKGI and nitric-oxide synthetase are required for A₃AR stimulation of SERT activity, and proteins PKGI and NOS both form complexes with SERT, our findings suggest a mechanism by which signaling pathways coordinating A₃AR signaling to SERT can be spatially restricted and regulated, as well as compromised by neuropsychiatric disorders.

  11. 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.

  12. Effect of pulsed electromagnetic field exposure on adenosine receptors in rat brain.

    PubMed

    Varani, Katia; Vincenzi, Fabrizio; Targa, Martina; Corciulo, Carmen; Fini, Milena; Setti, Stefania; Cadossi, Ruggero; Borea, Pier Andrea

    2012-05-01

    Different effects of pulsed electromagnetic field (PEMF) exposure on brain tissue have been described in pre-clinical models and in clinical settings. Nevertheless, the mechanism of action and the possible interaction with membrane receptors such as adenosine receptors (ARs) has not been investigated. The present study focused on the effect of PEMFs on A1 and A2A ARs in the rat cerebral cortex and cortical neurons. Affinity and density of ARs were evaluated by means of saturation binding experiments while mRNA expression was investigated through retro-transcription polymerase chain reaction (RT-PCR). PEMF treatment of the intact rat cerebral cortex or cortical neurons at 1.5 mT mediated a transient and significant increase in A2A ARs after 4 h (2.0-fold increase) and 6 h (1.4- and 1.8-fold increase, respectively) of exposure. In addition, PEMF treatment of the rat cerebral cortex and rat cortical neurons at 3 mT upregulated A2A ARs after 2 h (2.0- and 2.2-fold increase, respectively) and 4 h (1.6- and 1.9-fold increase, respectively). The treatment of rat cortex membranes with PEMFs at 1.5 and 3 mT induced an increase in A2A AR density after 2 h (1.9- and 2.2-fold increase, respectively) and was constant at all incubation times investigated. In rat cortical neurons, mRNA levels of A1 and A2A ARs were not affected by PEMF exposure for the times and intensities used. These results suggest that PEMF treatment has different biological effects in whole organs or cells in comparison with isolated membranes.

  13. Presynaptic facilitatory adenosine A2A receptors mediate fade induced by neuromuscular relaxants that exhibit anticholinesterase activity.

    PubMed

    Bornia, Elaine Cs; Correia-de-Sá, Paulo; Alves-Do-Prado, Wilson

    2011-03-01

    1. Pancuronium, cisatracurium and vecuronium are antinicotinic agents that, in contrast with d-tubocurarine and hexamethonium, exhibit anticholinesterase activity. Pancuronium-, cisatracurium- and vecuronium-induced fade results from blockade of facilitatory nicotinic receptors on motor nerves, but fade produced by such agents also depends on the presynaptic activation of inhibitory muscarinic M2 receptors by acetylcholine released from motor nerve terminals and activation of inhibitory adenosine A1 receptors by adenosine released from motor nerves and muscles. The participation of presynaptic facilitatory A2A receptors in fade caused by pancuronium, cisatracurium and vecuronium has not yet been investigated. In the present study, we determined the effects of ZM241385, an antagonist of presynaptic facilitatory A2A receptors, on fade produced by these neuromuscular relaxants in the rat phrenic nerve-diaphragm (PND) preparation. 2. The muscles were stimulated indirectly at 75±3Hz to induce a sustained tetanizing muscular contraction. The lowest concentration at which each antinicotinic agent produced fade without modifying initial tetanic tension (presynaptic action) was determined. 3. d-Tubocurarine-induced fade occurred only at 55 nmol/L, a concentration that also reduced maximal tetanic tension (post-synaptic action). At 10 nmol/L, ZM 241385 alone did not produce fade, but it did attenuate pancuronium (0.32 μmol/L)-, cisatracurium (0.32 μmol/L)- and vecuronium (0.36 μmol/L)-induced fade. 4. The fade induced by the 'pure' antinicotinic agents d-tubocurarine (55 nmol/L) and hexamethonium (413 μmol/L) was not altered by 10 nmol/L ZM 241385, indicating that presynaptic adenosine A2A receptors play a significant role in the fade produced by antinicotinic agents when such agents have anticholinesterase activity.

  14. Blockade of adenosine A2A receptors prevents protein phosphorylation in the striatum induced by cortical stimulation.

    PubMed

    Quiroz, César; Gomes, Catarina; Pak, Arlene C; Ribeiro, Joaquim A; Goldberg, Steven R; Hope, Bruce T; Ferré, Sergi

    2006-10-18

    Previous studies have shown that cortical stimulation selectively activates extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and immediate early gene expression in striatal GABAergic enkephalinergic neurons. In the present study, we demonstrate that blockade of adenosine A2A receptors with caffeine or a selective A2A receptor antagonist counteracts the striatal activation of cAMP-protein kinase A cascade (phosphorylation of the Ser845 residue of the glutamate receptor 1 subunit of the AMPA receptor) and mitogen-activated protein kinase (ERK1/2 phosphorylation) induced by the in vivo stimulation of corticostriatal afferents. The results indicate that A2A receptors strongly modulate the efficacy of glutamatergic synapses on striatal enkephalinergic neurons.

  15. PD 115,199: an antagonist ligand for adenosine A2 receptors

    SciTech Connect

    Bruns, R.F.; Fergus, J.H.; Badger, E.W.; Bristol, J.A.; Santay, L.A.; Hays, S.J.

    1986-03-05

    PD 115,199, N-(2-(dimethylamino)ethyl)-N-methyl-4-((2,3,6,-7-tetrahydro)-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)benzenesulfonamide, is an adenosine (ado) antagonist with high affinity at both the A1 and A2 subtypes of ado receptor, with an IC50 of 24 nM in (/sup 3/H)CHA binding to A1 receptors and 23 nM in (/sup 3/H)NECA binding to A2 receptors. (/sup 3/H)PD 115,199 (126 Ci/mmol) was prepared by reduction of the diallyl analog. In rat striatal membranes, dose-inhibition curves for the A1-selective ado antagonist PD 116,948 vs 0.5 nM (/sup 3/H)PD 115,199 were markedly biphasic, with 21% of specific binding representing A1 sites with an IC50 for PD 116,948 of 0.52 nM, and the remaining 79% representing apparent A2 sites with an IC50 for PD 116,948 of 163 nM. Subsequent experiments were done in the presence of 20 nM PD 116,948 to eliminate A1 binding. Under these conditions, about 80% of binding was specific. (/sup 3/H)PD 115,199 bound with high affinity (K/sub d/ 10 nM) to a limited number of sites (Bmax 120 pmol/g wet weight). Affinities of compounds in (/sup 3/H)PD 115,199 binding closely paralleled their affinities in (/sup 3/H)NECA binding. Ado antagonists were generally several fold more active vs (/sup 3/H)PD 115,199 than vs (/sup 3/H)NECA, whereas the reverse was the case for agonists. Binding was much higher in striatum than in other brain areas. These results indicate that (/sup 3/H)PD 115,199 binds to a high-affinity A2 receptor.

  16. 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

  17. Adenosine A(2A) receptor modulation of hippocampal CA3-CA1 synapse plasticity during associative learning in behaving mice.

    PubMed

    Fontinha, Bruno M; Delgado-García, José M; Madroñal, Noelia; Ribeiro, Joaquim A; Sebastião, Ana M; Gruart, Agnès

    2009-06-01

    Previous in vitro studies have characterized the electrophysiological and molecular signaling pathways of adenosine tonic modulation on long-lasting synaptic plasticity events, particularly for hippocampal long-term potentiation (LTP). However, it remains to be elucidated whether the long-term changes produced by endogenous adenosine in the efficiency of synapses are related to those required for learning and memory formation. Our goal was to understand how endogenous activation of adenosine excitatory A(2A) receptors modulates the associative learning evolution in conscious behaving mice. We have studied here the effects of the application of a highly selective A(2A) receptor antagonist, SCH58261, upon a well-known associative learning paradigm-classical eyeblink conditioning. We used a trace paradigm, with a tone as the conditioned stimulus (CS) and an electric shock presented to the supraorbital nerve as the unconditioned stimulus (US). A single electrical pulse was presented to the Schaffer collateral-commissural pathway to evoke field EPSPs (fEPSPs) in the pyramidal CA1 area during the CS-US interval. In vehicle-injected animals, there was a progressive increase in the percentage of conditioning responses (CRs) and in the slope of fEPSPs through conditioning sessions, an effect that was completely prevented (and lost) in SCH58261 (0.5 mg/kg, i.p.) -injected animals. Moreover, experimentally evoked LTP was impaired in SCH58261-injected mice. In conclusion, the endogenous activation of adenosine A(2A) receptors plays a pivotal effect on the associative learning process and its relevant hippocampal circuits, including activity-dependent changes at the CA3-CA1 synapse.

  18. Selective ligands for rat A3 adenosine receptors: structure-activity relationships of 1,3-dialkylxanthine 7-riboside derivatives.

    PubMed

    Kim, H O; Ji, X D; Melman, N; Olah, M E; Stiles, G L; Jacobson, K A

    1994-11-11

    1,3-Dibutylxanthine 7-riboside has been found to be a partial agonist at A3 adenosine receptors (van Galen et al. Mol. Pharmacol. 1994, 45, 1101-1111). 1,3-Dialkylxanthine 7-riboside analogues modified at the 1-, 3-, and 8-purine positions and at the ribose 5'-position were synthesized. The nucleoside analogues were examined for affinity in radioligand binding assays at rat brain A3 adenosine receptors stably expressed in CHO cells, using the radioligand [[125I]-4-amino-3-iodobenzyl]adenosine-5'-N-methyluronamide (AB-MECA). Affinity was assayed at rat brain A1 and A2a receptors using [3H]PIA and [3H]CGS 21680, respectively. The affinity of xanthine 7-ribosides at A3 receptors depended on the 1,3-dialkyl substituents in the order: Pent > or = Bu > Hx > Pr approximately Me. 1,3-Dipentylxanthine 7-riboside was slightly selective for A3 receptors (2-fold vs A1 and 10-fold vs A2a). 8-Methoxy substitution was tolerated at A3 receptors. 2-Thio vs 2-oxo substitution increased potency at all three subtypes and slightly increased A3 vs A1 selectivity. The 5'-uronamide modification, which was previously found to enhance A3 selectivity in N6-benzyladenosine derivatives, was also incorporated into the xanthine 7-ribosides, with similar results. The affinity of 1,3-dialkylxanthine 7-riboside 5'-uronamides at A3 receptors depended on the N-alkyluronamide substituent in the order: MeNH > EtNH > NH2 > Me2N. Affinity of the 5'-uronamides at A3 receptors was dependent on the 1,3-dialkyl substitution in the order: Bu > Pent > Hex. 1,3-Dibutylxanthine 7-riboside 5'-N-methylcarboxamide, with a Ki value of 229 nM at A3 receptors, was 160-fold selective for rat A3 vs A1 receptors and > 400-fold selective vs A2a receptors. This derivative acted as a full agonist in the A3 receptor-mediated inhibition of adenylate cyclase.

  19. Identification of a retinal aldosterone system and the protective effects of mineralocorticoid receptor antagonism on retinal vascular pathology.

    PubMed

    Wilkinson-Berka, Jennifer L; Tan, Genevieve; Jaworski, Kassie; Miller, Antonia G

    2009-01-01

    Blockade of the renin-angiotensin-aldosterone system (RAAS) is being evaluated as a treatment for diabetic retinopathy; however, whether the mineralocorticoid receptor (MR) and aldosterone influence retinal vascular pathology is unknown. We examined the effect of MR antagonism on pathological angiogenesis in rats with oxygen-induced retinopathy (OIR). To determine the mechanisms by which the MR and aldosterone may influence retinal angiogenesis; inflammation and glucose-6-phosphate dehydrogenase (G6PD) were evaluated in OIR and cultured bovine retinal endothelial cells (BRECs) and bovine retinal pericytes (BRPs). In OIR, MR antagonism (spironolactone) was antiangiogenic. Aldosterone may mediate the pathogenic actions of MR in the retina, with 11beta-hydroxysteroid dehydrogenase type 2 mRNA being detected and with aldosterone stimulating proliferation and tubulogenesis in BRECs and exacerbating angiogenesis in OIR, which was attenuated with spironolactone. The MR and aldosterone modulated retinal inflammation, with leukostasis and monocyte chemoattractant protein-1 mRNA and protein in OIR being reduced by spironolactone and increased by aldosterone. A reduction in G6PD may be an early response to aldosterone. In BRECs, BRPs, and early OIR, aldosterone reduced G6PD mRNA, and in late OIR, aldosterone increased mRNA for the NAD(P)H oxidase subunit Nox4. A functional retinal MR-aldosterone system was evident with MR expression, translocation of nuclear MR, and aldosterone synthase expression, which was modulated by RAAS blockade. We make the first report that MR and aldosterone influence retinal vasculopathy, which may involve inflammatory and G6PD mechanisms. MR antagonism may be relevant when developing treatments for retinopathies that target the RAAS.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. RADIOLABELING AND EFFICIENT SYNTHESIS OF TRITIATED 2-CHLORO-N6-(3-IODOBENZYL)ADENOSINE-5'-N-METHYLURON-AMIDE, A POTENT, SELECTIVE A3 ADENOSINE RECEPTOR AGONIST

    PubMed Central

    Kim, Hea O.; Hawes, Calvin; Towers, Pat; Jacobson, Kenneth A.

