Science.gov

Sample records for a2b adenosine receptor

  1. Elevated Adenosine Induces Placental DNA Hypomethylation Independent of A2B Receptor Signaling in Preeclampsia.

    PubMed

    Huang, Aji; Wu, Hongyu; Iriyama, Takayuki; Zhang, Yujin; Sun, Kaiqi; Song, Anren; Liu, Hong; Peng, Zhangzhe; Tang, Lili; Lee, Minjung; Huang, Yun; Ni, Xin; Kellems, Rodney E; Xia, Yang

    2017-07-01

    Preeclampsia is a prevalent pregnancy hypertensive disease with both maternal and fetal morbidity and mortality. Emerging evidence indicates that global placental DNA hypomethylation is observed in patients with preeclampsia and is linked to altered gene expression and disease development. However, the molecular basis underlying placental epigenetic changes in preeclampsia remains unclear. Using 2 independent experimental models of preeclampsia, adenosine deaminase-deficient mice and a pathogenic autoantibody-induced mouse model of preeclampsia, we demonstrate that elevated placental adenosine not only induces hallmark features of preeclampsia but also causes placental DNA hypomethylation. The use of genetic approaches to express an adenosine deaminase minigene specifically in placentas, or adenosine deaminase enzyme replacement therapy, restored placental adenosine to normal levels, attenuated preeclampsia features, and abolished placental DNA hypomethylation in adenosine deaminase-deficient mice. Genetic deletion of CD73 (an ectonucleotidase that converts AMP to adenosine) prevented the elevation of placental adenosine in the autoantibody-induced preeclampsia mouse model and ameliorated preeclampsia features and placental DNA hypomethylation. Immunohistochemical studies revealed that elevated placental adenosine-mediated DNA hypomethylation predominantly occurs in spongiotrophoblasts and labyrinthine trophoblasts and that this effect is independent of A2B adenosine receptor activation in both preeclampsia models. Extending our mouse findings to humans, we used cultured human trophoblasts to demonstrate that adenosine functions intracellularly and induces DNA hypomethylation without A2B adenosine receptor activation. Altogether, both mouse and human studies reveal novel mechanisms underlying placental DNA hypomethylation and potential therapeutic approaches for preeclampsia. © 2017 American Heart Association, Inc.

  2. Adenosine A2A receptor ligand recognition and signaling is blocked by A2B receptors.

    PubMed

    Hinz, Sonja; Navarro, Gemma; Borroto-Escuela, Dasiel; Seibt, Benjamin F; Ammon, York-Christoph; de Filippo, Elisabetta; Danish, Azeem; Lacher, Svenja K; Červinková, Barbora; Rafehi, Muhammad; Fuxe, Kjell; Schiedel, Anke C; Franco, Rafael; Müller, Christa E

    2018-03-02

    The adenosine receptor (AR) subtypes A 2A and A 2B are rhodopsin-like G s protein-coupled receptors whose expression is highly regulated under pathological, e.g. hypoxic, ischemic and inflammatory conditions. Both receptors play important roles in inflammatory and neurodegenerative diseases, are blocked by caffeine, and have now become major drug targets in immuno-oncology. By Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation (BiFC) and proximity ligation assays (PLA) we demonstrated A 2A -A 2B AR heteromeric complex formation. Moreover we observed a dramatically altered pharmacology of the A 2A AR when co-expressed with the A 2B AR (A 2B ≥ A 2A ) in recombinant as well as in native cells. In the presence of A 2B ARs, A 2A -selective ligands lost high affinity binding to A 2A ARs and displayed strongly reduced potency in cAMP accumulation and dynamic mass redistribution (DMR) assays. These results have major implications for the use of A 2A AR ligands as drugs as they will fail to modulate the receptor in an A 2A -A 2B heteromer context. Accordingly, A 2A -A 2B AR heteromers represent novel pharmacological targets.

  3. 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.

  4. Characterization of the A2B adenosine receptor from mouse, rabbit, and dog.

    PubMed

    Auchampach, John A; Kreckler, Laura M; Wan, Tina C; Maas, Jason E; van der Hoeven, Dharini; Gizewski, Elizabeth; Narayanan, Jayashree; Maas, Garren E

    2009-04-01

    We have cloned and pharmacologically characterized the A(2B) adenosine receptor (AR) from the dog, rabbit, and mouse. The full coding regions of the dog and mouse A(2B)AR were obtained by reverse transcriptase-polymerase chain reaction, and the rabbit A(2B)AR cDNA was obtained by screening a rabbit brain cDNA library. It is noteworthy that an additional clone was isolated by library screening that was identical in sequence to the full-length rabbit A(2B)AR, with the exception of a 27-base pair deletion in the region encoding amino acids 103 to 111 (A(2B)AR(103-111)). This 9 amino acid deletion is located in the second intracellular loop at the only known splice junction of the A(2B)AR and seems to result from the use of an additional 5' donor site found in the rabbit and dog but not in the human, rat, or mouse sequences. [(3)H]3-Isobutyl-8-pyrrolidinoxanthine and 8-[4-[((4-cyano-[2,6-(3)H]-phenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine ([(3)H]MRS 1754) bound with high affinity to membranes prepared from human embryonic kidney (HEK) 293 cells expressing mouse, rabbit, and dog A(2B)ARs. Competition binding studies performed with a panel of agonist (adenosine and 2-amino-3,5-dicyano-4-phenylpyridine analogs) and antagonist ligands identified similar potency orders for the A(2B)AR orthologs, although most xanthine antagonists displayed lower binding affinity for the dog A(2B)AR compared with A(2B)ARs from rabbit and mouse. No specific binding could be detected with membranes prepared from HEK 293 cells expressing the rabbit A(2B)AR(103-111) variant. Furthermore, the variant failed to stimulate adenylyl cyclase or calcium mobilization. We conclude that significant differences in antagonist pharmacology of the A(2B)AR exist between species and that some species express nonfunctional variants of the A(2B)AR due to "leaky" splicing.

  5. A2B adenosine receptor induces protective antihelminth type 2 immune responses.

    PubMed

    Patel, Nirav; Wu, Wenhui; Mishra, Pankaj K; Chen, Fei; Millman, Ariel; Csóka, Balázs; Koscsó, Balázs; Eltzschig, Holger K; Haskó, György; Gause, William C

    2014-03-12

    The type 2 immune response evoked by intestinal nematode parasites contributes to worm expulsion and tolerance to associated tissue damage. We investigated whether this host response is affected by blocking signaling by the putative endogenous danger signal adenosine, which can be released during inflammation and host cell damage. Specific blockade of the A2B adenosine receptor (A2BAR) inhibited worm elimination and the development of innate and adaptive components of the type 2 primary and memory response. Infected mice lacking A2BAR exhibited decreased M2 macrophage and eosinophil recruitment and reduced IL-4 and IL-13 cytokine production. Additionally, shortly after infection, upregulation of the alarmin IL-33, which drives type 2 immunity, and activation of innate lymphoid type 2 (ILC2) cells was inhibited, while exogenous IL-33 restored ILC2 cell activation and type 2 cytokine expression. Thus, adenosine acts as a danger-associated molecular pattern (DAMP) that initiates helminth-induced type 2 immune responses through A2BAR. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. 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

  7. Links Between Insulin Resistance, Adenosine A2B Receptors, and Inflammatory Markers in Mice and Humans

    PubMed Central

    Figler, Robert A.; Wang, Guoquan; Srinivasan, Susseela; Jung, Dae Young; Zhang, Zhiyou; Pankow, James S.; Ravid, Katya; Fredholm, Bertil; Hedrick, Catherine C.; Rich, Stephen S.; Kim, Jason K.; LaNoue, Kathryn F.; Linden, Joel

    2011-01-01

    OBJECTIVE To determine the mechanisms by which blockade of adenosine A2B receptors (A2BRs) reduces insulin resistance. RESEARCH DESIGN AND METHODS We investigated the effects of deleting or blocking the A2BR on insulin sensitivity using glucose tolerance tests (GTTs) and hyperinsulinemic-euglycemic clamps in mouse models of type 2 diabetes. The effects of diabetes on A2BR transcription and signaling were measured in human and mouse macrophages and mouse endothelial cells. In addition, tag single nucleotide polymorphisms (SNPs) in ∼42 kb encompassing the A2BR gene, ADORA2B, were evaluated for associations with markers of diabetes and inflammation. RESULTS Treatment of mice with the nonselective adenosine receptor agonist 5′-N-ethylcarboxamidoadensoine (NECA) increased fasting blood glucose and slowed glucose disposal during GTTs. These responses were inhibited by A2BR deletion or blockade and minimally affected by deletion of A1Rs or A2ARs. During hyperinsulinemic-euglycemic clamp of diabetic KKAY mice, A2BR antagonism increased glucose infusion rate, reduced hepatic glucose production, and increased glucose uptake into skeletal muscle and brown adipose tissue. Diabetes caused a four- to sixfold increase in A2BR mRNA in endothelial cells and macrophages and resulted in enhanced interleukin (IL)-6 production in response to NECA due to activation of protein kinases A and C. Five consecutive tag SNPs in ADORA2B were highly correlated with IL-6 and C-reactive protein (CRP). Diabetes had a highly significant independent effect on variation in inflammatory markers. The strength of associations between several ADORA2B SNPs and inflammatory markers was increased when accounting for diabetes status. CONCLUSIONS Diabetes affects the production of adenosine and the expression of A2BRs that stimulate IL-6 and CRP production, insulin resistance, and the association between ADORA2B SNPs and inflammatory markers. We hypothesize that increased A2BR signaling in diabetes

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

    PubMed

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

    2012-01-04

    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.