    2012-01-01

    SUMMARY We recently reported that 2-substitution of N6-benzyladenosine-5'-uronamides greatly enhances selectivity of agonists for rat A3 adenosine receptors J. Med. Chem. 1994, 37, 3614–3621). Specifically, 2-Chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (2-CI-IB-MECA), which displayed a K1 value of 0.33 nM, is the most selective for A3 receptors yet reported with selectivity versus A1 and A2a receptors of 2500- and 1400-fold, respectively. In order to obtain pharmacological tools for the study of A3 adenosine receptors, two routes for radiolabeling of 2-CI-IB-MECA through incorporation of tritium at the 5'-methylamido group were compared. One route formed a 2',3'-protected nucleoside 5'-carboxylic acid (9), which was condensed with methylamine and deprotected. The more efficient synthesis started from D-ribose and provided 2-CI-IB-MECA (12) in six steps with an overall yield of 5.6 %. Tritium was introduced in the penultimate step by heating N6-(3-iodobenzyl)-2-chloro-2',3'-di-O-acetyl-5'-(methoxycarbonyl)adenosine (17) with [3H]methylamine in methanol at 60 °C for 2 h. The specific activity of [3H]2-CI-IB-MECA was 29 Ci/mmol with a radiochemical purity of 99%. PMID:23598401

  5. Role of A2B Adenosine Receptors in Regulation of Paracrine Functions of Stem Cell Antigen 1-Positive Cardiac Stromal Cells

    PubMed Central

    Ryzhov, Sergey; Goldstein, Anna E.; Novitskiy, Sergey V.; Blackburn, Michael R.; Biaggioni, Italo

    2012-01-01

    The existence of multipotent cardiac stromal cells expressing stem cell antigen (Sca)-1 has been reported, and their proangiogenic properties have been demonstrated in myocardial infarction models. In this study, we tested the hypothesis that stimulation of adenosine receptors on cardiac Sca-1+ cells up-regulates their secretion of proangiogenic factors. We found that Sca-1 is expressed in subsets of mouse cardiac stromal CD31− and endothelial CD31+ cells. The population of Sca-1+CD31+ endothelial cells was significantly reduced, whereas the population of Sca-1+CD31− stromal cells was increased 1 week after myocardial infarction, indicating their relative functional importance in this pathophysiological process. An increase in adenosine levels in adenosine deaminase-deficient mice in vivo significantly augmented vascular endothelial growth factor (VEGF) production in cardiac Sca-1+CD31− stromal cells but not in Sca-1+CD31+ endothelial cells. We found that mouse cardiac Sca-1+CD31− stromal cells predominantly express mRNA encoding A2B adenosine receptors. Stimulation of adenosine receptors significantly increased interleukin (IL)-6, CXCL1 (a mouse ortholog of human IL-8), and VEGF release from these cells. Using conditionally immortalized Sca-1+CD31− stromal cells obtained from wild-type and A2B receptor knockout mouse hearts, we demonstrated that A2B receptors are essential for adenosine-dependent up-regulation of their paracrine functions. We found that the human heart also harbors a population of stromal cells similar to the mouse cardiac Sca-1+CD31− stromal cells that increase release of IL-6, IL-8, and VEGF in response to A2B receptor stimulation. Thus, our study identified A2B adenosine receptors on cardiac stromal cells as potential targets for up-regulation of proangiogenic factors in the ischemic heart. PMID:22431204

  6. Potential role of A2A adenosine receptor in traumatic optic neuropathy.

    PubMed

    Ahmad, Saif; Fatteh, Nadeem; El-Sherbiny, Nehal M; Naime, Mohammad; Ibrahim, Ahmed S; El-Sherbini, Ahmed M; El-Shafey, Sally A; Khan, Sohail; Fulzele, Sadanand; Gonzales, Joyce; Liou, Gregory I

    2013-11-15

    In traumatic optic neuropathy (TON), apoptosis of retinal ganglion cells is closely related to the local production of reactive oxygen species and inflammatory mediators from activated microglial cells. Adenosine receptor A2A (A2AAR) has been shown to possess anti-inflammatory properties that have not been studied in TON. In the present study, we examined the role of A2AAR in retinal complications associated with TON. Initial studies in wild-type mice revealed that treatment with the A2AAR agonist resulted in marked decreases in the TON-induced microglial activation, retinal cell death and releases of reactive oxygen species and pro-inflammatory cytokines TNF-α and IL-6. To further assess the role of A2AAR in TON, we studied the effects of A2AAR ablation on the TON-induced retinal abnormalities. A2AAR-/- mice with TON showed a significantly higher mRNA level of TNF-α, Iba1-1 in retinal tissue, and ICAM-1 expression in retinal sections compared with wild-type mice with TON. To explore a potential mechanism by which A2AAR-signaling regulates inflammation in TON, we performed additional studies using hypoxia- or LPS-treated microglial cells as an in vitro model for TON. Activation of A2AAR attenuates hypoxia or LPS-induced TNF-α release and significantly repressed the inflammatory signaling, ERK in the activated microglia. Collectively, this work provides pharmacological and genetic evidence for A2AAR signaling as a control point of cell death in TON and suggests that the retinal protective effect of A2AAR is mediated by attenuating the inflammatory response that occurs in microglia via interaction with MAPKinase pathway.

  7. 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

  8. Adenosine A(2A) receptor stimulation reduces inflammation and neointimal growth in a murine carotid ligation model.

    PubMed

    McPherson, J A; Barringhaus, K G; Bishop, G G; Sanders, J M; Rieger, J M; Hesselbacher, S E; Gimple, L W; Powers, E R; Macdonald, T; Sullivan, G; Linden, J; Sarembock, I J

    2001-05-01

    Endothelial activation and leukocyte recruitment are early events in atherosclerosis and the vascular response to injury. Adenosine has anti-inflammatory effects on leukocytes and endothelial cells mediated through its A(2A) receptor. We tested the hypothesis that A(2A) activation would reduce inflammation and neointimal formation in a murine carotid ligation model. Before injury, mice were randomized to a 7-day subcutaneous infusion of a specific A(2A) receptor agonist (ATL-146e, 0.004 microg/kg per minute), vehicle control, ATL-146e plus ZM241385 (a selective A(2A) antagonist), or ZM241385 alone. Leukocyte recruitment and adhesion molecule expression were assessed at early time points, and the neointimal area was measured at 14 and 28 days after injury. Compared with control mice, ATL-146e-treated mice had significantly less neutrophil and macrophage recruitment and vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and P-selectin expression in the first 7 days after injury. Neointimal area was markedly and persistently reduced by 80% at 14 and 28 days, despite termination of ATL infusion at 7 days. ATL-146e+ZM241385-treated and ZM241385-treated animals had neointimal areas similar to those of control animals, confirming that the observed effects of ATL-146e were mediated specifically by the A(2A) receptor. These data demonstrate that novel stimulation of adenosine A(2A) receptors can inhibit early inflammatory processes that are important in neointimal formation after vascular injury.

  9. 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

  10. 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…

  11. ATP-induced vasodilation and purinergic receptors in the human leg: roles of nitric oxide, prostaglandins, and adenosine.

    PubMed

    Mortensen, Stefan P; González-Alonso, José; Bune, Laurids T; Saltin, Bengt; Pilegaard, Henriette; Hellsten, Ylva

    2009-04-01

    Plasma ATP is thought to contribute to the local regulation of skeletal muscle blood flow. Intravascular ATP infusion can induce profound limb muscle vasodilatation, but the purinergic receptors and downstream signals involved in this response remain unclear. This study investigated: 1) the role of nitric oxide (NO), prostaglandins, and adenosine as mediators of ATP-induced limb vasodilation and 2) the expression and distribution of purinergic P(2) receptors in human skeletal muscle. Systemic and leg hemodynamics were measured before and during 5-7 min of femoral intra-arterial infusion of ATP [0.45-2.45 micromol/min] in 19 healthy male subjects with and without coinfusion of N(G)-monomethyl-l-arginine (l-NMMA; NO formation inhibitor; 12.3 +/- 0.3 (SE) mg/min), indomethacin (INDO; prostaglandin formation blocker; 613 +/- 12 microg/min), and/or theophylline (adenosine receptor blocker; 400 +/- 26 mg). During control conditions, ATP infusion increased leg blood flow (LBF) from baseline conditions by 1.82 +/- 0.14 l/min. When ATP was coinfused with either l-NMMA, INDO, or l-NMMA + INDO combined, the increase in LBF was reduced by 14 +/- 6, 15 +/- 9, and 39 +/- 8%, respectively (all P < 0.05), and was associated with a parallel lowering in leg vascular conductance and cardiac output and a compensatory increase in leg O(2) extraction. Infusion of theophylline did not alter the ATP-induced leg hyperemia or systemic variables. Real-time PCR analysis of the mRNA content from the vastus lateralis muscle of eight subjects showed the highest expression of P(2Y2) receptors of the 10 investigated P(2) receptor subtypes. Immunohistochemistry showed that P(2Y2) receptors were located in the endothelium of microvessels and smooth muscle cells, whereas P(2X1) receptors were located in the endothelium and the sacrolemma. Collectively, these results indicate that NO and prostaglandins, but not adenosine, play a role in ATP-induced vasodilation in human skeletal muscle. The expression

  12. Adenosine administration produces an antidepressant-like effect in mice: evidence for the involvement of A1 and A2A receptors.

    PubMed

    Kaster, Manuella P; Rosa, Angelo Oscar; Rosso, Matheus M; Goulart, Eduardo C; Santos, Adair R S; Rodrigues, Ana Lúcia S

    2004-01-23

    This study investigated the effect of adenosine in the forced swimming test (FST) and the tail suspension test (TST) in mice, and the contribution of adenosine A1 and A2A receptors to adenosine's antidepressant-like effect. The immobility time in the FST was reduced by adenosine given either by i.p. (5-10 mg/kg) or i.c.v. (0.01-10 microg/site) route. Adenosine (1-10 mg/kg, i.p.) also produced an antidepressant-like effect in the TST. No treatment affected locomotion in an open-field. The anti-immobility effect of adenosine (10 mg/kg, i.p.) in the FST was prevented by i.p. pretreatment of mice with caffeine (3 mg/kg), DPCPX (2 mg/kg) and ZM241385 (1 mg/kg). CHA (0.05 mg/kg, i.p.) and DPMA (1-5 mg/kg, i.p.) also produced an antidepressant-like effect in the FST. This is the first report of an antidepressant-like effect of adenosine in mice, apparently mediated through an interaction with A1 and A2A receptors.

  13. Update on the Mechanism of Action of Aripiprazole: Translational Insights into Antipsychotic Strategies Beyond Dopamine Receptor Antagonism.

    PubMed

    de Bartolomeis, Andrea; Tomasetti, Carmine; Iasevoli, Felice

    2015-09-01

    Dopamine partial agonism and functional selectivity have been innovative strategies in the pharmacological treatment of schizophrenia and mood disorders and have shifted the concept of dopamine modulation beyond the established approach of dopamine D2 receptor (D2R) antagonism. Despite the fact that aripiprazole was introduced in therapy more than 12 years ago, many questions are still unresolved regarding the complexity of the effects of this agent on signal transduction and intracellular pathways, in part linked to its pleiotropic receptor profile. The complexity of the mechanism of action has progressively shifted the conceptualization of this agent from partial agonism to functional selectivity. From the induction of early genes to modulation of scaffolding proteins and activation of transcription factors, aripiprazole has been shown to affect multiple cellular pathways and several cortical and subcortical neurotransmitter circuitries. Growing evidence shows that, beyond the consequences of D2R occupancy, aripiprazole has a unique neurobiology among available antipsychotics. The effect of chronic administration of aripiprazole on D2R affinity state and number has been especially highlighted, with relevant translational implications for long-term treatment of psychosis. The hypothesized effects of aripiprazole on cell-protective mechanisms and neurite growth, as well as the differential effects on intracellular pathways [i.e. extracellular signal-regulated kinase (ERK)] compared with full D2R antagonists, suggest further exploration of these targets by novel and future biased ligand compounds. This review aims to recapitulate the main neurobiological effects of aripiprazole and discuss the potential implications for upcoming improvements in schizophrenia therapy based on dopamine modulation beyond D2R antagonism.

  14. 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

  15. 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

  16. [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

  17. Regulation of lung surfactant secretion by the A sub 2 adenosine receptor

    SciTech Connect

    Gilfillan, A.M.; Gobran, L.I.; Rooney, S.A. )

    1987-05-01

    The authors previously reported that adenosine (A) stimulates secretion of phosphatidylcholine (PC), the major component of surfactant, in type II pneumocytes. To determine how this effect is mediated we examined the effect of P{sub 1} purinoceptor agonists -N{sup 6}-phenylisoprpyl-A (PIA), 5{prime}-N-ethylcarboxyamido-A (NECA), 2-chloro-A (CA) - and antagonists - theophylline (T) and 8-phenyltheophylline (8PT) - on PC secretion and cAMP levels in type II cells isolated from the adult rat. The cells were preincubated with {sup 3}H-choline for 20 h, transferred to fresh medium and incubated {plus minus} test agents for 1.5 h after which {sup 3}H-PC in the cells and medium was measured. A and its analogs stimulated PC secretion in a dose-dependent manner. At the optimal concentration (A, 1 mM; analogs, 0.01 mM) secretion was stimulated approx. 2-fold from a basal rate of 0.08-1.02% of total PC in the medium after 1.5 h. The potency order was NECA>CA=L-PIA>A>D-PIA. The EC{sub 50} for NECA was 8.9 {times} 10{sup {minus}8}M. The effect of NECA was significantly inhibited by 8PT (0.01 mM) and T (0.05 mM). NECA, A and L-PIA increased cellular cAMP levels 34, 12 and 8 fold, respectively, from a basal level of 0.23-0.28 pmol/10{sup 6} cells. These data suggest that the A{sub 2} subtype of the P{sub 1} receptor mediates the effect of A. In newborn rabbits, lung lavage PC increased form 24.1 {plus minus} 1.6 ug P/g lung dry wt at 0 h to 62.6 {plus minus} 7.7 after breathing for 3 h (n=13). 8PT (15 mg/kg, i.m.) at 0 h decreased the PC content at 3 h by 29% to 44.4 {plus minus} 5.1 ug/g. This suggests a functional role for the P{sub 1} receptor in lung surfactant secretion.

  18. 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

  19. Physiological roles of A1 and A2A adenosine receptors in regulating heart rate, body temperature, and locomotion as revealed using knockout mice and caffeine.

    PubMed

    Yang, Jiang-Ning; Chen, Jiang-Fan; Fredholm, Bertil B

    2009-04-01

    Heart rate (HR), body temperature (Temp), locomotor activity (LA), and oxygen consumption (O(2)C) were studied in awake mice lacking one or both of the adenosine A(1) or A(2A) receptors (A(1)R or A(2A)R, respectively) using telemetry and respirometry, before and after caffeine administration. All parameters were lower during day than night and higher in females than males. When compared with wild-type (WT) littermates, HR was higher in male A(1)R knockout (A(1)RKO) mice but lower in A(2A)RKO mice and intermediate in A(1)-A(2A)R double KO mice. A single dose of an unselective beta-blocker (timolol; 1 mg/kg) abolished the HR differences between these genotypes. Deletion of A(1)Rs had little effect on Temp, whereas deletion of A(2A)Rs increased it in females and decreased it in males. A(1)-A(2A)RKO mice had lower Temp than WT mice. LA was unaltered in A(1)RKO mice and lower in A(2A)RKO and A(1)-A(2A)RKO mice than in WT mice. Caffeine injection increased LA but only in mice expressing A(2A)R. Caffeine ingestion also increased LA in an A(2A)R-dependent manner in male mice. Caffeine ingestion significantly increased O(2)C in WT mice, but less in the different KO mice. Injection of 30 mg/kg caffeine decreased Temp, especially in KO mice, and hence in a manner unrelated to A(1)R or A(2A)R blockade. Selective A(2B) antagonism had little or no effect. Thus A(1)R and A(2A)R influence HR, Temp, LA, and O(2)C in mice in a sex-dependent manner, indicating effects of endogenous adenosine. The A(2A)R plays an important role in the modulation of O(2)C and LA by acute and chronic caffeine administration. There is also evidence for effects of higher doses of caffeine being independent of both A(1)R and A(2A)R.

  20. Alix/AIP1 antagonizes epidermal growth factor receptor downregulation by the Cbl-SETA/CIN85 complex.

    PubMed

    Schmidt, Mirko H H; Hoeller, Daniela; Yu, Jiuhong; Furnari, Frank B; Cavenee, Webster K; Dikic, Ivan; Bögler, Oliver

    2004-10-01

    The assembly of the Cbl-SETA/CIN85-endophilin complex at the C terminus of the epidermal growth factor receptor (EGFR) following ligand activation mediates its internalization and ubiquitination. We found that the SETA/CIN85-interacting protein Alix/AIP1, which also binds endophilins, modulates this complex. Alix was found to associate indirectly with EGFR, regardless of its activation state, and with DeltaEGFR, which signals at low intensity and does not bind Cbls or SETA/CIN85. In agreement with this, Alix interaction did not occur via SETA/CIN85. However, SETA/CIN85 and Alix were capable of mutually promoting their interaction with the EGFR. Increasing the level of Alix weakened the interaction between SETA/CIN85 and Cbl and reduced the tyrosine phosphorylation of c-Cbl and the level of ubiquitination of EGFR, SETA/CIN85, and Cbls. This antagonism of the Cbl-SETA/CIN85 complex by Alix was reflected in its diminution of EGFR internalization. In agreement with this, small interfering RNA-mediated knockdown of Alix promoted EGFR internalization and downregulation. It has been suggested that SETA/CIN85 promotes receptor internalization by recruiting endophilins. However, Alix was also capable of increasing the level of endophilin associated with EGFR, implying that this is not sufficient to promote receptor internalization. We propose that Alix inhibits EGFR internalization by attenuating the interaction between Cbl and SETA/CIN85 and by inhibiting Cbl-mediated ubiquitination of the EGFR.

  1. Role of adenosine A2A receptor signaling in the nicotine-evoked attenuation of reflex cardiac sympathetic control.

    PubMed

    El-Mas, Mahmoud M; El-Gowilly, Sahar M; Fouda, Mohamed A; Saad, Evan I

    2011-08-01

    Baroreflex dysfunction contributes to increased cardiovascular risk in cigarette smokers. Given the importance of adenosinergic pathways in baroreflex control, the hypothesis was tested that defective central adenosinergic modulation of cardiac autonomic activity mediates the nicotine-baroreflex interaction. Baroreflex curves relating changes in heart rate (HR) to increases or decreases in blood pressure (BP) evoked by i.v. doses (1-16μg/kg) of phenylephrine (PE) and sodium nitroprusside (SNP), respectively, were constructed in conscious rats; slopes of the curves were taken as measures of baroreflex sensitivity (BRS). Nicotine (25 and 100μg/kg i.v.) dose-dependently reduced BRS(SNP) in contrast to no effect on BRS(PE). BRS(SNP) was also attenuated after intracisternal (i.c.) administration of nicotine. Similar reductions in BRS(SNP) were observed in rats pretreated with atropine or propranolol. The combined treatment with nicotine and atropine produced additive inhibitory effects on BRS, an effect that was not demonstrated upon concurrent exposure to nicotine and propranolol. BRS(SNP) was reduced in preparations treated with i.c. 8-phenyltheophylline (8-PT, nonselective adenosine receptor antagonist), 8-(3-Chlorostyryl) caffeine (CSC, A(2A) antagonist), or VUF5574 (A(3) antagonist). In contrast, BRS(SNP) was preserved after blockade of A(1) (DPCPX) or A(2B) (alloxazine) receptors or inhibition of adenosine uptake by dipyridamole. CSC or 8-PT abrogated the BRS(SNP) depressant effect of nicotine whereas other adenosinergic antagonists were without effect. Together, nicotine preferentially impairs reflex tachycardia via disruption of adenosine A(2A) receptor-mediated facilitation of reflex cardiac sympathoexcitation. Clinically, the attenuation by nicotine of compensatory sympathoexcitation may be detrimental in conditions such as hypothalamic defense response, posture changes, and ventricular rhythms. PMID:21550361

  2. Expression pattern of FOS in orexin neurons during sleep induced by an adenosine A2A receptor agonist.

    PubMed

    Satoh, Shinsuke; Matsumura, Hitoshi; Kanbayashi, Takashi; Yoshida, Yasushi; Urakami, Takahito; Nakajima, Tomoko; Kimura, Nobuko; Nishino, Seiji; Yoneda, Hiroshi

    2006-06-30

    The present study examined the expression pattern of FOS in the hypothalamic peptide neurons during the sleep-dominant state induced by an adenosine A2A receptor agonist. The control rats, those that received the microdialysis-perfusion of their ventral striatum with artificial cerebrospinal fluid in the dark-active phase, spent 24% of the 90-min period prior to sacrifice in non-rapid eye movement (non-REM) sleep and 2.3% of that in REM sleep. These rats exhibited FOS, a transcription factor, in 21% of their orexin neurons and in 1.0% of their melanin-concentrating hormone (MCH) neurons in the perifornical/lateral hypothalamic areas. However, the rats perfused with 50 microM CGS21680, an adenosine A2A receptor agonist, spent 60% of the 90-min period prior to sacrifice in non-REM sleep and 11% of that in REM sleep. These rats exhibited FOS in 1.7% of their orexin neurons and FOS in 0.5% of their MCH neurons. When the sleep-dominant state was disturbed by mild stimulation and the rats were kept in the sleepy state by treatment with a sleep-inducing dose of CGS21680, the rats exhibited FOS in 13.3% of their orexin neurons, which percentage was about half of that for the control rats. These results suggest that the sleep-promoting process induced by this adenosine A2A receptor agonist was associated with a decline in the activity of orexin neurons. MCH neurons are not likely to change their activities during this sleep-promoting process.

  3. 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

  4. 1,2,4-Triazolo[1,5-a]quinoxaline derivatives: synthesis and biological evaluation as adenosine receptor antagonists.

    PubMed

    Catarzi, Daniela; Colotta, Vittoria; Varano, Flavia; Filacchioni, Guido; Martini, Claudia; Trincavelli, Letizia; Lucacchini, Antonio

    2004-02-01

    Since most of the reported adenosine receptor antagonists are 2-(hetero)aryl-substituted tricyclic heteroaromatic derivatives, in the present study we report the synthesis and the biological evaluation of a new set of 4-amino-1,2,4-triazolo[1,5-a]quinoxalines containing at position-2 an ethyl carboxylate group or a hydrogen atom. The structure-activity relationships on these compounds were in accordance with those of a previously reported series of analogous size and shape, thus suggesting a similar A(1)-binding mode. In particular, the binding data indicate that alkylation of the 4-amino group of these derivatives lead to potent A(1)-receptor antagonists. Moreover, as new results, this study has pointed out that the ethyl 2-carboxylate group can advantageously replace the 2-(hetero)aryl ring of previously reported triazoloquinoxaline derivatives, affording an ameliorated interaction with the A(1)-receptor subtype.

  5. 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.

  6. Regulation of Hippocampal Cannabinoid CB1 Receptor Actions by Adenosine A1 Receptors and Chronic Caffeine Administration: Implications for the Effects of Δ9-Tetrahydrocannabinol on Spatial Memory

    PubMed Central

    Sousa, Vasco C; Assaife-Lopes, Natália; Ribeiro, Joaquim A; Pratt, Judith A; Brett, Ros R; Sebastião, Ana M

    2011-01-01

    The cannabinoid CB1 receptor-mediated modulation of γ-aminobutyric acid (GABA) release from inhibitory interneurons is important for the integrity of hippocampal-dependent spatial memory. Although adenosine A1 receptors have a central role in fine-tuning excitatory transmission in the hippocampus, A1 receptors localized in GABAergic cells do not directly influence GABA release. CB1 and A1 receptors are the main targets for the effects of two of the most heavily consumed psychoactive substances worldwide: Δ9-tetrahydrocannabinol (THC, a CB1 receptor agonist) and caffeine (an adenosine receptor antagonist). We first tested the hypothesis that an A1–CB1 interaction influences GABA and glutamate release in the hippocampus. We found that A1 receptor activation attenuated the CB1-mediated inhibition of GABA and glutamate release and this interaction was manifested at the level of G-protein activation. Using in vivo and in vitro approaches, we then investigated the functional implications of the adenosine–cannabinoid interplay that may arise following chronic caffeine consumption. Chronic administration of caffeine in mice (intraperitoneally, 3 mg/kg/day, for 15 days, >12 h before trials) led to an A1-mediated enhancement of the CB1-dependent acute disruptive effects of THC on a short-term spatial memory task, despite inducing a reduction in cortical and hippocampal CB1 receptor number and an attenuation of CB1 coupling with G protein. A1 receptor levels were increased following chronic caffeine administration. This study shows that A1 receptors exert a negative modulatory effect on CB1-mediated inhibition of GABA and glutamate release, and provides the first evidence of chronic caffeine-induced alterations on the cannabinoid system in the cortex and hippocampus, with functional implications in spatial memory. PMID:20927050

  7. [3H]SCH 58261, a selective adenosine A2A receptor antagonist, is a useful ligand in autoradiographic studies.

    PubMed

    Fredholm, B B; Lindström, K; Dionisotti, S; Ongini, E

    1998-03-01

    We have characterized the new potent and selective nonxanthine adenosine A2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A2A receptors ([3H]CGS 21680) or A1 receptors (N6-[3H]cyclohexyladenosine), it was found that SCH 58261 is close to 800-fold selective for rat brain A2A versus A1 receptors (Ki values of 1.2 nM versus 0.8 microM). Moreover, receptor autoradiography showed that [3H]SCH 58261, in concentrations below 2 nM, binds only to the dopamine-rich regions of the rat brain, with a K(D) value of 1.4 (0.8-1.8) nM. The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 nM, the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [3H] SCH 58261 with the following estimated Ki values (nM): 2-hex-1-ynyl-5'-N-ethylcarboxamidoadenosine, 3.9 (1.8-8.4); CGS 21680, 130 (42-405); N6-cyclohexyladenosine, 9,985 (3,169-31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 microM) or Mg2+ (10 mM). The present results show that in its tritium-labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [3H]CGS 21680.

  8. A selective sigma-2 receptor ligand antagonizes cocaine-induced hyperlocomotion in mice.

    PubMed

    Lever, John R; Miller, Dennis K; Green, Caroline L; Fergason-Cantrell, Emily A; Watkinson, Lisa D; Carmack, Terry L; Fan, Kuo-Hsien; Lever, Susan Z

    2014-02-01

    Cocaine functions, in part, through agonist actions at sigma-1 (σ1 ) receptors, while roles played by sigma-2 (σ2 ) receptors are less established. Attempts to discriminate σ2 receptor-mediated effects of cocaine in locomotor hyperactivity assays have been hampered by the lack of potent and selective antagonists. Certain tetrahydroisoquinolinyl benzamides display high σ2 receptor affinity, and excellent selectivity for binding to σ2 over σ1 receptors. The behavioral properties of this structural class of σ ligands have not yet been investigated. The present study evaluated 5-bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl)]-2,3-dimethoxy-benzamide, 1, a ligand shown by others to bind preferentially to σ2 over σ1 receptors, as well as dopamine D2 and D3 sites. First, we determined binding to monoamine transporters and opioid receptors, and noted 57-fold selectivity for σ2 receptors over the serotonin transporter, and >800-fold selectivity for σ2 receptors over the other sites tested. We then examined 1 in locomotor activity studies using male CD-1® mice, and saw no alteration of basal activity at doses up to 31.6 µmol/kg. Cocaine produced a fivefold increase in locomotor activity, which was attenuated by 66% upon pretreatment of mice with 1 at 31.6 µmol/kg. In vivo radioligand binding studies also were performed, and showed no occupancy of σ1 receptors or the dopamine transporter by 1, or its possible metabolites, at the 31.6 µmol/kg dose. Thus, ligand 1 profiles behaviorally as a σ2 receptor-selective antagonist that is able to counteract cocaine's motor stimulatory effects.

  9. 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.

  10. 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

  11. 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.