  9. 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

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

    PubMed Central

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

    2008-01-01

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

  11. Adenosine receptor A2b on hematopoietic cells mediates LPS-induced migration of PMNs into the lung interstitium.

    PubMed

    Konrad, Franziska M; Witte, Esther; Vollmer, Irene; Stark, Stefanie; Reutershan, Jörg

    2012-09-01

    Uncontrolled transmigration of polymorphonuclear leukocytes (PMNs) into the different compartments of the lungs (intravascular, interstitial, alveolar) is a critical event in the early stage of acute lung injury and acute respiratory distress syndrome. Adenosine receptor A(2b) is highly expressed in the inflamed lungs and has been suggested to mediate cell trafficking. In a murine model of LPS-induced lung inflammation, we investigated the role of A(2b) on migration of PMNs into the different compartments of the lung. In A(2b)(-/-) mice, LPS-induced accumulation of PMNs was significantly higher in the interstitium, but not in the alveolar space. In addition, pulmonary clearance of PMNs was delayed in A(2b)(-/-) mice. Using chimeric mice, we identified A(2b) on hematopoietic cells as crucial for PMN migration. A(2b) did not affect the release of relevant chemokines into the alveolar space. LPS-induced microvascular permeability was under the control of A(2b) on both hematopoietic and nonhematopoietic cells. Activation of A(2b) on endothelial cells also reduced formation of LPS-induced stress fibers, highlighting its role for endothelial integrity. A specific A(2b) agonist (BAY 60-6583) was effective in decreasing PMN migration into the lung interstitium and microvascular permeability. In addition, in vitro transmigration of human PMNs through a layer of human endothelial or epithelial cells was A(2b) dependent. Activation of A(2b) on human PMNs reduced oxidative burst activity. Together, our results demonstrate anti-inflammatory effects of A(2b) on two major characteristics of acute lung injury, with a distinct role of hematopoietic A(2b) for cell trafficking and endothelial A(2b) for microvascular permeability.

  12. Epithelial-specific A2B adenosine receptor signaling protects the colonic epithelial barrier during acute colitis

    PubMed Central

    Aherne, CM; Saeedi, B; Collins, CB; Masterson, JC; McNamee, EN; Perrenoud, L; Rapp, CR; Curtis, VF; Bayless, A; Fletcher, A; Glover, LE; Evans, CM; Jedlicka, P; Furuta, GT; de Zoeten, EF; Colgan, SP; Eltzschig, HK

    2015-01-01

    Central to inflammatory bowel disease (IBD) pathogenesis is loss of mucosal barrier function. Emerging evidence implicates extracellular adenosine signaling in attenuating mucosal inflammation. We hypothesized that adenosine-mediated protection from intestinal barrier dysfunction involves tissue-specific signaling through the A2B adenosine receptor (Adora2b) at the intestinal mucosal surface. To address this hypothesis, we combined pharmacologic studies and studies in mice with global or tissue-specific deletion of the Adora2b receptor. Adora2b−/− mice experienced a significantly heightened severity of colitis, associated with a more acute onset of disease and loss of intestinal epithelial barrier function. Comparison of mice with Adora2b deletion on vascular endothelial cells (Adora2bfl/flVeCadCre+) or intestinal epithelia (Adora2bfl/flVillinCre+) revealed a selective role for epithelial Adora2b signaling in attenuating colonic inflammation. In vitro studies with Adora2b knockdown in intestinal epithelial cultures or pharmacologic studies highlighted Adora2b-driven phosphorylation of vasodilator-stimulated phosphoprotein (VASP) as a specific barrier repair response. Similarly, in vivo studies in genetic mouse models or treatment studies with an Adora2b agonist (BAY 60-6583) recapitulate these findings. Taken together, our results suggest that intestinal epithelial Adora2b signaling provides protection during intestinal inflammation via enhancing mucosal barrier responses. PMID:25850656

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

    PubMed Central

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

    2010-01-01

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

  14. Neutrophil-derived 5′-Adenosine Monophosphate Promotes Endothelial Barrier Function via CD73-mediated Conversion to Adenosine and Endothelial A2B Receptor Activation

    PubMed Central

    Lennon, Paul F.; Taylor, Cormac T.; Stahl, Gregory L.; Colgan, Sean P.

    1998-01-01

    During episodes of inflammation, polymorphonuclear leukocyte (PMN) transendothelial migration has the potential to disturb vascular barrier function and give rise to intravascular fluid extravasation and edema. However, little is known regarding innate mechanisms that dampen fluid loss during PMN-endothelial interactions. Using an in vitro endothelial paracellular permeability model, we observed a PMN-mediated decrease in endothelial paracellular permeability. A similar decrease was elicited by cell-free supernatants from activated PMN (FMLP 10−6 M), suggesting the presence of a PMN-derived soluble mediator(s). Biophysical and biochemical analysis of PMN supernatants revealed a role for PMN-derived 5′-adenosine monophosphate (AMP) and its metabolite, adenosine, in modulation of endothelial paracellular permeability. Supernatants from activated PMN contained micromolar concentrations of bioactive 5′-AMP and adenosine. Furthermore, exposure of endothelial monolayers to authentic 5′-AMP and adenosine increased endothelial barrier function more than twofold in both human umbilical vein endothelial cells and human microvascular endothelial cells. 5′-AMP bioactivity required endothelial CD73-mediated conversion of 5′-AMP to adenosine via its 5′-ectonucleotidase activity. Decreased endothelial paracellular permeability occurred through adenosine A2B receptor activation and was accompanied by a parallel increase in intracellular cAMP. We conclude that activated PMN release soluble mediators, such as 5′-AMP and adenosine, that promote endothelial barrier function. During inflammation, this pathway may limit potentially deleterious increases in endothelial paracellular permeability and could serve as a basic mechanism of endothelial resealing during PMN transendothelial migration. PMID:9782120

  15. Stimulation of adenosine A2b receptors blocks apoptosis in the non-infarcted myocardium even when administered after the onset of infarction.

    PubMed

    Simonis, Gregor; Wiedemann, Stephan; Joachim, Dirk; Weinbrenner, Christof; Marquetant, Rainer; Strasser, Ruth H

    2009-08-01

    Chronic adenosine A2b receptor stimulation has been shown to prevent ventricular remodelling after myocardial infarction (MI). We hypothesized that this effect is due to the inhibition of cardiac myocyte apoptosis in the myocardium remote from the infarction. Rats were subjected to MI by LAD ligation in situ. Some animals were pre-treated with the stable adenosine analogue 2-chloro-adenosine (CADO). After 24 h, pro- and anti-apoptotic signals (protein kinase C isoforms, p38, g proteins, Bcl-2/Bax ratio, Akt, Bad), and marker of apoptosis execution (caspase-3, TUNEL) were quantified in the remote myocardium. CADO prevented the occurrence of apoptosis in the remote myocardium of an infarcted heart. This effect occured not only when CADO was started before the onset of ischemia but also when it started 3 h after the infarction. The anti-apoptotic effect of CADO was blocked by simultaneous administration of the selective adenosine A2b receptor antagonist MRS1754 (1 mg/kg). The anti-apoptotic effect of CADO seems to be mediated by g(alphaq) and by the activation of survival kinases (Bad) and by inhibition of the pro-apoptotic PKC-delta/p38-MAPK-pathway. Chronic adenosine A2b receptor stimulation blocks cardiac myocyte apoptosis in the remote myocardium even when started after the onset of infarction. This may explain the anti-remodelling-effect of the A2b receptor stimulation after infarction.