  12. A role for adenosine in coronary vasoregulation in man. Effects of theophylline and enprofylline.

    PubMed

    Edlund, A; Conradsson, T; Sollevi, A

    1995-11-01

    Adenosine has been suggested to have a role in regulation of the tone of the cardiac resistance vessels. To elucidate the coronary vasoregulatory role of endogenous adenosine in man, we studied the effects of adenosine receptor antagonism by theophylline on coronary blood flow at rest and during light exercise. However, theophylline may also exert pharmacological effects not related to adenosine antagonism. To clarify the contribution of endogenous adenosine in coronary hyperaemia, the effect of theophylline was compared to that of enprofylline, a xanthine which exerts similar pharmacological effects as theophylline while lacking antagonistic action at adenosine receptors. Twenty healthy subjects (10 males) aged 22-39 years were examined. Coronary sinus (CS) blood flow and blood oxygen content were determined at rest and during supine bicycle exercise, at a load of 50 watts, for 10 min. Thereafter, stepwise infusion of adenosine (30 to 60 micrograms/kg/min into the subclavian vein) was performed. Theophylline or enprofylline treatment was instituted randomly and double-blind (10 in each group), and the procedures (i.e. determinations at rest, during exercise and during infusion of adenosine) were repeated. In all 20 subjects, basal CS flow was 70 +/- 6 ml/min and the cardiac oxygen extraction ((A-CS)O2D) was 123 +/- 3 ml/l. During exercise, CS flow and (A-CS)O2D increased to 135 +/- 17 ml/min and 132 +/- 3 ml/l, respectively. Adenosine increased CS flow dose dependently to 161 +/- 27 ml/min, while (A-CS)O2D decreased to 66 +/- 7 ml/l. The vasodilatory effect of adenosine was readily counteracted by theophylline, the increase in CS flow being 33% vs. 133% in the control situation. Enprofylline, on the other hand, enhanced the response to exogenous adenosine. Theophylline, at a dose lacking effect on heart rate and blood pressure, decreased CS flow at rest by 14% (P < 0.05) and during exercise by 18% (P < 0.05). ((A-CS)O2D increased by 14% at rest and during exercise

  13. 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

  14. 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

  15. 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

  16. Inosine induces presynaptic inhibition of acetylcholine release by activation of A3 adenosine receptors at the mouse neuromuscular junction

    PubMed Central

    Cinalli, A R; Guarracino, J F; Fernandez, V; Roquel, L I; Losavio, A S

    2013-01-01

    Background and Purpose The role of inosine at the mammalian neuromuscular junction (NMJ) has not been clearly defined. Moreover, inosine was classically considered to be the inactive metabolite of adenosine. Hence, we investigated the effect of inosine on spontaneous and evoked ACh release, the mechanism underlying its modulatory action and the receptor type and signal transduction pathway involved. Experimental Approach End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the mouse phrenic-nerve diaphragm preparations using conventional intracellular electrophysiological techniques. Key Results Inosine (100 μM) reduced MEPP frequency and the amplitude and quantal content of EPPs; effects inhibited by the selective A3 receptor antagonist MRS-1191. Immunohistochemical assays confirmed the presence of A3 receptors at mammalian NMJ. The voltage-gated calcium channel (VGCC) blocker Cd2+, the removal of extracellular Ca2+ and the L-type and P/Q-type VGCC antagonists, nitrendipine and ω-agatoxin IVA, respectively, all prevented inosine-induced inhibition. In the absence of endogenous adenosine, inosine decreased the hypertonic response. The effects of inosine on ACh release were prevented by the Gi/o protein inhibitor N-ethylmaleimide, PKC antagonist chelerytrine and calmodulin antagonist W-7, but not by PKA antagonists, H-89 and KT-5720, or the inhibitor of CaMKII KN-62. Conclusion and Implications Our results suggest that, at motor nerve terminals, inosine induces presynaptic inhibition of spontaneous and evoked ACh release by activating A3 receptors through a mechanism that involves L-type and P/Q-type VGCCs and the secretory machinery downstream of calcium influx. A3 receptors appear to be coupled to Gi/o protein. PKC and calmodulin may be involved in these effects of inosine. PMID:23731236

  17. Platelet-activating factor receptor antagonism targets neuroinflammation in experimental epilepsy

    PubMed Central

    Musto, Alberto E.; Samii, Mark

    2010-01-01

    Purpose Temporal lobe epilepsy is associated with the inflammatory process related to the basic mechanisms that lead to seizure susceptibility and brain damage. Platelet-activating factor (PAF), a potent, short-lived phospholipid mediator of inflammation participates in physiological signaling in the brain. However, after seizures PAF accumulates in the brain and activates intracellular signaling related with inflammation-mediated excitotoxicity and hippocampal hyperexcitability. The objective of this study is to evaluate the effect of PAF antagonism on hippocampal hyperexcitability, seizure susceptibility and neuroprotection using the kindling paradigm and pilocarpine-induced seizure damage models. Methods The PAF antagonist, LAU-0901 (60 mg/kg, i.p.), or vehicle was administrated each day of kindling or daily during the four weeks after status epilepticus (SE). We analyzed seizure severity, electrical activity, cellular damage and inflammation in the hippocampi of both treated groups. Results LAU-0901 limits the progression of kindling and attenuates seizure susceptibility one week after the kindling procedure. Also, under the seizure-damage conditions studied here, we observed that LAU-0901 induces hippocampal neuroprotection and limits somatostatin interneuronal cell loss and inflammation. Discussion Our results indicate that modulation of PAF over-activity attenuates seizure susceptibility, hippocampal hyperexcitability and neuroinflammation. PMID:21204830

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. Transfected adenosine A1 receptor-mediated modulation of thrombin-stimulated phospholipase C and phospholipase A2 activity in CHO cells.

    PubMed

    Dickenson, J M; Hill, S J

    1997-02-19

    Thrombin receptor activation in Chinese hamster ovary (CHO) cells stimulates the hydrolysis of inositol phospholipids and the release of arachidonic acid. Our previous studies have shown that activation of the human transfected adenosine A1 receptor in CHO cells (CHO-A1) potentiates the accumulation of inositol phosphates elicited by endogenous P2U purinoceptors and CCKA receptors. In this study we have investigated whether adenosine A1 receptor activation can modulate thrombin-stimulated arachidonic acid release and/or inositol phospholipid hydrolysis in CHO-A1 cells. Thrombin stimulated [3H]arachidonic acid release and total [3H]inositol phosphate accumulation in CHO-A1 cells. Both these responses to thrombin were were insensitive to pertussis toxin. The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), potentiated thrombin-stimulated [3H]arachidonic acid. In marked contrast, PMA inhibited thrombin-stimulated [3H]inositol phosphate accumulation. The selective protein kinase C inhibitor Ro 31-8220 (3-¿1-[3-(2-isothioureido)propyl] indol-3-yl¿-4-(1-methylindol-3-yl)-3-pyrrolin-2,5-dione) had no effect on thrombin-stimulated [3H]arachidonic acid release but reversed the potentiation of thrombin-stimulated [3H]arachidonic acid release elicited by PMA. The selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) augmented the release of [3H]arachidonic acid produced by thrombin. Co-activation of the adenosine A1 receptor also potentiated thrombin-stimulated [3H]inositol phosphate accumulation. The synergistic interactions between the adenosine A1 receptor and thrombin were abolished in pertussis-toxin-treated cells. The potentiation of [3H]arachidonic acid release by CPA was blocked by the protein kinase C inhibitors Ro 31-8220 and GF 109203X (3-[1-[3-(dimethylamino)propyl]-1 H-indol-3-yl]-4-(1 H-indol-3-yl)- 1H-pyrrole-2,5-dione). In conclusion, thrombin receptor activation in CHO-A1 cells stimulates the accumulation of [3H

  4. The effects of methylmercury on motor activity are sex- and age-dependent, and modulated by genetic deletion of adenosine receptors and caffeine administration.

    PubMed

    Björklund, Olga; Kahlström, Johan; Salmi, Peter; Ogren, Sven Ove; Vahter, Marie; Chen, Jiang-Fan; Fredholm, Bertil B; Daré, Elisabetta

    2007-11-30

    Adenosine and its receptors are, as part of the brain stress response, potential targets for neuroprotective drugs. We have investigated if the adenosine receptor system affects the developmental neurotoxicity caused by the fish pollutant methylmercury (MeHg). Behavioral outcomes of low dose perinatal MeHg exposure were studied in mice where the A(1) and A(2A) adenosine receptors were either partially blocked by caffeine treatment or eliminated by genetic modification (A(1)R and A(2A)R knock-out mice). From gestational day 7 to day 7 of lactation dams were administered doses that mimic human intake via normal diet, i.e. 1microM MeHg and/or 0.3g/l caffeine in the drinking water. This exposure to MeHg resulted in a doubling of brain Hg levels in wild type females and males at postnatal day 21 (PND21). Open field analysis was performed at PND21 and 2 months of age. MeHg caused time-dependent behavioral alterations preferentially in male mice. A decreased response to amphetamine in 2-month-old males pointed to disturbances in dopaminergic functions. Maternal caffeine intake induced long-lasting changes in the offspring evidenced by an increased motor activity and a modified response to psychostimulants in adult age, irrespectively of sex. Similar alterations were observed in A(1)R knock-out mice, suggesting that adenosine A(1) receptors are involved in the alterations triggered by caffeine exposure during development. Perinatal caffeine treatment and, to some extent, genetic elimination of adenosine A(1) receptors, attenuated the behavioral consequences of MeHg in males. Importantly, also deletion of the A(2A) adenosine receptor reduced the vulnerability to MeHg, consistent with the neuroprotective effects of adenosine A(2A) receptor inactivation observed in hypoxia and Parkinson's disease. Thus, the consequences of MeHg toxicity during gestation and lactation can be reduced by adenosine A(1) and A(2A) receptor inactivation, either via their genetic deletion or by

  5. Endomorphin analogues with mixed μ-opioid (MOP) receptor agonism/δ-opioid (DOP) receptor antagonism and lacking β-arrestin2 recruitment activity.

    PubMed

    Cai, Jun; Song, Bowen; Cai, Yunxin; Ma, Yu; Lam, Ai-Leen; Magiera, Julia; Sekar, Sunder; Wyse, Bruce D; Ambo, Akihiro; Sasaki, Yusuke; Lazarus, Lawrence H; Smith, Maree T; Li, Tingyou

    2014-04-01

    Analogues of endomorphin (Dmt-Pro-Xaa-Xaa-NH2) modified at position 4 or at positions 4 and 3, and tripeptides (Dmt-Pro-Xaa-NH2) modified at position 3, with various phenylalanine analogues (Xaa=Trp, 1-Nal, 2-Nal, Tmp, Dmp, Dmt) were synthesized and their effects on in vitro opioid activity were investigated. Most of the peptides exhibited high μ-opioid (MOP) receptor binding affinity (KiMOP=0.13-0.81nM), modest MOP-selectivity (Kiδ-opioid (DOP)/KiMOP=3.5-316), and potent functional MOP agonism (GPI, IC50=0.274-249nM) without DOP and κ-opioid (KOP) receptor agonism. Among them, compounds 7 (Dmt-Pro-Tmp-Tmp-NH2) and 9 (Dmt-Pro-1-Nal-NH2) were opioids with potent mixed MOP receptor agonism/DOP receptor antagonism and devoid of β-arrestin2 recruitment activity. They may offer a unique template for the discovery of potent analgesics that produce less respiratory depression, less gastrointestinal dysfunction and that have a lower propensity to induce tolerance and dependence compared with morphine.

  6. 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

  7. Evaluation of age-dependent response to NMDA receptor antagonism in zebrafish.

    PubMed

    Menezes, Fabiano Peres; Kist, Luiza Wilges; Bogo, Maurício Reis; Bonan, Carla Denise; Da Silva, Rosane Souza

    2015-04-01

    Imbalances in glutamatergic signaling have been proposed as the cause of several neurological disturbances. The use of MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist, to mimic features of these neurological disorders is effective both in mammals and in fish. However, the variability of the subunits comprising the NMDA receptor during development alters the pharmacokinetic properties of the receptor and leads to different responses to this drug. Here, we evaluated the locomotor response of zebrafish to MK-801 (1, 5, and 20 μM) through the development (30 days postfertilization [dpf] to 2 years postfertilization [ypf]). The NMDA receptor subunit gene expression was also analyzed through the development (7 dpf to 2 ypf). Zebrafish displayed an age-related response to MK-801 with a higher response at 60 and 120 dpf. The magnitude of hyperlocomotion promoted by MK-801 seems to be less powerful for zebrafish in relation to rodents. The verification of expression levels in zebrafish NMDA receptor subunits shows that NR1.1 had a slight reduction throughout the development, while the NR2 subunits, especially NR2A.2 and NR2C.1, vary their expression levels according to the stage of development. The time-specific locomotor response to MK-801 through the development could be a consequence of differential NMDA receptor subunit expression. This result of developmental response to MK-801 is a crucial component in the consolidation of zebrafish as a suitable model to study glutamatergic neurotransmission in early phases.

  8. 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

  9. Antagonism of rat orexin receptors by almorexant attenuates central chemoreception in wakefulness in the active period of the diurnal cycle.

    PubMed

    Li, Aihua; Nattie, Eugene

    2010-08-01

    Central chemoreception, the highly sensitive ventilatory response to small changes in CO(2)/pH, involves many sites. Hypothalamic orexin neurons are CO(2) sensitive in vitro, prepro-orexin knockout mice have a reduced CO(2) response prominently in wakefulness, and focal antagonism of the orexin receptor 1 (OX(1)R) in two central chemoreceptor sites, the retrotrapezoid nucleus (RTN) or the medullary raphé, results in a reduction of the CO(2) response predominately in wakefulness (-30% and -16%, respectively). Here we hypothesize that acute and selective inhibition of both orexin receptors (OX(1)R and OX(2)R) at all central locations by an orally administered dual orexin receptor antagonist, almorexant, will substantially attenuate the CO(2) response in a vigilance-state- and diurnal-cycle-dependent manner. We found that almorexant attenuated the CO(2) response by 26% only in wakefulness during the dark period of the diurnal cycle to a level observed during NREM sleep in the light period in controls suggesting that the sleep-wake difference in the CO(2) response can be in large part attributed to orexin. Almorexant also decreased wakefulness and increased NREM and REM sleep during the dark period, as previously reported, and unexpectedly decreased the number of sighs and post-sigh apnoeas during wakefulness in both the light and the dark period and during both wakefulness and NREM sleep in the dark period. The results support our hypothesis that the orexin system participates importantly in central chemoreception in a vigilance-state- and diurnal-cycle-dependent manner and indicate a role for orexin in the important process of sighing.

  10. 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

  11. Dorsal versus ventral hippocampal contributions to trace and contextual conditioning: differential effects of regionally selective NMDA receptor antagonism on acquisition and expression.

    PubMed

    Czerniawski, Jennifer; Ree, Fredrick; Chia, Chester; Otto, Tim

    2012-07-01

    The dorsal and ventral subregions of the hippocampus likely play dissociable roles in some forms of learning. For example, we have previously demonstrated that temporary inactivation of ventral, but not dorsal, hippocampus dramatically impaired the acquisition of trace fear conditioning, while temporary inactivation of dorsal, but not ventral, hippocampus impaired spatially guided reinforced alternation (Czerniawski et al. (2009) Hippocampus 19:20-32). Importantly, emerging data suggest that lesions, temporary inactivation, and NMDA receptor antagonism within these subregions can produce quite different patterns of behavioral effects when administered into the same region. Specifically, while neither lesions nor temporary inactivation of dorsal hippocampus impair the acquisition of trace fear conditioning, learning in this paradigm is severely impaired by pre-training administration of the NMDA receptor antagonist dl-2-phosphonovaleric acid (APV) in dorsal hippocampus; the effect of NMDA receptor antagonism within ventral hippocampus on the acquisition and expression of trace conditioning, or on learning in general, has not yet been systematically explored. The present study extends our previous work examining the differential effect of lesions or inactivation of the dorsal and ventral hippocampal subregions by systematically examining the effect of regionally selective pre-training or pre-testing administration of APV on the acquisition and expression of trace and contextual fear conditioning. The results of these studies demonstrate that while pre-training NMDA receptor antagonism within either the dorsal or ventral subregion of the hippocampus impaired the acquisition of both trace and contextual conditioning, pre-testing NMDA receptor antagonism within ventral, but not dorsal, hippocampus impaired the expression of previously-acquired trace and contextual fear conditioning. These data suggest that selectively manipulating the integrity of individual subregions

  12. Adenosine in the tuberomammillary nucleus inhibits the histaminergic system via A1 receptors and promotes non-rapid eye movement sleep.

    PubMed

    Oishi, Yo; Huang, Zhi-Li; Fredholm, Bertil B; Urade, Yoshihiro; Hayaishi, Osamu

    2008-12-16

    Adenosine has been proposed to promote sleep through A(1) receptors (A(1)R's) and/or A(2A) receptors in the brain. We previously reported that A(2A) receptors mediate the sleep-promoting effect of prostaglandin D(2), an endogenous sleep-inducing substance, and that activation of these receptors induces sleep and blockade of them by caffeine results in wakefulness. On the other hand, A(1)R has been suggested to increase sleep by inhibition of the cholinergic region of the basal forebrain. However, the role and target sites of A(1)R in sleep-wake regulation remained controversial. In this study, immunohistochemistry revealed that A(1)R was expressed in histaminergic neurons of the rat tuberomammillary nucleus (TMN). In vivo microdialysis showed that the histamine release in the frontal cortex was decreased by microinjection into the TMN of N(6)-cyclopentyladenosine (CPA), an A(1)R agonist, adenosine or coformycin, an inhibitor of adenosine deaminase, which catabolizes adenosine to inosine. Bilateral injection of CPA into the rat TMN significantly increased the amount and the delta power density of non-rapid eye movement (non-REM; NREM) sleep but did not affect REM sleep. CPA-promoted sleep was observed in WT mice but not in KO mice for A(1)R or histamine H(1) receptor, indicating that the NREM sleep promoted by A(1)R-specific agonist depended on the histaminergic system. Furthermore, the bilateral injection of adenosine or coformycin into the rat TMN increased NREM sleep, which was completely abolished by coadministration of 1,3-dimethyl-8-cyclopenthylxanthine, a selective A(1)R antagonist. These results indicate that endogenous adenosine in the TMN suppresses the histaminergic system via A(1)R to promote NREM sleep.

  13. Adenosine and the Auditory System

    PubMed Central

    Vlajkovic, Srdjan M; Housley, Gary D; Thorne, Peter R

    2009-01-01

    Adenosine is a signalling molecule that modulates cellular activity in the central nervous system and peripheral organs via four G protein-coupled receptors designated A1, A2A, A2B, and A3. This review surveys the literature on the role of adenosine in auditory function, particularly cochlear function and its protection from oxidative stress. The specific tissue distribution of adenosine receptors in the mammalian cochlea implicates adenosine signalling in sensory transduction and auditory neurotransmission although functional studies have demonstrated that adenosine stimulates cochlear blood flow, but does not alter the resting and sound-evoked auditory potentials. An interest in a potential otoprotective role for adenosine has recently evolved, fuelled by the capacity of A1 adenosine receptors to prevent cochlear injury caused by acoustic trauma and ototoxic drugs. The balance between A1 and A2A receptors is conceived as critical for cochlear response to oxidative stress, which is an underlying mechanism of the most common inner ear pathologies (e.g. noise-induced and age-related hearing loss, drug ototoxicity). Enzymes involved in adenosine metabolism, adenosine kinase and adenosine deaminase, are also emerging as attractive targets for controlling oxidative stress in the cochlea. Other possible targets include ectonucleotidases that generate adenosine from extracellular ATP, and nucleoside transporters, which regulate adenosine concentrations on both sides of the plasma membrane. Developments of selective adenosine receptor agonists and antagonists that can cross the blood-cochlea barrier are bolstering efforts to develop therapeutic interventions aimed at ameliorating cochlear injury. Manipulations of the adenosine signalling system thus hold significant promise in the therapeutic management of oxidative stress in the cochlea. PMID:20190966

  14. Differential role of nitric oxide in regional sympathetic responses to stimulation of NTS A2a adenosine receptors.

    PubMed

    Scislo, Tadeusz J; Tan, Nobusuke; O'Leary, Donal S

    2005-02-01

    Our previous studies showed that preganglionic adrenal (pre-ASNA), renal (RSNA), lumbar, and postganglionic adrenal sympathetic nerve activities (post-ASNA) are inhibited after stimulation of arterial baroreceptors, nucleus of the solitary tract (NTS), and glutamatergic and P2x receptors and are activated after stimulation of adenosine A1 receptors. However, stimulation of adenosine A2a receptors inhibited RSNA and post-ASNA, whereas it activated pre-ASNA. Because the effects evoked by NTS A2a receptors may be mediated via activation of nitric oxide (NO) mechanisms in NTS neurons, we tested the hypothesis that NO synthase (NOS) inhibitors would attenuate regional sympathetic responses to NTS A2a receptor stimulation, whereas NO donors would evoke contrasting responses from pre-ASNA versus RSNA and post-ASNA. Therefore, in chloralose/urethane-anesthetized rats, we compared hemodynamic and regional sympathetic responses to microinjections of selective A2a receptor agonist (CGS-21680, 20 pmol/50 nl) after pretreatment with NOS inhibitors Nomega-nitro-L-arginine methyl ester (10 nmol/100 nl) and 1-[2-(trifluoromethyl)phenyl]imidazole (100 pmol/100 nl) versus pretreatment with vehicle (100 nl). In addition, responses to microinjections into the NTS of different NO donors [40 and 400 pmol/50 nl sodium nitroprusside (SNP); 0.5 and 5 nmol/50 nl 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA NONOate, also known as NOC-18), and 2 nmol/50 nl 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PAPA NONOate, also known as NOC-15)], the NO precursor L-arginine (10-50 nmol/50 nl), and sodium glutamate (500 pmol/50 nl) were evaluated. SNP, DETA NONOate, and PAPA NONOate activated pre-ASNA and inhibited RSNA and post-ASNA, whereas l-arginine and glutamate microinjected into the same site of the NTS inhibited all these sympathetic outputs. Decreases in heart rate and depressor or biphasic responses accompanied the neural responses. Pretreatment with NOS inhibitors

  15. Mutagenesis Reveals Structure–Activity Parallels between Human A2A Adenosine Receptors and Biogenic Amine G Protein-Coupled Receptors

    PubMed Central

    Jiang, Qiaoling; Lee, Brian X.; Glashofer, Marc; van Rhee, A. Michiel; Jacobson, Kenneth A.

    2012-01-01

    Structure–affinity relationships for ligand binding at the human A2A adenosine receptor have been probed using site-directed mutagenesis in the transmembrane helical domains (TMs). The mutant receptors were expressed in COS-7 cells and characterized by binding of the radioligands [3H]CGS21680, [3H]NECA, and [3H]XAC. Three residues, at positions essential for ligand binding in other G protein-coupled receptors, were individually mutated. The residue V(3.32) in the A2A receptor that is homologous to the essential aspartate residue of TM3 in the biogenic amine receptors, i.e., V84(3.32), may be substituted with L (present in the A3 receptor) but not with D (in biogenic amine receptors) or A. H250(6.52), homologous to the critical N507 of rat m3 muscarinic acetylcholine receptors, may be substituted with other aromatic residues or with N but not with A (Kim et al. J. Biol. Chem. 1995, 270, 13987–13997). H278(7.43), homologous to the covalent ligand anchor site in rhodopsin, may not be substituted with either A, K, or N. Both V84L(3.32) and H250N(6.52) mutant receptors were highly variable in their effect on ligand competition depending on the structural class of the ligand. Adenosine-5′-uronamide derivatives were more potent at the H250N(6.52) mutant receptor than at wild type receptors. Xanthines tended to be close in potency (H250N(6.52)) or less potent (V84L-(3.32)) than at wild type receptors. The affinity of CGS21680 increased as the pH was lowered to 5.5 in both the wild type and H250N(6.52) mutant receptors. Thus, protonation of H250-(6.52) is not involved in this pH dependence. These data are consistent with a molecular model predicting the proximity of bound agonist ligands to TM3, TM5, TM6, and TM7. PMID:9258366

  16. Coupling of a transfected human brain A1 adenosine receptor in CHO-K1 cells to calcium mobilisation via a pertussis toxin-sensitive mechanism.

    PubMed Central

    Iredale, P. A.; Alexander, S. P.; Hill, S. J.

    1994-01-01

    1. The presence of A1 adenosine receptors in CHO-K1 cells transfected with the human brain A1 sequence was confirmed by ligand binding studies using 8-cyclopentyl-[3H] 1,3-dipropylxanthine ([3H]-DPCPX). 2. Alterations in intracellular calcium ([Ca2+]i) were measured with the calcium-sensitive dye, fura-2. 3. N6-cyclopentyladenosine (CPA), the selective A1 agonist, and 5'-N-ethylcarboxaminoadenosine (NECA), a relatively non-selective adenosine receptor agonist, elicited rapid, biphasic increases in [Ca2+]i which involved both mobilisation from intracellular stores and calcium entry. 4. The calcium response to CPA was significantly inhibited by the selective A1 antagonist DPCPX. The non-selective adenosine receptor, xanthine amino congener (XAC), was less potent. 5. The calcium response to CPA was completely prevented by pretreatment of the cells with pertussis toxin implicating the involvement of Gi in the receptor-mediated response. 6. In summary, we present evidence for the coupling of transfected human brain A1 adenosine receptors in CHO-K1 cells to mobilisation of [Ca2+]i via a pertussis toxin-sensitive G protein. PMID:8032613

  17. 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

  18. 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

  19. HIV-1 Env gp120 Structural Determinants for Peptide Triazole Dual Receptor Site Antagonism

    PubMed Central

    Tuzer, Ferit; Madani, Navid; Kamanna, Kantharaju; Zentner, Isaac; LaLonde, Judith; Holmes, Andrew; Upton, Elizabeth; Rajagopal, Srivats; McFadden, Karyn; Contarino, Mark; Sodroski, Joseph; Chaiken, Irwin

    2013-01-01

    Despite advances in HIV therapy, viral resistance and side-effects with current drug regimens require targeting new components of the virus. Dual antagonist peptide triazoles (PT) are a novel class of HIV-1 inhibitors that specifically target the gp120 component of the viral spike and inhibit its interaction with both of its cell surface protein ligands, namely the initial receptor CD4 and the co-receptor (CCR5/CXCR4), thus preventing viral entry. Following an initial survey of 19 gp120 alanine mutants by ELISA, we screened 11 mutants for their importance in binding to, and inhibition by the PT KR21 using surface plasmon resonance. Key mutants were purified and tested for their effects on the peptide’s affinity and its ability to inhibit binding of CD4 and the co-receptor surrogate mAb 17b. Effects of the mutations on KR21 viral neutralization were measured by single-round cell infection assays. Two mutations, D474A and T257A, caused large-scale loss of KR21 binding, as well as losses in both CD4/17b and viral inhibition by KR21. A set of other Ala mutants revealed more moderate losses in direct binding affinity and inhibition sensitivity to KR21. The cluster of sensitive residues defines a PT functional epitope. This site is in a conserved region of gp120 that overlaps the CD4 binding site and is distant from the co-receptor/17b binding site, suggesting an allosteric mode of inhibition for the latter. The arrangement and sequence conservation of the residues in the functional epitope explain the breadth of antiviral activity, and improve the potential for rational inhibitor development. PMID:23011758

  20. Animal models of cognitive dysfunction and negative symptoms of schizophrenia: focus on NMDA receptor antagonism.

    PubMed

    Neill, Joanna C; Barnes, Samuel; Cook, Samantha; Grayson, Ben; Idris, Nagi F; McLean, Samantha L; Snigdha, Shikha; Rajagopal, Lakshmi; Harte, Michael K

    2010-12-01

    Cognitive deficits in schizophrenia remain an unmet clinical need. Improved understanding of the neuro- and psychopathology of these deficits depends on the availability of carefully validated animal models which will assist the development of novel therapies. There is much evidence that at least some of the pathology and symptomatology (particularly cognitive and negative symptoms) of schizophrenia results from a dysfunction of the glutamatergic system which may be modelled in animals through the use of NMDA receptor antagonists. The current review examines the validity of this model in rodents. We review the ability of acute and sub-chronic treatment with three non-competitive NMDA antagonists; phencyclidine (PCP), ketamine and MK801 (dizocilpine) to produce cognitive deficits of relevance to schizophrenia in rodents and their subsequent reversal by first- and second-generation antipsychotic drugs. Effects of NMDA receptor antagonists on the performance of rodents in behavioural tests assessing the various domains of cognition and negative symptoms are examined: novel object recognition for visual memory, reversal learning and attentional set shifting for problem solving and reasoning, 5-Choice Serial Reaction Time for attention and speed of processing; in addition to effects on social behaviour and neuropathology. The evidence strongly supports the use of NMDA receptor antagonists to model cognitive deficit and negative symptoms of schizophrenia as well as certain pathological disturbances seen in the illness. This will facilitate the evaluation of much-needed novel pharmacological agents for improved therapy of cognitive deficits and negative symptoms in schizophrenia.