  16. The aminopyridine-3,5-dicarbonitrile core for the design of new non-nucleoside-like agonists of the human adenosine A2B receptor.

    PubMed

    Betti, Marco; Catarzi, Daniela; Varano, Flavia; Falsini, Matteo; Varani, Katia; Vincenzi, Fabrizio; Dal Ben, Diego; Lambertucci, Catia; Colotta, Vittoria

    2018-04-25

    A new series of amino-3,5-dicyanopyridines (3-28) as analogues of the adenosine hA 2B receptor agonist BAY60-6583 (compound 1) was synthesized. All the compounds that interact with the hA 2B adenosine receptor display EC 50 values in the range 9-350 nM behaving as partial agonists, with the only exception being the 2-{[4-(4-acetamidophenyl)-6-amino-3,5-dicyanopyridin-2-yl]thio}acetamide (8) which shows a full agonist profile. Moreover, the 2-[(1H-imidazol-2-yl)methylthio)]-6-amino-4-(4-cyclopropylmethoxy-phenyl)pyridine-3,5-dicarbonitrile (15) turns out to be 3-fold more active than 1 although less selective. This result can be considered a real breakthrough due to the currently limited number of non-adenosine hA 2B AR agonists reported in literature. To simulate the binding mode of nucleoside and non-nucleoside agonists at the hA 2B AR, molecular docking studies were performed at homology models of this AR subtype developed by using two crystal structures of agonist-bound A 2A AR as templates. These investigations allowed us to represent a hypothetical binding mode of hA 2B receptor agonists belonging to the amino-3,5-dicyanopyridine series and to rationalize the observed SAR. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  17. Myeloid-derived suppressor cells contribute to A2B adenosine receptor-induced VEGF production and angiogenesis in a mouse melanoma model.

    PubMed

    Sorrentino, Claudia; Miele, Lucio; Porta, Amalia; Pinto, Aldo; Morello, Silvana

    2015-09-29

    Vascular endothelial growth factor (VEGF) is an angiogenic factor critically involved in tumor progression. Adenosine A2B receptor plays a pivotal role in promoting tumor growth. The aim of this study was to investigate the role of myeloid-derived suppressor cells (MDSCs) in the pro-angiogenic effects of A2B and to determine whether A2B blockade could enhance the effectiveness of anti-VEGF treatment. Mice treated with Bay60-6583, a selective A2B receptor agonist, showed enhanced tumor VEGF-A expression and vessel density. This effect was associated with accelerated tumor growth, which could be reversed with anti-VEGF treatment. Bay60-6583 increased the accumulation of tumor CD11b+Gr1+ cells. Depletion of MDSCs in mice significantly reduced A2B-induced VEGF production. However, A2B receptor stimulation did not directly regulate VEGF expression in isolated tumor myeloid cells. Mechanistically, Bay60-6583-treated melanoma tissues showed increased STAT3 activation. Inhibition of STAT3 significantly decreased the pro-tumor activity of Bay60-6583 and reduced tumor VEGF expression. Pharmacological blockade of A2B receptor with PSB1115 significantly reduced tumor growth by inhibiting tumor angiogenesis and increasing T cells numbers within the tumor microenvironment. These effects are, at least in part, dependent on impaired tumor accumulation of Gr1+ cells upon A2B receptor blockade. PSB1115 increased the effectiveness of anti-VEGF treatment.

  18. 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

  19. Blockade of A2b Adenosine Receptor Reduces Tumor Growth and Immune Suppression Mediated by Myeloid-Derived Suppressor Cells in a Mouse Model of Melanoma12

    PubMed Central

    Iannone, Raffaella; Miele, Lucio; Maiolino, Piera; Pinto, Aldo; Morello, Silvana

    2013-01-01

    The A2b receptor (A2bR) belongs to the adenosine receptor family. Emerging evidence suggest that A2bR is implicated in tumor progression in some murine tumor models, but the therapeutic potential of targeting A2bR in melanoma has not been examined. This study first shows that melanoma-bearing mice treated with Bay 60-6583, a selective A2bR agonist, had increased melanoma growth. This effect was associated with higher levels of immune regulatory mediators interleukin-10 (IL-10) and monocyte chemoattractant protein 1 (MCP-1) and accumulation of tumor-associated CD11b positive Gr1 positive cells (CD11b+Gr1+) myeloid-derived suppressor cells (MDSCs). Depletion of CD11b+Gr1+ cells completely reversed the protumor activity of Bay 60-6583. Conversely, pharmacological blockade of A2bR with PSB1115 reversed immune suppression in the tumor microenvironment, leading to a significant melanoma growth delay. PSB1115 treatment reduced both levels of IL-10 and MCP-1 and CD11b+Gr1+ cell number in melanoma lesions. These effects were associated with higher frequency of tumor-infiltrating CD8 positive (CD8+) T cells and natural killer T (NKT) cells and increased levels of T helper 1 (Th1)-like cytokines. Adoptive transfer of CD11b+Gr1+ cells abrogated the antitumor activity of PSB1115. These data suggest that the antitumor activity of PSB1115 relies on its ability to lower accumulation of tumor-infiltrating MDSCs and restore an efficient antitumor T cell response. The antitumor effect of PSB1115 was not observed in melanoma-bearing nude mice. Furthermore, PSB1115 enhanced the antitumor efficacy of dacarbazine. These data indicate that A2bR antagonists such as PSB1115 should be investigated as adjuvants in the treatment of melanoma. PMID:24403862

  20. Caffeine Reduces 11β-Hydroxysteroid Dehydrogenase Type 2 Expression in Human Trophoblast Cells through the Adenosine A2B Receptor

    PubMed Central

    Sharmin, Saina; Guan, Haiyan; Williams, Andrew Scott; Yang, Kaiping

    2012-01-01

    Maternal caffeine consumption is associated with reduced fetal growth, but the underlying molecular mechanisms are unknown. Since there is evidence that decreased placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) is linked to fetal growth restriction, we hypothesized that caffeine may inhibit fetal growth partly through down regulating placental 11β-HSD2. As a first step in examining this hypothesis, we studied the effects of caffeine on placental 11β-HSD2 activity and expression using our established primary human trophoblast cells as an in vitro model system. Given that maternal serum concentrations of paraxanthine (the primary metabolite of caffeine) were greater in women who gave birth to small-for-gestational age infants than to appropriately grown infants, we also studied the effects of paraxanthine. Our main findings were: (1) both caffeine and paraxanthine decreased placental 11β-HSD2 activity, protein and mRNA in a concentration-dependent manner; (2) this inhibitory effect was mediated by the adenosine A2B receptor, since siRNA-mediated knockdown of this receptor prevented caffeine- and paraxanthine-induced inhibition of placental 11β-HSD2; and (3) forskolin (an activator of adenyl cyclase and a known stimulator of 11β-HSD2) abrogated the inhibitory effects of both caffeine and paraxanthine, which provides evidence for a functional link between exposure to caffeine and paraxanthine, decreased intracellular levels of cAMP and reduced placental 11β-HSD2. Taken together, these findings reveal that placental 11β-HSD2 is a novel molecular target through which caffeine may adversely affect fetal growth. They also uncover a previously unappreciated role for the adenosine A2B receptor signaling in regulating placental 11β-HSD2, and consequently fetal development. PMID:22701600

  1. An A2B Adenosine Receptor Agonist Promotes Th17 Autoimmune Responses in Experimental Autoimmune Uveitis (EAU) via Dendritic Cell Activation.

    PubMed

    Chen, Mingjiazi; Liang, Dongchun; Zuo, Aijun; Shao, Hui; Kaplan, Henry J; Sun, Deming

    2015-01-01

    We have recently reported that, although adenosine receptor (AR) agonists have a suppressive effect on Th1 autoreactive T cells, their effect on Th17 autoreactive T cells and γδ T cells is stimulatory and this effect is mainly mediated via A2A adenosine receptors (A2ARs). In this study, we further demonstrate that treatment of C57BL/6 (B6) mice with a selective A2B adenosine receptor (A2BR) agonist greatly enhanced the development of experimental autoimmune uveitis (EAU), whereas treatment with an A2BR antagonist significantly ameliorated severity of EAU. The A2BR agonist-treated mice showed augmented Th17, but not Th1, responses. Mechanistic studies showed that the A2BR agonist-induced enhancement of the Th17 response was significantly lower when TCR-δ-/- mice received the same treatment and that transfer of γδ T cells into TCR-δ-/- mice partially restored this effect. We also showed that dendritic cells (DCs) from A2BR agonist-treated mice showed a significantly increased ability to activate γδ T cells and Th17 autoreactive T cells. Thus, our previous studies have shown that, in EAU, activated γδ T cells possess greatly increased ability to enhance Th17 autoimmune responses. In the present study, we showed that exposure of DCs to A2BR agonist facilitated γδ T cell activation, leading to augmented Th17 responses and progressive EAU development. Our results further support our previous finding that AR agonists have distinct effects on Th1 and Th17 autoimmune responses.