  1. 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

  2. P2X7 receptor antagonism improves renal blood flow and oxygenation in angiotensin-II infused rats

    PubMed Central

    Menzies, Robert I.; Howarth, Amelia R.; Unwin, Robert J.; Tam, Frederick W.K.; Mullins, John J.; Bailey, Matthew A.

    2015-01-01

    Chronic activation of the renin angiotensin system promotes hypertension, renal microvascular dysfunction, tissue hypoxia and inflammation. We found previously that the injurious response to excess angiotensin II (ANGII) is greater in F344 rats, whereas Lewis rats are renoprotected, despite similar hypertension. We further identified p2rx7, encoding the P2X7 receptor (P2X7R), as a candidate gene for differential susceptibility and here we have tested the hypothesis that activation of P2X7R promotes vascular dysfunction under high ANGII tone. 14-day infusion of ANGII at 30ng/min into F344 rats increased blood pressure by ~15mmHg without inducing fibrosis or albuminuria. In vivo pressure natriuresis was suppressed, medullary perfusion reduced by ~50% and the cortico-medullary oxygenation gradient disrupted. Selective P2X7R antagonism restored pressure natriuresis, promoting a significant leftward shift in the intercept and increasing the slope. Sodium excretion was increased 6 fold and blood pressure normalized. The specific P2X7R antagonist AZ11657312 increased renal medullary perfusion, but only in ANGII-treated rats. Tissue oxygenation was improved by P2X7R blockade, particularly in poorly oxygenated regions of the kidney. Activation of P2X7R induces microvascular dysfunction and regional hypoxia when ANGII is elevated. These pro-inflammatory effects may contribute to progression of renal injury induced by chronic ANGII. PMID:26108066

  3. 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

  4. Basal adenosine modulates the functional properties of AMPA receptors in mouse hippocampal neurons through the activation of A1R A2AR and A3R

    PubMed Central

    Di Angelantonio, Silvia; Bertollini, Cristina; Piccinin, Sonia; Rosito, Maria; Trettel, Flavia; Pagani, Francesca; Limatola, Cristina; Ragozzino, Davide

    2015-01-01

    Adenosine is a widespread neuromodulator within the CNS and its extracellular level is increased during hypoxia or intense synaptic activity, modulating pre- and postsynaptic sites. We studied the neuromodulatory action of adenosine on glutamatergic currents in the hippocampus, showing that activation of multiple adenosine receptors (ARs) by basal adenosine impacts postsynaptic site. Specifically, the stimulation of both A1R and A3R reduces AMPA currents, while A2AR has an opposite potentiating effect. The effect of ARs stimulation on glutamatergic currents in hippocampal cultures was investigated using pharmacological and genetic approaches. A3R inhibition by MRS1523 increased GluR1-Ser845 phosphorylation and potentiated AMPA current amplitude, increasing the apparent affinity for the agonist. A similar effect was observed blocking A1R with DPCPX or by genetic deletion of either A3R or A1R. Conversely, impairment of A2AR reduced AMPA currents, and decreased agonist sensitivity. Consistently, in hippocampal slices, ARs activation by AR agonist NECA modulated glutamatergic current amplitude evoked by AMPA application or afferent fiber stimulation. Opposite effects of AR subtypes stimulation are likely associated to changes in GluR1 phosphorylation and represent a novel mechanism of physiological modulation of glutamatergic transmission by adenosine, likely acting in normal conditions in the brain, depending on the level of extracellular adenosine and the distribution of AR subtypes. PMID:26528137

  5. 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.

  6. Adenosine signaling inhibits CIITA-mediated MHC class II transactivation in lung fibroblast cells.

    PubMed

    Fang, Mingming; Xia, Jun; Wu, Xiaoyan; Kong, Hui; Wang, Hong; Xie, Weiping; Xu, Yong

    2013-08-01

    Efficient antigen presentation by major histocompatibility complex (MHC) molecules represents a critical process in adaptive immunity. Class II transactivator (CIITA) is considered the master regulator of MHC class II (MHC II) transcription. Previously, we have shown that CIITA expression is upregulated in smooth muscle cells deficient in A2b adenosine receptor. Here, we report that treatment with the adenosine receptor agonist adenosine-5'N-ethylcarboxamide (NECA) attenuated MHC II transcription in lung fibro-blast cells as a result of CIITA repression. Further analysis revealed that NECA preferentially abrogated CIITA transcription through promoters III and IV. Blockade with a selective A2b receptor antagonist MRS-1754 restored CIITA-dependent MHC II transactivation. Forskolin, an adenylyl cyclase activator, achieved the same effect as NECA. A2b signaling repressed CIITA transcription by altering histone modifications and recruitment of key factors on the CIITA promoters in a STAT1-dependent manner. MRS-1754 blocked the antagonism of transforming growth factor beta (TGF-β) in CIITA induction by interferon gamma (IFN-γ), alluding to a potential dialogue between TGF-β and adenosine signaling pathways. Finally, A2b signaling attenuated STAT1 phosphorylation and stimulated TGF-β synthesis. In conclusion, we have identified an adenosine-A2b receptor-adenylyl cyclase axis that influences CIITA-mediated MHC II transactivation in lung fibroblast cells and as such have provided invaluable insights into the development of novel immune-modulatory strategies.

  7. Antagonism of N-methyl-D-aspartate receptors reduces the vulnerability of the immune system to stress after chronic morphine.

    PubMed

    Alonzo, Norma C; Bayer, Barbara M

    2003-11-01

    It has been shown that morphine-tolerant animals have an altered immunological sensitivity to stress. Although the glutamatergic system has been implicated in the neuroadaptive process underlying this tolerant state, its potential role in development of the altered immunological sensitivity consequent to chronic morphine treatment is not known. To determine this, a morphine-tolerant state was induced by 10-day administration of an escalating dose of morphine from 10 to 40 mg/kg (s.c., b.i.d.), and lymphocyte proliferative response to a T-cell mitogen was measured. Morphine challenge (10 mg/kg s.c.) after days of treatment was gradually less immunosuppressive, and this tolerance progression was delayed by concurrent administration of the N-methyl-D-aspartate (NMDA) receptor antagonist (-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (0.1 mg/kg s.c.) with chronic morphine. The effect was independent of glucocorticoid level changes and was not a result of an acute interaction of the drugs or the prolonged presence of the antagonist alone. Subsequent to chronic treatment, animals were subjected to opioid withdrawal and water stress. Both stressors induced 50% immunosuppression in morphine-tolerant animals compared with saline-treated controls. Increased immunological sensitivity to these stressors was attenuated when MK-801 was administered with chronic morphine as demonstrated by an accelerated recovery rate and lack of immunosuppression from opioid withdrawal and water stress, respectively. Together, these findings provide the first evidence that the neuroadapted state of the immune response after chronic morphine can be modified by NMDA receptor antagonism, as illustrated by a temporal deceleration of the development of immunological tolerance during chronic treatment that is associated with an attenuation of the immunological vulnerability of morphine-tolerant animals to stress. PMID:12966157

  8. Glucocorticoids antagonize cAMP-induced Star transcription in Leydig cells through the orphan nuclear receptor NR4A1.

    PubMed

    Martin, Luc J; Tremblay, Jacques J

    2008-09-01

    It is well established that stress, either physical or psychosocial, causes a decrease in testosterone production by Leydig cells. Glucocorticoids (Gc) are the main mediators of stress response and they convey their repressive effect on Leydig cells through the glucocorticoid receptor (GR). So far, various mechanisms have been proposed to explain the mechanism of action of Gc on Leydig cell steroidogenesis including repression of genes involved in testosterone biosynthesis. Several steroidogenic genes, including steroidogenic acute regulatory (STAR) protein, have been shown to be repressed by Gc in a GR-dependent manner but the underlying mechanisms remain to be fully elucidated. Here, we found that dexamethasone (Dex), a potent synthetic Gc, partly antagonizes the cAMP-dependent stimulation of the mouse Star promoter in MA-10 Leydig cells as revealed by transient transfection assays. This repression requires an element located at -95 bp previously implicated in the activation of the Star promoter by the nuclear receptors, NR4A1 and NR5A1. Dex was found to inhibit NR4A1-dependent transactivation of the Star promoter in Leydig cells by decreasing NR4A1, but not NR5A1, recruitment to the proximal Star promoter as determined by chromatin immunoprecipitation assay. Western blots revealed that Dex did not affect NR4A1 or NR5A1 expression in response to cAMP. These data suggest that NR4A1 would be associated with the GR in a transcriptionally inactive complex as previously demonstrated in pituitary corticotrope cells. Thus, our data provide new molecular insights into the stress-mediated suppression of testosterone production in testicular Leydig cells.

  9. 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.

  10. 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

  11. 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.

  12. Antagonism of Human Formyl Peptide Receptor 1 (FPR1) by Chromones and Related Isoflavones

    PubMed Central

    Schepetkin, Igor A.; Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Cheng, Ni; Ye, Richard D.; Quinn, Mark T.

    2014-01-01

    Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) expressed on a variety of cell types. Because FPRs play an important role in the regulation of inflammatory reactions implicated in disease pathogenesis, FPR antagonists may represent novel therapeutics for modulating innate immunity. Previously, 4H-chromones were reported to be potent and competitive FPR1 antagonists. In the present studies, 96 additional chromone analogs, including related synthetic and natural isoflavones were evaluated for FPR1 antagonist activity. We identified a number of novel competitive FPR1 antagonists that inhibited fMLF-induced intracellular Ca2+ mobilization in FPR1-HL60 cells and effectively competed with WKYMVm-FITC for binding to FPR1 in FPR1-HL60 and FPR1-RBL cells. Compound 10 (6-hexyl-2-methyl-3-(1-methyl-1H-benzimidazol-2-yl)-4-oxo-4H-chromen-7-yl acetate) was found to be the most potent FPR1-specific antagonist, with binding affinity Ki~100 nM. These chromones inhibited Ca2+ flux and chemotaxis in human neutrophils with nanomolar-micromolar IC50 values. In addition, the most potent novel FPR1 antagonists inhibited fMLF-induced phosphorylation of extracellular signal-regulated kinases (ERK1/2) in FPR1-RBL cells. These antagonists were specific for FPR1 and did not inhibit WKYMVM/WKYMVm-induced intracellular Ca2+ mobilization in FPR2-HL60 cells, FPR3-HL60 cells, RBL cells transfected with murine Fpr1, or interleukin 8-induced Ca2+ flux in human neutrophils and RBL cells transfected with CXC chemokine receptor 1 (CXCR1). Moreover, pharmacophore modeling showed that the active chromones had a significantly higher degree of similarity with the pharmacophore template as compared to inactive analogs. Thus, the chromone/isoflavone scaffold represents a relevant backbone for development of novel FPR1 antagonists. PMID:25450672

  13. Endothelin-a receptor antagonism after renal angioplasty enhances renal recovery in renovascular disease.

    PubMed

    Chade, Alejandro R; Tullos, Nathan; Stewart, Nicholas J; Surles, Bret

    2015-05-01

    Percutaneous transluminal renal angioplasty/stenting (PTRAS) is frequently used to treat renal artery stenosis and renovascular disease (RVD); however, renal function is restored in less than one half of the cases. This study was designed to test a novel intervention that could refine PTRAS and enhance renal recovery in RVD. Renal function was quantified in pigs after 6 weeks of chronic RVD (induced by unilateral renal artery stenosis), established renal damage, and hypertension. Pigs with RVD then underwent PTRAS and were randomized into three groups: placebo (RVD+PTRAS), chronic endothelin-A receptor (ET-A) blockade (RVD+PTRAS+ET-A), and chronic dual ET-A/B blockade (RVD+PTRAS+ET-A/B) for 4 weeks. Renal function was again evaluated after treatments, and then, ex vivo studies were performed on the stented kidney. PTRAS resolved renal stenosis, attenuated hypertension, and improved renal function but did not resolve renal microvascular rarefaction, remodeling, or renal fibrosis. ET-A blocker therapy after PTRAS significantly improved hypertension, microvascular rarefaction, and renal injury and led to greater recovery of renal function. Conversely, combined ET-A/B blockade therapy blunted the therapeutic effects of PTRAS alone or PTRAS followed by ET-A blockade. These data suggest that ET-A receptor blockade therapy could serve as a coadjuvant intervention to enhance the outcomes of PTRAS in RVD. These results also suggest that ET-B receptors are important for renal function in RVD and may contribute to recovery after PTRAS. Using clinically available compounds and techniques, our results could contribute to both refinement and design of new therapeutic strategies in chronic RVD.

  14. Effects of adenosine A(3) receptor agonist on bone marrow granulocytic system in 5-fluorouracil-treated mice.

    PubMed

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

    2006-05-24

    The purpose of the experiments reported was to investigate effects of N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), a selective adenosine A(3) receptor agonist, on the granulocytic system in femoral marrow of mice depleted by the cytotoxic drug 5-fluorouracil. In the phase of the highest cell depletion IB-MECA was injected i.p. at single doses of 200 nmol/kg given either once or twice daily in 2- and 4-day regimens starting on day 1 after 5-fluorouracil administration; the effects were evaluated on days 3 and 5, respectively. The general effect of IB-MECA in all these experiments was an enhancement of the counts of morphologically recognizable proliferative granulocytic cells, interpreted as evidence of the differentiation of committed progenitor cells. A more expressive effect was observed after IB-MECA injected twice daily. It was found that the induction of the strong differentiation pressures by IB-MECA given twice daily shortly after 5-fluorouracil treatment can be counterproductive due to the preponderance of differentiaton processes over the proliferation control. In additional experiments, it has been shown that the use of the 2-day administration of IB-MECA given twice daily in the recovery phase, i.e., on days 5 and 6 after 5-fluorouracil administration, does not induce stimulatory effects. Thus, the dosing and timing of IB-MECA treatment determines its effectivity in stimulating granulopoiesis under conditions of myelosuppression.

  15. Rational Design of Sulfonated A3 Adenosine Receptor-Selective Nucleosides as Pharmacological Tools to Study Chronic Neuropathic Pain

    PubMed Central

    Paoletta, Silvia; Tosh, Dilip K.; Finley, Amanda; Gizewski, Elizabeth T.; Moss, Steven M.; Gao, Zhan-Guo; Auchampach, John A.; Salvemini, Daniela; Jacobson, Kenneth A.

    2013-01-01

    (N)-Methanocarba (bicyclo[3.1.0]hexane)-adenosine derivatives were probed for sites of charged sulfonate substitution, which precludes diffusion across biological membranes, e.g. blood brain barrier. Molecular modeling predicted that sulfonate groups on C2-phenylethynyl substituents would provide high affinity at both mouse (m) and human (h) A3 adenosine receptors (ARs), while a N6-p-sulfo-phenylethyl substituent would determine higher hA3AR vs. mA3AR affinity. These modeling predictions, based on steric fitting of the binding cavity and crucial interactions with key residues, were confirmed by binding/efficacy studies of synthesized sulfonates. N6-3-Chlorobenzyl-2-(3-sulfophenylethynyl) derivative 7 (MRS5841) bound selectively to h/m A3ARs (Ki hA3AR 1.9 nM) as agonist, while corresponding p-sulfo isomer 6 (MRS5701) displayed mixed A1/A3AR agonism. Both nucleosides administered i.p. reduced mouse chronic neuropathic pain that was ascribed to either A3 or A1/A3ARs using A3AR genetic deletion. Thus, rational design methods based on A3AR homology models successfully predicted sites for sulfonate incorporation, for delineating adenosine’s CNS vs. peripheral actions. PMID:23789857

  16. Inhibition of muscarinic K+ current in guinea-pig atrial myocytes by PD 81,723, an allosteric enhancer of adenosine binding to A1 receptors

    PubMed Central

    Brandts, B; Bünemann, M; Hluchy, J; Sabin, G V; Pott, L

    1997-01-01

    PD 81,723 has been shown to enhance binding of adenosine to A1 receptors by stabilizing G protein-receptor coupling (‘allosteric enhancement'). Evidence has been provided that in the perfused hearts and isolated atria PD 81,723 causes a sensitization to adenosine via this mechanism. We have studied the effect of PD 81,723 in guinea-pig isolated atrial myocytes by use of whole-cell measurement of the muscarinic K+ current (IK(ACh)) activated by different Gi-coupled receptors (A1, M2, sphingolipid). PD 81,273 caused inhibition of IK(ACh) (IC50≃5 μM) activated by either of the three receptors. Receptor-independent IK(ACh) in cells loaded with GTP-γ-S and background IK(ACh), which contributes to the resting conductance of atrial myocytes, were equally sensitive to PD 81,723. At no combination of concentrations of adenosine and PD 81,723 could an enhancing effect be detected. The compound was active from the outside only. Loading of the cells with PD 81,723 (50 μM) via the patch pipette did not affect either IK(ACh) or its sensitivity to adenosine. We suggest that PD 81,723 acts as an inhibitor of inward rectifying K+ channels; this is supported by the finding that ventricular IK1, which shares a large degree of homology with the proteins (GIRK1/GIRK4) forming IK(ACh) but is not G protein-gated, was also blocked by this compound. It is concluded that the functional effects of PD 81,723 described in the literature are not mediated by the A1 adenosine receptor-Gi-IK(ACh) pathway. PMID:9249260

  17. 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

  18. 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

  19. An Update on Adenosine A2A-Dopamine D2 receptor interactions. Implications for the Function of G Protein-Coupled Receptors

    PubMed Central

    Ferré, S.; Quiroz, C.; Woods, A. S.; Cunha, R.; Popoli, P.; Ciruela, F.; Lluis, C.; Franco, R.; Azdad, K.; Schiffmann, S. N.

    2008-01-01

    Adenosine A2A-dopamine D2 receptor interactions play a very important role in striatal function. A2A-D2 receptor interactions provide an example of the capabilities of information processing by just two different G protein-coupled receptors. Thus, there is evidence for the coexistence of two reciprocal antagonistic interactions between A2A and D2 receptors in the same neurons, the GABAergic enkephalinergic nens. An antagonistic A2A-D2 intramembrane receptor interaction, which depends on A2A-D2 receptor heteromerization and Gq/11-PLC signaling, modulates neuronal excitability and neurotransmitter release. On the other hand, an antagonistic A2A-D2 receptor interaction at the adenylyl-cyclase level, which depends on Gs/olf- and Gi/o- type V adenylyl-cyclase signaling, modulates protein phosphorylation and gene expression. Finally, under conditions of upregulation of an activator of G protein signaling (AGS3), such as during chronic treatment with addictive drugs, a synergistic A2A-D2 receptor interaction can also be demonstrated. AGS3 facilitates a synergistic interaction between Gs/olf- and Gi/o- coupled receptors on the activation of types II/IV adenylyl cyclase, leading to a paradoxical increase in protein phosphorylation and gene expression upon co-activation of A2A and D2 receptors. The analysis of A2-D2 receptor interactions will have implications for the pathophysiology and treatment of basal ganglia disorders and drug addiction. PMID:18537670

  20. The role of 5-HT7 receptor antagonism in the amelioration of MK-801-induced learning and memory deficits by the novel atypical antipsychotic drug lurasidone.

    PubMed

    Horisawa, Tomoko; Nishikawa, Hiroyuki; Toma, Satoko; Ikeda, Atsushi; Horiguchi, Masakuni; Ono, Michiko; Ishiyama, Takeo; Taiji, Mutsuo

    2013-05-01

    Lurasidone is a novel atypical antipsychotic with high affinity for dopamine D2, serotonin 5-HT7 and 5-HT2A receptors. We previously reported that lurasidone and the selective 5-HT7 receptor antagonist, SB-656104-A improved learning and memory deficits induced by MK-801, an N-methyl-d-aspartate (NMDA) receptor antagonist, in the rat passive avoidance test. In this study, we first examined the role of the 5-HT7 receptor antagonistic activity of lurasidone in its pro-cognitive effect to ameliorate MK-801-induced deficits in the rat passive avoidance test. The 5-HT7 receptor agonist, AS19, (2S)-(+)-5-(1,3,5-trimethylpyrazol-4-yl)-2-(dimethylamino) tetralin, (3 mg/kg, s.c.) completely blocked the attenuating effects of lurasidone (3 mg/kg, p.o.), highlighting the importance of 5-HT7 receptor antagonism in the pro-cognitive effect of lurasidone. AS19 (3 mg/kg, s.c.) also blocked the ameliorating effect of SB-656104-A (10 mg/kg, i.p.) in the same experimental paradigm. To further extend our observation, we next tested whether 5-HT7 receptor antagonism still led to the amelioration of MK-801-induced deficits when combined with D2 and 5-HT2A receptor antagonists, and found that SB-656104-A (10 mg/kg, i.p.) significantly ameliorated MK-801-induced deficits even in the presence of the D2 receptor antagonist raclopride (0.1 mg/kg, s.c.) and 5-HT2A receptor antagonist ketanserin (1 mg/kg, s.c.). Taken together, these results suggest that the 5-HT7 receptor antagonistic activity of lurasidone plays an important role in its effectiveness against MK-801-induced deficits, and may contribute to its pharmacological actions in patients with schizophrenia.

  1. 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

  2. GABAA receptor antagonism ameliorates behavioral and synaptic impairments associated with MeCP2 overexpression.

    PubMed

    Na, Elisa S; Morris, Michael J; Nelson, Erika D; Monteggia, Lisa M

    2014-07-01

    Methyl-CpG-binding protein 2 (MeCP2) is a ubiquitously expressed transcriptional regulator with functional importance in the central nervous system. Loss-of-function mutations in MECP2 results in the neurodevelopmental disorder, Rett syndrome, whereas increased expression levels are associated with the neurological disorder, MECP2 duplication syndrome. Previous characterization of a mouse line overexpressing Mecp2 demonstrated that this model recapitulated key behavioral features of MECP2 duplication syndrome with specific deficits in synaptic plasticity and neurotransmission. Alterations in excitation/inhibition balance have been suggested to underlie neurodevelopmental disorders with recent data suggesting that picrotoxin (PTX), a GABAA receptor antagonist, rescues certain behavioral and synaptic phenotypes in a mouse model of Down syndrome. We therefore examined whether a similar treatment regimen would impact the behavioral and synaptic phenotypes in a mouse model of MECP2 duplication syndrome. We report that chronic treatment with low doses of PTX ameliorates specific behavioral phenotypes, including motor coordination, episodic memory impairments, and synaptic plasticity deficits. These findings suggest that GABAA receptor antagonists may offer a possible therapeutic target for the treatment of MECP2 duplication syndrome.

  3. 5-HT Receptor Antagonism Attenuates the Ischemia-Reperfusion Injury After Rabbit Lung Preservation.

    PubMed

    Arreola-Ramírez, J L; Alquicira-Mireles, J; Morales-Hernández, P E; Vargas, M H; Villalba-Caloca, J; Segura-Medina, P

    2015-01-01

    The success of lung transplantation is threatened by the appearance of ischemia-reperfusion injury, which is characterized by increased vascular permeability. 5-Hydroxytryptamine (5-HT; serotonin) is known to produce microvascular leakage in the systemic circulation, but its possible role in ischemia-reperfusion injury after lung preservation has not been reported. In this work we measured the release of 5-HT during a 24-hour rabbit lung preservation, and the effect of methiothepin (antagonist of the majority of 5-HT receptors) and SB204741 (antagonist of 5-HT2B/2C receptors) on the modified capillary filtration coefficient (mKf,c) was evaluated at the end of this period. Our results showed that the highest release rate of 5-HT occurred during the first 15 minutes after the lung harvesting and progressively decreased in the following time intervals. The baseline mKf,c greatly increased after 24 hours of lung preservation, and this increment was partially reduced by methiothepin and even more by SB204741. We concluded that 5-HT may play an important role in the ischemia-reperfusion process after lung preservation.

  4. 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

  5. FGF receptor antagonism does not affect adipose tissue development in nutritionally induced obesity.

    PubMed

    Scroyen, Ilse; Vranckx, Christine; Lijnen, Henri Roger

    2014-01-01

    The fibroblast growth factor (FGF)-FGF receptor (FGFR) system plays a role in angiogenesis and maintenance of vascular integrity, but its potential role in adipose tissue related angiogenesis and development is still unknown. Administration of SSR, a low molecular weight inhibitor of multiple FGFRs, did not significantly affect body weight nor weight of subcutaneous or gonadal (GON) fat, as compared with pair-fed control mice. Adipocyte hypertrophy and reduced adipocyte density were only observed in GON adipose tissues of treated mice. Adipose tissue angiogenesis was not affected by SSR treatment, as normalized blood vessel density was comparable in adipose tissues of both groups. Blocking the FGF-FGFR system in vivo does not markedly affect adipose tissue development in mice with nutritionally induced obesity.

  6. 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.

  7. Adenosine A2A-receptor blockade abolishes the roll-off respiratory response to hypoxia in awake lambs.

    PubMed

    Koos, Brian J; Kawasaki, Yoshikazu; Kim, Young-Han; Bohorquez, Fanor

    2005-05-01

    Adenosine (ADO) receptor antagonists (aminophylline, caffeine) blunt the respiratory roll-off response to hypoxia in the newborn. This study was designed to determine the ADO receptor subtype involved in the respiratory depression. Chronically catheterized lambs of 7-16 days of age breathed via face mask a gas mixture with a fraction of inspired O2 of 0.21 (normoxia) or 0.07 (hypoxia), while being infused intravascularly with 9-cyclopentyl-1,3-dipropylxanthine (DPCPX; ADO A1-receptor antagonist, n=8), ZM-241385 (ADO A2A-receptor antagonist, n=7), or vehicle. Ventilation was measured at 20 degrees C by a turbine transducer flowmeter. In normoxia [arterial Po2 (PaO2) of approximately 83 Torr], infusion of vehicle did not alter cardiorespiratory measurements, whereas hypoxia (PaO2 of approximately 31 Torr, 15 min) elicited biphasic effects on mean arterial pressure (transient increase), heart rate (HR; diminishing tachycardia), and minute ventilation. In the latter, hypoxia increased ventilation to a peak value of approximately 2.5 times control within the first 3 min, which was followed by a significant (P<0.05) decline to approximately 50% of the maximum increment over the subsequent 7 min. ZM-241385 abolished the hypoxic ventilatory roll-off and blunted the rate of rise in HR without affecting mean arterial pressure or rectal temperature responses. In normoxia, DPCPX increased ventilation and mean arterial pressure but did not change HR. Compared with vehicle, DPCPX did not significantly affect cardiorespiratory responses to hypoxemia (PaO2 of approximately 31 Torr, 10 min). It is concluded that 1) ADO A2A receptors are critically involved in the ventilatory roll-off and HR responses to hypoxia, and 2) ADO A1 receptors, which are tonically active in cardiorespiratory control in normoxia, appear to have little impact on hypoxic ventilatory depression.

  8. 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

  9. Direct influence of C-terminally substituted amino acids in the Dmt-Tic pharmacophore on delta-opioid receptor selectivity and antagonism.