  2. An A2B Adenosine Receptor Agonist Promotes Th17 Autoimmune Responses in Experimental Autoimmune Uveitis (EAU) via Dendritic Cell Activation

    PubMed Central

    Chen, Mingjiazi; Liang, Dongchun; Zuo, Aijun; Shao, Hui; Kaplan, Henry J.; Sun, Deming

    2015-01-01

    We have recently reported that, although adenosine receptor (AR) agonists have a suppressive effect on Th1 autoreactive T cells, their effect on Th17 autoreactive T cells and γδ T cells is stimulatory and this effect is mainly mediated via A2A adenosine receptors (A2ARs). In this study, we further demonstrate that treatment of C57BL/6 (B6) mice with a selective A2B adenosine receptor (A2BR) agonist greatly enhanced the development of experimental autoimmune uveitis (EAU), whereas treatment with an A2BR antagonist significantly ameliorated severity of EAU. The A2BR agonist-treated mice showed augmented Th17, but not Th1, responses. Mechanistic studies showed that the A2BR agonist-induced enhancement of the Th17 response was significantly lower when TCR-δ-/- mice received the same treatment and that transfer of γδ T cells into TCR-δ-/- mice partially restored this effect. We also showed that dendritic cells (DCs) from A2BR agonist-treated mice showed a significantly increased ability to activate γδ T cells and Th17 autoreactive T cells. Thus, our previous studies have shown that, in EAU, activated γδ T cells possess greatly increased ability to enhance Th17 autoimmune responses. In the present study, we showed that exposure of DCs to A2BR agonist facilitated γδ T cell activation, leading to augmented Th17 responses and progressive EAU development. Our results further support our previous finding that AR agonists have distinct effects on Th1 and Th17 autoimmune responses. PMID:26147733

  3. Leishmania amazonensis-Induced cAMP Triggered by Adenosine A2B Receptor Is Important to Inhibit Dendritic Cell Activation and Evade Immune Response in Infected Mice

    PubMed Central

    Figueiredo, Amanda Braga; Souza-Testasicca, Míriam Conceição; Mineo, Tiago Wilson Patriarca; Afonso, Luís Carlos Crocco

    2017-01-01

    Differently from others Leishmania species, infection by the protozoan parasite L. amazonensis is associated with a lack of antigen-specific T-cell responses. Dendritic cells (DC) are essential for the innate immune response and for directing the differentiation of T-helper lymphocytes. Previously, we showed that L. amazonensis infection impairs DC activation through the activation of adenosine A2B receptor, and here, we evaluated the intracellular events triggered by this receptor in infected cells. To this aim, bone marrow-derived DC from C57BL/6J mice were infected with metacyclic promastigotes of L. amazonensis. Our results show, for the first time, that L. amazonensis increases the production of cAMP and the phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in infected DC by a mechanism dependent on the A2B receptor. Furthermore, L. amazonensis impairs CD40 expression and IL-12 production by DC, and the inhibition of adenylate cyclase, phosphoinositide 3-kinase (PI3K), and ERK1/2 prevent these effects. The increase of ERK1/2 phosphorylation and the inhibition of DC activation by L. amazonensis are independent of protein kinase A (PKA). In addition, C57BL/6J mice were inoculated in the ears with metacyclic promastigotes, in the presence of PSB1115, an A2B receptor antagonist. PSB1115 treatment increases the percentage of CD40+ DC on ears and draining lymph nodes. Furthermore, this treatment reduces lesion size and tissue parasitism. Lymph node cells from treated mice produce higher levels of IFN-γ than control mice, without altering the production of IL-10. In conclusion, we suggest a new pathway used by the parasite (A2B receptor → cAMP → PI3K → ERK1/2) to suppress DC activation, which may contribute to the decrease of IFN-γ production following by the deficiency in immune response characteristic of L. amazonensis infection. PMID:28791011

  4. Leishmania amazonensis-Induced cAMP Triggered by Adenosine A2B Receptor Is Important to Inhibit Dendritic Cell Activation and Evade Immune Response in Infected Mice.

    PubMed

    Figueiredo, Amanda Braga; Souza-Testasicca, Míriam Conceição; Mineo, Tiago Wilson Patriarca; Afonso, Luís Carlos Crocco

    2017-01-01

    Differently from others Leishmania species, infection by the protozoan parasite L. amazonensis is associated with a lack of antigen-specific T-cell responses. Dendritic cells (DC) are essential for the innate immune response and for directing the differentiation of T-helper lymphocytes. Previously, we showed that L. amazonensis infection impairs DC activation through the activation of adenosine A 2B receptor, and here, we evaluated the intracellular events triggered by this receptor in infected cells. To this aim, bone marrow-derived DC from C57BL/6J mice were infected with metacyclic promastigotes of L. amazonensis . Our results show, for the first time, that L. amazonensis increases the production of cAMP and the phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in infected DC by a mechanism dependent on the A 2B receptor. Furthermore, L. amazonensis impairs CD40 expression and IL-12 production by DC, and the inhibition of adenylate cyclase, phosphoinositide 3-kinase (PI3K), and ERK1/2 prevent these effects. The increase of ERK1/2 phosphorylation and the inhibition of DC activation by L. amazonensis are independent of protein kinase A (PKA). In addition, C57BL/6J mice were inoculated in the ears with metacyclic promastigotes, in the presence of PSB1115, an A 2B receptor antagonist. PSB1115 treatment increases the percentage of CD40 + DC on ears and draining lymph nodes. Furthermore, this treatment reduces lesion size and tissue parasitism. Lymph node cells from treated mice produce higher levels of IFN-γ than control mice, without altering the production of IL-10. In conclusion, we suggest a new pathway used by the parasite (A 2B receptor → cAMP → PI3K → ERK1/2) to suppress DC activation, which may contribute to the decrease of IFN-γ production following by the deficiency in immune response characteristic of L. amazonensis infection.

  5. Ligand-, structure- and pharmacophore-based molecular fingerprints: a case study on adenosine A1, A2A, A2B, and A3 receptor antagonists

    NASA Astrophysics Data System (ADS)

    Sirci, Francesco; Goracci, Laura; Rodríguez, David; van Muijlwijk-Koezen, Jacqueline; Gutiérrez-de-Terán, Hugo; Mannhold, Raimund

    2012-11-01

    FLAP fingerprints are applied in the ligand-, structure- and pharmacophore-based mode in a case study on antagonists of all four adenosine receptor (AR) subtypes. Structurally diverse antagonist collections with respect to the different ARs were constructed by including binding data to human species only. FLAP models well discriminate "active" (=highly potent) from "inactive" (=weakly potent) AR antagonists, as indicated by enrichment curves, numbers of false positives, and AUC values. For all FLAP modes, model predictivity slightly decreases as follows: A2BR > A2AR > A3R > A1R antagonists. General performance of FLAP modes in this study is: ligand- > structure- > pharmacophore- based mode. We also compared the FLAP performance with other common ligand- and structure-based fingerprints. Concerning the ligand-based mode, FLAP model performance is superior to ECFP4 and ROCS for all AR subtypes. Although focusing on the early first part of the A2A, A2B and A3 enrichment curves, ECFP4 and ROCS still retain a satisfactory retrieval of actives. FLAP is also superior when comparing the structure-based mode with PLANTS and GOLD. In this study we applied for the first time the novel FLAPPharm tool for pharmacophore generation. Pharmacophore hypotheses, generated with this tool, convincingly match with formerly published data. Finally, we could demonstrate the capability of FLAP models to uncover selectivity aspects although single AR subtype models were not trained for this purpose.

  6. 2-Alkynyl-8-aryladenines possessing an amide moiety: their synthesis and structure-activity relationships of effects on hepatic glucose production induced via agonism of the A(2B) adenosine receptor.