    PubMed

    Balboni, Gianfranco; Salvadori, Severo; Guerrini, Remo; Negri, Lucia; Giannini, Elisa; Bryant, Sharon D; Jinsmaa, Yunden; Lazarus, Lawrence H

    2004-07-29

    A series of 17 analogues were developed on the basis of the general formula H-Dmt-Tic-NH-CH(R)-R' (denotes chirality; R = charged, neutral, or aromatic functional group; R' = -OH or -NH(2)). These compounds were designed to test the following hypothesis: the physicochemical properties of third-residue substitutions C-terminal to Tic in the Dmt-Tic pharmacophore modify delta-opioid receptor selectivity and delta-opioid receptor antagonism through enhanced interactions with the mu-opioid receptor. The data substantiate the following conclusions: (i) all compounds had high receptor affinity [K(i)(delta) = 0.034-1.1 nM], while that for the mu-opioid receptor fluctuated by orders of magnitude [K(i)(mu) = 15.1-3966 nM]; (ii) delta-opioid receptor selectivity [K(i)(mu)/K(i)(delta)] declined 1000-fold from 22,600 to 21; (iii) a C-terminal carboxyl group enhanced selectivity but only as a consequence of the specific residue; (iv) amidated, positive charged residues [Lys-NH(2) (6), Arg-NH(2) (7)], and a negatively charged aromatic residue [Trp-OH (11)] enhanced mu-opioid affinity [K(i)(mu) = 17.0, 15.1, and 15.7 nM, respectively], while Gly-NH(2) (8), Ser-NH(2) (10), and His-OH (12) were nearly one-tenth as active; and (v) D-isomers exhibited mixed effects on mu-opioid receptor affinity (2' < 3' < 4' < 1' < 5') and decreased delta-selectivity in D-Asp-NH(2) (1') and D-Lys(Ac)-OH (5'). The analogues exhibited delta-opioid receptor antagonism (pA(2) = 6.9-10.07) and weak mu-opioid receptor agonism (IC(50) > 1 microM) except H-Dmt-Tic-Glu-NH(2) (3), which was a partial delta-opioid receptor agonist (IC(50) = 2.5 nM). Thus, these C-terminally extended analogues indicated that an amino acid residue containing a single charge, amino or guanidino functionality, or aromatic group substantially altered the delta-opioid receptor activity profile (selectivity and antagonism) of the Dmt-Tic pharmacophore, which suggests that the C-terminal constituent plays a major role in determining

  10. Diminished adenosine A1 receptor expression on macrophages in brain and blood of patients with multiple sclerosis.

    PubMed

    Johnston, J B; Silva, C; Gonzalez, G; Holden, J; Warren, K G; Metz, L M; Power, C

    2001-05-01

    The nucleoside adenosine has been shown to control the production of proinflammatory molecules through its actions on cell surface purine receptors. Previously, we have reported that the adenosine A1 receptor (A1AR) regulates tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) expression and exhibits diminished function in patients with multiple sclerosis (MS; Mayne et al., Ann Neurol 1999;45:633-639). In the present study, A1AR expression in both brain and peripheral blood mononuclear cells (PBMC) from MS and control groups was characterized by fluorescence-activated cell sorting (FACS), reverse transcriptase-polymerase chain reaction (RT-PCR), and immunohistochemical analyses. FACS analyses of PBMC revealed that A1AR expression was chiefly detectable on CD14-positive cells and was reduced by 53.1% (p < 0.01) in MS patients compared to controls. A1AR mRNA levels were reduced by 43.1% (p < 0.001) in the brains of MS patients compared to patients with other neurological diseases and controls. A1AR protein expression in brain was detected primarily in CD45-positive glial cells and was markedly diminished in MS patients. The analysis of A1AR transcripts in the brain revealed that the A1AR-beta transcript was diminished (49.2%) in MS patients compared to controls (p < 0.002). These results indicate that the A1AR, expressed principally on cells of monocyte/macrophage lineage in both brain and blood, is selectively diminished in MS patients. Reduction of the A1AR-beta transcript in MS patients suggests that dysregulated splicing may influence A1AR protein levels, potentially leading to increased macrophage activation and central nervous system inflammation. PMID:11357956

  11. 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.

  12. 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.

  13. 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

  14. Circannual rhythm in body temperature, torpor, and sensitivity to A₁ adenosine receptor agonist in arctic ground squirrels.

    PubMed

    Olson, Jasmine M; Jinka, Tulasi R; Larson, Lindy K; Danielson, Jeffrey J; Moore, Jeanette T; Carpluck, Joanna; Drew, Kelly L

    2013-06-01

    A₁ adenosine receptor (A₁AR) activation within the central nervous system induces torpor, but in obligate hibernators such as the arctic ground squirrel (AGS; Urocitellus parryii), A₁AR stimulation induces torpor only during the hibernation season, suggesting a seasonal increase in sensitivity to A₁AR signaling. The purpose of this research was to investigate the relationship between body temperature (Tb) and sensitivity to an adenosine A1 receptor agonist in AGS. We tested the hypothesis that increased sensitivity in A₁AR signaling would lead to lower Tb in euthermic animals during the hibernation season when compared with the summer season. We further predicted that if a decrease in euthermic Tb reflects increased sensitivity to A₁AR activation, then it should likewise predict spontaneous torpor. We used subcutaneous IPTT-300 transponders to monitor Tb in AGS housed under constant ambient conditions (12:12 L:D, 18 °C) for up to 16 months. These animals displayed an obvious rhythm in euthermic Tb that cycled with a period of approximately 8 months. Synchrony in the Tb rhythm within the group was lost after several months of constant L:D conditions; however, individual rhythms in Tb continued to show clear sine wave-like waxing and waning. AGS displayed spontaneous torpor only during troughs in euthermic Tb. To assess sensitivity to A₁AR activation, AGS were administered the A₁AR agonist N(6)-cyclohexyladenosine (CHA, 0.1 mg/kg, ip), and subcutaneous Tb was monitored. AGS administered CHA during a seasonal minimum in euthermic Tb showed a greater drug-induced decrease in Tb (1.6 ± 0.3 °C) than did AGS administered CHA during a peak in euthermic Tb (0.4 ± 0.3 °C). These results provide evidence for a circannual rhythm in Tb that is associated with increased sensitivity to A₁AR signaling and correlates with the onset of torpor.

  15. 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

  16. 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.

  17. Inhibition of angiogenesis by selective estrogen receptor modulators through blockade of cholesterol trafficking rather than estrogen receptor antagonism.

    PubMed

    Shim, Joong Sup; Li, Ruo-Jing; Lv, Junfang; Head, Sarah A; Yang, Eun Ju; Liu, Jun O

    2015-06-28

    Selective estrogen receptor modulators (SERM) including tamoxifen are known to inhibit angiogenesis. However, the underlying mechanism, which is independent of their action on the estrogen receptor (ER), has remained largely unknown. In the present study, we found that tamoxifen and other SERM inhibited cholesterol trafficking in endothelial cells, causing a hyper-accumulation of cholesterol in late endosomes/lysosomes. Inhibition of cholesterol trafficking by tamoxifen was accompanied by abnormal subcellular distribution of vascular endothelial growth factor receptor-2 (VEGFR2) and inhibition of the terminal glycosylation of the receptor. Tamoxifen also caused perinuclear positioning of lysosomes, which in turn trapped the mammalian target of rapamycin (mTOR) in the perinuclear region of endothelial cells. Abnormal distribution of VEGFR2 and mTOR and inhibition of VEGFR2 and mTOR activities by tamoxifen were significantly reversed by addition of cholesterol-cyclodextrin complex to the culture media of endothelial cells. Moreover, high concentrations of tamoxifen inhibited endothelial and breast cancer cell proliferation in a cholesterol-dependent, but ER-independent, manner. Together, these results unraveled a previously unrecognized mechanism of angiogenesis inhibition by tamoxifen and other SERM, implicating cholesterol trafficking as an attractive therapeutic target for cancer treatment.

  18. Effect of opioid receptor antagonism on proopiomelanocortin peptide levels and gene expression in the hypothalamus.

    PubMed

    Markowitz, C E; Berkowitz, K M; Jaffe, S B; Wardlaw, S L

    1992-06-01

    In order to determine how brain beta-endorphin (beta-EP) and its precursor proopiomelanocortin (POMC) adapt to chronic opioid blockade we have examined the effects of treatment with the opioid receptor antagonist naltrexone (NTX) on POMC gene expression and peptide levels in the hypothalamus. Male rats were treated with NTX by daily injection or constant minipump infusion. RNA was isolated from the medial basal hypothalamus (MBH) after an aliquot was removed for peptide RIA and the amount of POMC mRNA was measured by solution hybridization SI nuclease protection assay. beta-EP and several other POMC-derived peptides including alpha-melanocyte-stimulating hormone (alpha-MSH) and corticotropin-like intermediate lobe peptide (CLIP) or gamma(3)-MSH were measured in the MBH and anterior hypothalamus (AH) by RIA. In an initial experiment POMC peptide levels were measured after 7 days of NTX (4.8 mg/day) infusion. There was a marked fall in the concentrations of beta-EP, alpha-MSH, and CLIP; levels in the MBH declined by more than 60% (P < 0.001). In the next experiment NTX (1 mg) was injected daily and POMC peptides and mRNA were measured after 2 and 5 days of treatment. (beta-EP) and alpha-MSH levels fell progressively in the MBH and AH and were significantly less than those of the controls by 5 days of treatment (P < 0.02). POMC mRNA levels, however, did not change after 2 or 5 days. When NTX was infused for 3 weeks there was a decrease in the concentrations of beta-EP, alpha-MSH, and gamma(3)-MSH in the MBH (P < 0.001). The concentration of POMC mRNA in the MBH, however, was significantly higher in the NTX-treated animals, 0.99 +/- 0.06 pg/mug RNA vs 0.81 +/- 0.05 pg/mug RNA (P < 0.05). Since NTX can affect LH and testosterone release, the study was repeated in castrated rats. POMC peptide levels again fell after 3 weeks of NTX. POMC mRNA levels were higher in the castrated rats than in the intact rats, 1.14 +/- 0.06 pg/mug RNA vs 0.85 +/- 0.09 pg/mug RNA (P < 0

  19. 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

  20. Antagonism of chemokine receptor CXCR3 inhibits osteosarcoma metastasis to lungs.

    PubMed

    Pradelli, Emmanuelle; Karimdjee-Soilihi, Babou; Michiels, Jean-François; Ricci, Jean-Ehrland; Millet, Marie-Ange; Vandenbos, Fanny; Sullivan, Timothy J; Collins, Tassie L; Johnson, Michael G; Medina, Julio C; Kleinerman, Eugenie S; Schmid-Alliana, Annie; Schmid-Antomarchi, Heidy

    2009-12-01

    Metastasis continues to be the leading cause of mortality for patients with cancer. Several years ago, it became clear that chemokines and their receptors could control the tumor progress. CXCR3 has now been identified in many cancers including osteosarcoma and CXCR3 ligands were expressed by lungs that are the primary sites to which this tumor metastasize. This study tested the hypothesis that disruption of the CXCR3/CXCR3 ligands complexes could lead to a decrease in lungs metastasis. The experimental design involved the use of the CXCR3 antagonist, AMG487 and 2 murine models of osteosarcoma lung metastases. After tail vein injection of osteosarcoma cells, mice that were systematically treated with AMG487 according to preventive or curative protocols had a significant reduction in metastatic disease. Treatment of osteosarcoma cells in vitro with AMG487 led to decreased migration, decreased matrix metalloproteinase activity, decreased proliferation/survival and increased caspase-independent death. Taken together, our results support the hypothesis that CXCR3 and their ligands intervene in the initial dissemination of the osteosarcoma cells to the lungs and stimulate the growth and expansion of the metastatic foci in later stages. Moreover, these studies indicate that targeting CXCR3 may specifically inhibit tumor metastasis without adversely affecting antitumoral host response. PMID:19544560

  1. Antagonism of chemokine receptor CXCR3 inhibits osteosarcoma metastasis to lungs

    PubMed Central

    Pradelli, Emmanuelle; Karimdjee-Soilihi, Babou; Michiels, Jean-François; Ricci, Jean-Ehrland; Millet, Marie-Ange; Vandenbos, Fanny; Sullivan, Timothy J.; Collins, Tassie L.; Johnson, Michael G.; Medina, Julio C.; Kleinerman, Eugenie S.; Schmid-Alliana, Annie; Schmid-Antomarchi, Heidy

    2009-01-01

    Metastasis continues to be the leading cause of mortality for patients with cancer. Several years ago, it became clear that chemokines and their receptors could control the tumor progress. CXCR3 has now been identified in many cancers including osteosarcoma and CXCR3 ligands were expressed by lungs that are the primary sites to which this tumor metastasize. This study tested the hypothesis that disruption of the CXCR3/CXCR3 ligands complexes could lead to a decrease in lungs metastasis. The experimental design involved the use of the CXCR3 antagonist, AMG487 and 2 murine models of osteosarcoma lung metastases. After tail vein injection of osteosarcoma cells, mice that were systematically treated with AMG487 according to preventive or curative protocols had a significant reduction in metastatic disease. Treatment of osteosarcoma cells in vitro with AMG487 led to decreased migration, decreased matrix metalloproteinase activity, decreased proliferation/survival and increased caspase-independent death. Taken together, our results support the hypothesis that CXCR3 and their ligands intervene in the initial dissemination of the osteosarcoma cells to the lungs and stimulate the growth and expansion of the metastatic foci in later stages. Moreover, these studies indicate that targeting CXCR3 may specifically inhibit tumor metastasis without adversely affecting antitumoral host response. PMID:19544560

  2. The Halicylindramides, Farnesoid X Receptor Antagonizing Depsipeptides from a Petrosia sp. Marine Sponge Collected in Korea.

    PubMed

    Hahn, Dongyup; Kim, Hiyoung; Yang, Inho; Chin, Jungwook; Hwang, Hoosang; Won, Dong Hwan; Lee, Byoungchan; Nam, Sang-Jip; Ekins, Merrick; Choi, Hyukjae; Kang, Heonjoong

    2016-03-25

    Three new structurally related depsipeptides, halicylindramides F-H (1-3), and two known halicylindramides were isolated from a Petrosia sp. marine sponge collected off the shore of Youngdeok-Gun, East Sea, Republic of Korea. Their planar structures were elucidated by extensive spectroscopic data analyses including 1D and 2D NMR data as well as MS data. The absolute configurations of halicylindramides F-H (1-3) were determined by Marfey's method in combination with Edman degradation. The absolute configurations at C-4 of the dioxyindolyl alanine (Dioia) residues of halicylindramides G (2) and H (3) were determined as 4S and 4R, respectively, based on ECD spectroscopy. The C-2 configurations of Dioia in 2 and 3 were speculated to both be 2R