    PubMed

    Harada, H; Asano, O; Kawata, T; Inoue, T; Horizoe, T; Yasuda, N; Nagata, K; Murakami, M; Nagaoka, J; Kobayashi, S; Tanaka, I; Abe, S

    2001-10-01

    A series of 2-alkynyl-8-aryladenine derivatives bearing an amide moiety at the 9-position of adenine was synthesized. These analogues were evaluated for inhibitory activity on N-ethylcarboxamidoadenosine (NECA)-induced glucose production in primary cultured rat hepatocytes. The m-primary benzamide derivative 15f was the most potent compound (IC(50)=0.017 microM), being 15-fold more active than the corresponding 9-methyl derivative (1). Compound 15f showed 72- and 5.2-fold selectivity for human A(2B) receptor versus human A(1) and A(2A) receptors, respectively. Structure-activity relationship (SAR) studies of the synthesized compounds indicated that a three-carbon linker, fixed in the form of a benzene ring, between the adenine core and the amide moiety is important for both A(2B) antagonistic activity and selectivity. The IC(50) values in rat hepatocyte glucose assay correlated well with the IC(50) values in cAMP assay using Chinese hamster ovary cells stably transfected with human A(2B) receptors (r(2)=0.94). The A(1) and A(2A) affinities showed no correlation with the potency to inhibit NECA-induced glucose production. These results strongly support our previous conclusion that adenosine agonist-induced hepatic glucose production in rat hepatocytes is mediated through the A(2B) receptor.

  7. Synthesis and biological evaluation of novel 1-deoxy-1-[6-[((hetero)arylcarbonyl)hydrazino]- 9H-purin-9-yl]-N-ethyl-beta-D-ribofuranuronamide derivatives as useful templates for the development of A2B adenosine receptor agonists.

    PubMed

    Baraldi, Pier Giovanni; Preti, Delia; Tabrizi, Mojgan Aghazadeh; Fruttarolo, Francesca; Romagnoli, Romeo; Carrion, Maria Dora; Cara, Luisa Carlota Lopez; Moorman, Allan R; Varani, Katia; Borea, Pier Andrea

    2007-01-25

    The lack of molecules endowed with selective and potent agonistic activity toward the hA2B adenosine receptors has limited the studies on this pharmacological target and consequently the evaluation of its therapeutic potential. We report the design and the synthesis of the first potent (EC50 in the nanomolar range) and selective hA2B adenosine receptor agonists consisting of 1-deoxy-1-[6-[((hetero)arylcarbonyl)hydrazino]-9H-purin-9-yl]-N-ethyl-beta-D-ribofuranuronamide derivatives. The concurrent effect of 6-substitution of the purine nucleus with a ((hetero)arylcarbonyl)hydrazino function and a 2-chloro substitution has been investigated in such NECA derivatives.

  8. Adenosine receptor neurobiology: overview.

    PubMed

    Chen, Jiang-Fan; Lee, Chien-fei; Chern, Yijuang

    2014-01-01

    Adenosine is a naturally occurring nucleoside that is distributed ubiquitously throughout the body as a metabolic intermediary. In the brain, adenosine functions as an important upstream neuromodulator of a broad spectrum of neurotransmitters, receptors, and signaling pathways. By acting through four G-protein-coupled receptors, adenosine contributes critically to homeostasis and neuromodulatory control of a variety of normal and abnormal brain functions, ranging from synaptic plasticity, to cognition, to sleep, to motor activity to neuroinflammation, and cell death. This review begun with an overview of the gene and genome structure and the expression pattern of adenosine receptors (ARs). We feature several new developments over the past decade in our understanding of AR functions in the brain, with special focus on the identification and characterization of canonical and noncanonical signaling pathways of ARs. We provide an update on functional insights from complementary genetic-knockout and pharmacological studies on the AR control of various brain functions. We also highlight several novel and recent developments of AR neurobiology, including (i) recent breakthrough in high resolution of three-dimension structure of adenosine A2A receptors (A2ARs) in several functional status, (ii) receptor-receptor heterodimerization, (iii) AR function in glial cells, and (iv) the druggability of AR. We concluded the review with the contention that these new developments extend and strengthen the support for A1 and A2ARs in brain as therapeutic targets for neurologic and psychiatric diseases. © 2014 Elsevier Inc. All rights reserved.

  9. AMP is an adenosine A1 receptor agonist.

    PubMed

    Rittiner, Joseph E; Korboukh, Ilia; Hull-Ryde, Emily A; Jin, Jian; Janzen, William P; Frye, Stephen V; Zylka, Mark J

    2012-02-17

    Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5'-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5'-monophosphonate, ACP) directly activated the adenosine A(1) receptor (A(1)R). In contrast, AMP only activated the adenosine A(2B) receptor (A(2B)R) after hydrolysis to adenosine by ecto-5'-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A(1)R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A(1)R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A(1)R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A(1)R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine.

  10. AMP and adenosine are both ligands for adenosine 2B receptor signaling.

    PubMed

    Holien, Jessica K; Seibt, Benjamin; Roberts, Veena; Salvaris, Evelyn; Parker, Michael W; Cowan, Peter J; Dwyer, Karen M

    2018-01-15

    Adenosine is considered the canonical ligand for the adenosine 2B receptor (A 2B R). A 2B R is upregulated following kidney ischemia augmenting post ischemic blood flow and limiting tubular injury. In this context the beneficial effect of A 2B R signaling has been attributed to an increase in the pericellular concentration of adenosine. However, following renal ischemia both kidney adenosine monophosphate (AMP) and adenosine levels are substantially increased. Using computational modeling and calcium mobilization assays, we investigated whether AMP could also be a ligand for A 2B R. The computational modeling suggested that AMP interacts with more favorable energy to A 2B R compared with adenosine. Furthermore, AMPαS, a non-hydrolyzable form of AMP, increased calcium uptake by Chinese hamster ovary (CHO) cells expressing the human A 2B R, indicating preferential signaling via the G q pathway. Therefore, a putative AMP-A 2B R interaction is supported by the computational modeling data and the biological results suggest this interaction involves preferential G q activation. These data provide further insights into the role of purinergic signaling in the pathophysiology of renal IRI. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Adenosine A(2A) agonist and A(2B) antagonist mediate an inhibition of inflammation-induced contractile disturbance of a rat gastrointestinal preparation.

    PubMed

    Michael, Sebastian; Warstat, Claudia; Michel, Fabien; Yan, Luo; Müller, Christa E; Nieber, Karen

    2010-03-01

    Adenosine can show anti-inflammatory as well as pro-inflammatory activities. The contribution of the specific adenosine receptor subtypes in various cells, tissues and organs is complex. In this study, we examined the effect of the adenosine A(2A) receptor agonist CGS 21680 and the A(2B)R antagonist PSB-1115 on acute inflammation induced experimentally by 2,4,6-trinitrobenzenesulfonic acid (TNBS) on rat ileum/jejunum preparations. Pre-incubation of the ileum/jejunum segments with TNBS for 30 min resulted in a concentration-dependent inhibition of acetylcholine (ACh)-induced contractions. Pharmacological activation of the A(2A)R with CGS 21680 (0.1-10 microM) pre-incubated simultaneously with TNBS (10 mM) prevented concentration-dependently the TNBS-induced inhibition of the ACh contractions. Stimulation of A(2B)R with the selective agonist BAY 60-6583 (10 microM) did neither result in an increase nor in a further decrease of ACh-induced contractions compared to the TNBS-induced inhibition. The simultaneous pre-incubation of the ileum/jejunum segments with TNBS (10 mM) and the selective A(2B)R antagonist PSB-1115 (100 microM) inhibited the contraction-decreasing effect of TNBS. The effects of the A(2A)R agonist and the A(2B)R antagonist were in the same range as the effect induced by 1 microM methotrexate. The combination of the A(2A)R agonist CGS 21680 and the A(2B)R antagonist PSB-1115 at subthreshold concentrations of both agents found a significant amelioration of the TNBS-diminished contractility. Our results demonstrate that the activation of A(2A) receptors or the blockade of the A(2B) receptors can prevent the inflammation-induced disturbance of the ACh-induced contraction in TNBS pre-treated small intestinal preparations. The combination of both may be useful for the treatment of inflammatory bowel diseases.

  12. Adenosine receptor antagonists effect on plasma-enhanced killing.

    PubMed

    Bauzá, Gustavo; Moitra, Rituparna; Remick, Daniel

    2014-01-01

    Previous studies demonstrated that naive plasma has inherent capabilities to enhance bacterial opsonization and phagocyte killing, but not all plasma is equally effective. This raised the question of whether plasma constituents other than opsonins may play a role. Adenosine receptor antagonists have been shown to modulate cytokine response and survival in mice after a bacterial challenge. We investigated whether selective adenosine receptor blockade would influence the ability of naive plasma to effectively control bacterial growth. Colonic bacteria- and thioglycollate-elicited peritoneal macrophages and neutrophils were obtained from naive mice. Stock murine plasma from naive was purchased and categorized as having high plasma-enhanced bacterial killing capacity using our previously described methods. Bacteria and plasma were incubated to allow for opsonization and then added to macrophages previously exposed to selected adenosine receptor antagonists: ZM 241385: A2A, MRS1754: A2B, DPCPX: A1, and MRS1220: A3. The final mixture was plated on blood agar plates in aerobic and anaerobic conditions and bacterial colony-forming units quantified after 24 h. This study demonstrated that exogenous adenosine was able to significantly decrease phagocyte killing of cecal bacteria. Blocking adenosine receptors with selective antagonists altered the bacterial killing capacity of plasma. Selectively blocking the A1, A2A, or A2B receptors proved most beneficial at reversing the effect of adenosine. Consistent with previous work, only macrophage killing of bacteria could be modulated by adenosine receptor blockade because neutrophils were unaffected. These data demonstrate that adenosine decreases macrophage killing of enteric bacteria and that this effect is mediated through the adenosine receptors.

  13. Xanthines as Adenosine Receptor Antagonists

    PubMed Central

    Jacobson, Kenneth A.

    2013-01-01

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

  14. AMP Is an Adenosine A1 Receptor Agonist*

    PubMed Central

    Rittiner, Joseph E.; Korboukh, Ilia; Hull-Ryde, Emily A.; Jin, Jian; Janzen, William P.; Frye, Stephen V.; Zylka, Mark J.

    2012-01-01

    Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5′-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5′-monophosphonate, ACP) directly activated the adenosine A1 receptor (A1R). In contrast, AMP only activated the adenosine A2B receptor (A2BR) after hydrolysis to adenosine by ecto-5′-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A1R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A1R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A1R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A1R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine. PMID:22215671

  15. Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors.

    PubMed

    Pleli, Thomas; Mondorf, Antonia; Ferreiros, Nerea; Thomas, Dominique; Dvorak, Karel; Biondi, Ricardo M; Heringdorf, Dagmar Meyer Zu; Zeuzem, Stefan; Geisslinger, Gerd; Zimmermann, Herbert; Waidmann, Oliver; Piiper, Albrecht

    2018-03-27

    Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear. Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA. Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors. Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors. © 2018 The Author(s). Published by S. Karger AG, Basel.

  16. Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytes

    PubMed Central

    Varani, K; Vincenzi, F; Tosi, A; Targa, M; Masieri, FF; Ongaro, A; De Mattei, M; Massari, L; Borea, PA

    2010-01-01

    Background and purpose: Adenosine is an endogenous modulator, interacting with four G-protein coupled receptors (A1, A2A, A2B and A3) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes. Experimental approach: mRNA, Western blotting, saturation and competition binding experiments, cyclic AMP, p38 mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB activation, tumour necrosis factor α (TNF-α) and interleukin-8 (IL-8) release were assessed in human synoviocytes isolated from patients with osteoarthritis. Key results: mRNA and protein for A1, A2A, A2B and A3 adenosine receptors are expressed in human synoviocytes. Standard adenosine agonists and antagonists showed affinity values in the nanomolar range and were coupled to stimulation or inhibition of adenylyl cyclase. Activation of A2A and A3 adenosine receptors inhibited p38 MAPK and NF-κB pathways, an effect abolished by selective adenosine antagonists. A2A and A3 receptor agonists decreased TNF-α and IL-8 production. The phosphoinositide 3-kinase or Gs pathways were involved in the functional responses of A3 or A2A adenosine receptors. Synoviocyte A1 and A2B adenosine receptors were not implicated in the inflammatory process whereas stimulation of A2A and A3 adenosine receptors was closely associated with a down-regulation of the inflammatory status. Conclusions and implications: These results indicate that A2A and A3 adenosine receptors may represent a potential target in therapeutic modulation of joint inflammation. PMID:20331607

  17. Adenosine A(2A) receptor dynamics studied with the novel fluorescent agonist Alexa488-APEC.

    PubMed

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

    2008-08-20

    G protein-coupled receptors, such as the adenosine A(2A) receptor, are dynamic proteins, which undergo agonist-dependent redistribution from the cell surface to intracellular membranous compartments, such as endosomes. In order to study the kinetics of adenosine A(2A) receptor redistribution in living cells, we synthesized a novel fluorescent agonist, Alexa488-APEC. Alexa488-APEC binds to adenosine A(2A) (K(i)=149+/-27 nM) as well as A(3) receptors (K(i)=240+/-160 nM) but not to adenosine A(1) receptors. Further, we characterized the dose-dependent increase in Alexa488-APEC-induced cAMP production as well as cAMP response element binding (CREB) protein phosphorylation, verifying the ligand's functionality at adenosine A(2A) but not A(2B) receptors. In live-cell imaging studies, Alexa488-APEC-induced adenosine A(2A) receptor internalization, which was blocked by the competitive reversible antagonist ZM 241385 and hyperosmolaric sucrose. Further, internalized adenosine A(2A) receptors co-localized with clathrin and Rab5, indicating that agonist stimulation promotes adenosine A(2A) receptor uptake through a clathrin-dependent mechanism to Rab5-positive endosomes. The basic characterization of Alexa488-APEC described here showed that it provides a useful tool for tracing adenosine A(2A) receptors in vitro.

  18. 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 ofmore » 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.« less

  19. Anti-inflammatory effects of purine nucleosides, adenosine and inosine, in a mouse model of pleurisy: evidence for the role of adenosine A2 receptors.

    PubMed

    da Rocha Lapa, Fernanda; da Silva, Morgana Duarte; de Almeida Cabrini, Daniela; Santos, Adair R S

    2012-12-01

    Adenosine and its metabolite, inosine, have been described as molecules that participate in regulation of inflammatory response. The aim of this study was to investigate the effect of adenosine and inosine in a mouse model of carrageenan-induced pleurisy as well as the participation of adenosine receptors in this response. Injection of carrageenan into the pleural cavity induced an acute inflammatory response characterized by leukocyte migration, pleural exudation, and increased release of interleukin-1β and tumor necrosis factor-α in pleural exudates. The treatment with adenosine (0.3-100 mg/kg, i.p.) and inosine (0.1-300 mg/kg, i.p.) 30 min before carrageenan injection reduced significantly all these parameters analyzed. Our results also demonstrated that A(2A) and A(2B) receptors seem to mediate the adenosine and inosine effects observed, since pretreatment with selective antagonists of adenosine A(2A) (ZM241385) and A(2B) (alloxazine) receptors, reverted the inhibitory effects of adenosine and inosine in pleural inflammation. The involvement of A(2) receptors was reinforced with adenosine receptor agonist CGS21680 treatment, since its anti-inflammatory effects were reversed completely and partially with ZM241385 and alloxazine injection, respectively. Moreover, the combined treatment with subeffective dose of adenosine (0.3 mg/kg) and inosine (1.0 mg/kg) induced a synergistic anti-inflammatory effect. Thus, based on these findings, we propose that inosine contributes with adenosine to exert anti-inflammatory effects in pleural inflammation, reinforcing the notion that endogenous nucleosides play an important role in controlling inflammatory diseases. This effect is likely mediated by the activation of adenosine A(2) subtype receptors and inhibition of production or release of pro-inflammatory cytokines.

  20. 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

  1. Characteristic molecular vibrations of adenosine receptor ligands.

    PubMed

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

    2015-02-13

    Although the regulation of membrane receptor activation is known to be crucial for molecular signal transduction, the molecular mechanism underlying receptor activation is not fully elucidated. Here we study the physicochemical nature of membrane receptor behavior by investigating the characteristic molecular vibrations of receptor ligands using computational chemistry and informatics methods. By using information gain, t-tests, and support vector machines, we have identified highly informative features of adenosine receptor (AdoR) ligand and corresponding functional amino acid residues such as Asn (6.55) of AdoR that has informative significance and is indispensable for ligand recognition of AdoRs. These findings may provide new perspectives and insights into the fundamental mechanism of class A G protein-coupled receptor activation. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  2. Estrogen stimulates adenosine receptor expression subtypes in human breast cancer MCF-7 cell line.

    PubMed

    Mohamadi, Azam; Aghaei, Mahmoud; Panjehpour, Mojtaba

    2018-02-01

    Estrogen is a steroid hormone that plays a key role in the development and regulation of reproductive system. It has been shown that estrogen is related to breast cancer development through binding to its receptors. In order to uncover the estrogen effects on adenosine receptor expression, estrogen-positive MCF-7 cells were used to treat with agonist and antagonist of estrogen and then the mRNA expression of adenosine receptor subtypes were evaluated. Estrogen-positive MCF-7 cells were treated with various concentrations of 17β estradiol (E2) as an estrogen agonist, and ICI 182,780 as an estrogen antagonist. The gene expression of adenosine receptor subtypes were detected by real time RT-PCR. The results of MTT assay showed that E2 increased cell viability in a dose dependent manner. The expression pattern of all adenosine receptor subtypes are as follow; A2b > A1 > A2a > A3 in untreated MCF-7 cells. Obtained results showed that E2 incubation at 0.001-0.01 μM led to up-regulation of A1ARs, A2aARs and A3ARs dose dependently. E2 at 0.001 μM also had no significant effect on A2bARs expression but, at higher doses induced a considerable decrease in mRNA A2bARs expression. Treatment with antagonist confirmed that up-regulation of these receptors is mediated by estrogen receptor. Taken together, our results indicate that treatment of MCF-7 cells with E2 led to up-regulation of adenosine receptors. However, these effects were partially restored by treatment with antagonist suggesting that such effects are mediated by estrogen receptors.

  3. Adenosine and adenosine receptors in the pathogenesis and treatment of rheumatic diseases.

    PubMed

    Cronstein, Bruce N; Sitkovsky, Michail

    2017-01-01

    Adenosine, a nucleoside derived primarily from the extracellular hydrolysis of adenine nucleotides, is a potent regulator of inflammation. Adenosine mediates its effects on inflammatory cells by engaging one or more cell-surface receptors. The expression and function of adenosine receptors on different cell types change during the course of rheumatic diseases, such as rheumatoid arthritis (RA). Targeting adenosine receptors directly for the treatment of rheumatic diseases is currently under study; however, indirect targeting of adenosine receptors by enhancing adenosine levels at inflamed sites accounts for most of the anti-inflammatory effects of methotrexate, the anchor drug for the treatment of RA. In this Review, we discuss the regulation of extracellular adenosine levels and the role of adenosine in regulating the inflammatory and immune responses in rheumatic diseases such as RA, psoriasis and other types of inflammatory arthritis. In addition, adenosine and its receptors are involved in promoting fibrous matrix production in the skin and other organs, and the role of adenosine in fibrosis and fibrosing diseases is also discussed.

  4. A2BR Adenosine Receptor Modulates Sweet Taste in Circumvallate Taste Buds

    PubMed Central

    Yang, Dan; Shultz, Nicole; Vandenbeuch, Aurelie; Ravid, Katya; Kinnamon, Sue C.; Finger, Thomas E.

    2012-01-01

    In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3) on taste nerves as well as metabotropic (P2Y) purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR) is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate), but not anterior (fungiform, palate) taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields. PMID:22253866

  5. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Adenosine receptors and asthma in humans.

    PubMed

    Wilson, C N

    2008-10-01

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

  7. Transient adenosine release is modulated by NMDA and GABAB receptors

    PubMed Central

    Nguyen, Michael D.; Wang, Ying; Ganesana, Mallikarjunarao; Venton, B. Jill

    2017-01-01

    Adenosine is a neuroprotective agent that modulates neurotransmission and is modulated by other neurotransmitters. Spontaneous, transient adenosine is a recently discovered mode of signaling where adenosine is released and cleared from the extracellular space quickly, in less than three seconds. Spontaneous adenosine release is regulated by adenosine A1 and A2a receptors, but regulation by other neurotransmitter receptors has not been studied. Here, we examined the effect of glutamate and GABA receptors on the concentration and frequency of spontaneous, transient adenosine release by measuring adenosine with fast-scan cyclic voltammetry in the rat caudate-putamen. The glutamate NMDA antagonist, 3-(R-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 6 mg/kg i.p.), increased the frequency of adenosine transients and the concentration of individual transients but NMDA (agonist, 50 mg/kg, i.p.) did not change the frequency. In contrast, antagonists of other glutamate receptors had no effect on the frequency or concentration of transient adenosine release, including the AMPA antagonist NBQX (15 mg/kg i.p.) and the mGlu2/3 glutamate receptor antagonist LY 341495 (5 mg/kg i.p.). The GABAB antagonist CGP 52432 (30 mg/kg i.p.) significantly decreased the number of adenosine release events while the GABAB agonist baclofen (4 mg/kg i.p.) increased the frequency of adenosine release. The GABAA antagonist bicuculline (5 mg/kg i.p.) had no significant effects on adenosine. NMDA and GABAB likely act presynaptically, affecting the overall cell excitability for vesicular release. The ability to regulate adenosine with NMDA and GABAB receptors will help control the modulatory effects of transient adenosine release. PMID:28071892

  8. Evidence that endogenous inosine and adenosine-mediated hyperglycaemia during ischaemia-reperfusion through A3 adenosine receptors.

    PubMed

    Cortés, D; Guinzberg, R; Villalobos-Molina, R; Piña, E

    2009-10-01

    1 The molecular mechanism underlying stress-induced hyperglycemia has not been comprehensively clarified. Recently, we demonstrated in ischaemia-reperfusion (I-R) stress-subjected liver that inosine and adenosine are mainly responsible for the hyperglycemia observed. 2 We aimed to advance in the knowledge of the role of inosine plus adenosine as mediators of hepatic-induced hyperglycemia detected after I-R in lower limbs. 3 Acute ischaemia was conducted in anesthetized rats by occluding downstream abdominal aorta and cava vein; then, reperfusion was allowed. Blood samples from hepatic or abdominal cava veins were taken throughout the experiments to measure glucose, inosine and adenosine. Antagonists to adenosine (AdoR) and adrenergic receptors (AdrR) were administered during ischaemia to analyze their effect on hepatic glucose release. 4 Ischaemia up to 60 min produced minor increase of glucose and nucleosides blood values, but 5 min of ischaemia followed by 2- (or 10-) min reperfusion increased glucose 23%, and those of inosine or adenosine by 100%. After 60 min of ischaemia and 10 min of reperfusion, glycemia rose 2-fold and blood inosine and adenosine, 3.3- and 2.7-fold, respectively. A linear positive correlation, r(2), as high as 0.839 between glucose and either nucleoside blood values was calculated. The hyperglycemia response to I-R decreased by 0, 25, 33, 45 and 100% after selective inhibition of A(2B) AdoR, A(2A) AdoR, a(1B) AdrR, A(1) AdoR, and A(3) AdoR, respectively. 5 Inosine-adenosine couple through activation of hepatic A(3) AdoR is the main signal for releasing glucose from liver glycogen and for promoting hyperglycemia following experimental injury of I-R from lower limbs.

  9. Adenosine receptor subtypes in the airways responses to 5'-adenosine monophosphate inhalation of sensitized guinea-pigs.

    PubMed

    Smith, N; Broadley, K J

    2008-09-01

    Endogenous adenosine levels are raised in the lungs during asthma attacks. 5'-adenosine monophosphate (5'-AMP) inhalation in asthmatics causes bronchoconstriction and in sensitized guinea-pigs induces early (EAR) and late asthmatic responses (LAR), airway hyper-reactivity (AHR) and inflammatory cell recruitment to the lungs. The aim of this study was to investigate the roles of A(1), A(2A), A(2B) and A(3) adenosine receptors in these responses to inhaled 5'-AMP in sensitized guinea-pigs. Comparisons were made with the effect of dexamethasone treatment on 5'-AMP-induced responses. Functional airways responses to inhaled 5'-AMP (3 and 300 mM) of actively sensitized, conscious guinea-pigs were determined by whole-body plethysmography following administration of selective adenosine receptor antagonists or their vehicles. AHR to inhaled histamine (1 mM) and inflammatory cell influx in bronchoalveolar lavage fluid were determined. 5'-AMP at 3 mM caused an immediate bronchoconstriction (EAR), whereas 300 mM caused bronchodilatation. Both responses were followed at 6 h by a LAR, together with inflammatory cell influx and AHR to histamine. The A(2A) receptor antagonist, ZM241385, further enhanced cell influx after 5'-AMP inhalation (3 and 300 mM), and blocked the immediate bronchodilator response to 300 mM 5'-AMP, exposing an EAR. The A(2B) receptor antagonist, MRS1706 (in the presence of ZM241385), inhibited the LAR, AHR and cell influx, following inhalation of 5'-AMP (300 mM). The A(3) receptor antagonist, MRS1220, inhibited 5'-AMP-induced inflammatory cell influx. The A(1) receptor antagonist, DPCPX (in the presence of ZM241385), inhibited the EAR following 5'-AMP inhalation (300 mM). Dexamethasone inhibited the LAR, AHR and cell influx following inhalation of 5'-AMP (300 mM). All four adenosine receptor subtypes play various roles in the airways responses to inhaled 5'-AMP in sensitized guinea-pigs.

  10. Selective upregulation of the A3 adenosine receptor in eyes with pseudoexfoliation syndrome and glaucoma.

    PubMed

    Schlötzer-Schrehardt, Ursula; Zenkel, Matthias; Decking, Ulrich; Haubs, Daniela; Kruse, Friedrich E; Jünemann, Anselm; Coca-Prados, Miguel; Naumann, Gottfried O H

    2005-06-01

    Adenosine is increasingly released in metabolic stress conditions, like hypoxia or ischemia, and regulates many physiologic processes, such as aqueous humor secretion and intraocular pressure, via activation of four adenosine receptors. In the current study, the role of the adenosine system in the pathophysiology of pseudoexfoliation (PEX) syndrome, which is typically associated with anterior chamber hypoxia and elevated intraocular pressure, was examined. RT-PCR, Northern hybridization, in situ hybridization, and immunohistochemistry were applied to analyze the mRNA and protein expression of the adenosine receptor subtypes A1, A2A, A2B, and A3 in anterior segment tissues of PEX eyes, without and with glaucoma, in comparison to eyes with primary open-angle or angle-closure glaucoma and normal control eyes. Real-time PCR was used to study the effect of hypoxia and oxidative stress on adenosine receptor expression by nonpigmented ciliary epithelial cells in vitro. Levels of adenosine and its catabolites inosine, hypoxanthine, and xanthine were measured in cell culture supernatants and aqueous humor samples by HPLC. All four adenosine receptor subtypes (A2A > A1 > A2B > A3) were coexpressed but differently distributed in the ciliary epithelium of control eyes, with the A3 receptor being localized to the basolateral membrane infoldings of the nonpigmented epithelial cells. A selective, approximately 10-fold upregulation of A3 receptor mRNA and protein was consistently found in the nonpigmented ciliary epithelium of all PEX eyes, with and without glaucoma, compared with the normal and glaucomatous control eyes. Significant upregulation of A3 receptor message in nonpigmented epithelial cells was induced by both hypoxia and oxidative stress in vitro, together with increased levels of inosine, hypoxanthine, and xanthine in the supernatants. Levels of adenosine and its catabolites, however, were not significantly elevated in the aqueous humor of patients with PEX

  11. Neurabin scaffolding of adenosine receptor and RGS4 regulates anti-seizure effect of endogenous adenosine.

    PubMed

    Chen, Yunjia; Liu, Yin; Cottingham, Christopher; McMahon, Lori; Jiao, Kai; Greengard, Paul; Wang, Qin

    2012-02-22

    Endogenous adenosine is an essential protective agent against neural damage by various insults to the brain. However, the therapeutic potential of adenosine receptor-directed ligands for neuroprotection is offset by side effects in peripheral tissues and organs. An increase in adenosine receptor responsiveness to endogenous adenosine would enhance neuroprotection while avoiding the confounding effects of exogenous ligands. Here we report novel regulation of adenosine-evoked responses by a neural tissue-specific protein, neurabin. Neurabin attenuated adenosine A(1) receptor (A1R) signaling by assembling a complex between the A1R and the regulator of G-protein signaling 4 (RGS4), a protein known to turn off G-protein signaling. Inactivation of the neurabin gene enhanced A1R signaling and promoted the protective effect of adenosine against excitotoxic seizure and neuronal death in mice. Furthermore, administration of a small molecule inhibitor of RGS4 significantly attenuated seizure severity in mice. Notably, the dose of kainate capable of inducing an ∼50% rate of death in wild-type (WT) mice did not affect neurabin-null mice or WT mice cotreated with an RGS4 inhibitor. The enhanced anti-seizure and neuroprotective effect achieved by disruption of the A1R/neurabin/RGS4 complex is elicited by the on-site and on-demand release of endogenous adenosine, and does not require administration of A1R ligands. These data identify neurabin-RGS4 as a novel tissue-selective regulatory mechanism for fine-tuning adenosine receptor function in the nervous system. Moreover, these findings implicate the A1R/neurabin/RGS4 complex as a valid therapeutic target for specifically manipulating the neuroprotective effects of endogenous adenosine.

  12. Identification of A3 adenosine receptor agonists as novel non‐narcotic analgesics

    PubMed Central

    Janes, K; Symons‐Liguori, AM; Jacobson, K A

    2016-01-01

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

  13. A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

    PubMed

    Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

    2011-05-01

    A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

  14. The role of adenosine receptors and endogenous adenosine in citalopram-induced cardiovascular toxicity

    PubMed Central

    Oransay, Kubilay; Hocaoglu, Nil; Buyukdeligoz, Mujgan; Tuncok, Yesim; Kalkan, Sule

    2014-01-01

    Aim: We investigated the role of adenosine in citalopram-induced cardiotoxicity. Materials and Methods: Protocol 1: Rats were randomized into four groups. Sodium cromoglycate was administered to rats. Citalopram was infused after the 5% dextrose, 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX; A1 receptor antagonist), 8-(-3-chlorostyryl)-caffeine (CSC; A2a receptor antagonist), or dimethyl sulfoxide (DMSO) administrations. Protocol 2: First group received 5% dextrose intraperitoneally 1 hour prior to citalopram. Other rats were pretreated with erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; inhibitor of adenosine deaminase) and S-(4-Nitrobenzyl)-6-thioinosine (NBTI; inhibitor of facilitated adenosine transport). After pretreatment, group 2 received 5% dextrose and group 3 received citalopram. Adenosine concentrations, mean arterial pressure (MAP), heart rate (HR), QRS duration and QT interval were evaluated. Results: In the dextrose group, citalopram infusion caused a significant decrease in MAP and HR and caused a significant prolongation in QRS and QT. DPCPX infusion significantly prevented the prolongation of the QT interval when compared to control. In the second protocol, citalopram infusion did not cause a significant change in plasma adenosine concentrations, but a significant increase observed in EHNA/NBTI groups. In EHNA/NBTI groups, citalopram-induced MAP and HR reductions, QRS and QT prolongations were more significant than the dextrose group. Conclusions: Citalopram may lead to QT prolongation by stimulating adenosine A1 receptors without affecting the release of adenosine. PMID:25097274

  15. The role of adenosine receptors and endogenous adenosine in citalopram-induced cardiovascular toxicity.

    PubMed

    Oransay, Kubilay; Hocaoglu, Nil; Buyukdeligoz, Mujgan; Tuncok, Yesim; Kalkan, Sule

    2014-01-01

    We investigated the role of adenosine in citalopram-induced cardiotoxicity. Protocol 1: Rats were randomized into four groups. Sodium cromoglycate was administered to rats. Citalopram was infused after the 5% dextrose, 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX; A1 receptor antagonist), 8-(-3-chlorostyryl)-caffeine (CSC; A2a receptor antagonist), or dimethyl sulfoxide (DMSO) administrations. Protocol 2: First group received 5% dextrose intraperitoneally 1 hour prior to citalopram. Other rats were pretreated with erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; inhibitor of adenosine deaminase) and S-(4-Nitrobenzyl)-6-thioinosine (NBTI; inhibitor of facilitated adenosine transport). After pretreatment, group 2 received 5% dextrose and group 3 received citalopram. Adenosine concentrations, mean arterial pressure (MAP), heart rate (HR), QRS duration and QT interval were evaluated. In the dextrose group, citalopram infusion caused a significant decrease in MAP and HR and caused a significant prolongation in QRS and QT. DPCPX infusion significantly prevented the prolongation of the QT interval when compared to control. In the second protocol, citalopram infusion did not cause a significant change in plasma adenosine concentrations, but a significant increase observed in EHNA/NBTI groups. In EHNA/NBTI groups, citalopram-induced MAP and HR reductions, QRS and QT prolongations were more significant than the dextrose group. Citalopram may lead to QT prolongation by stimulating adenosine A1 receptors without affecting the release of adenosine.

  16. Adenosine receptors and caffeine in retinopathy of prematurity

    PubMed Central

    Chen, Jiang-Fan; Zhang, Shuya; Zhou, Rong; Lin, Zhenlang; Cai, Xiaohong; Lin, Jing; Huo, Yuqing; Liu, Xiaoling

    2017-01-01

    Retinopathy of prematurity (ROP) is a major cause of childhood blindness in the world and is caused by oxygen-induced damage to the developing retinal vasculature, resulting in hyperoxia-induced vaso-obliteration and subsequent delayed retinal vascularization and hypoxia-induced pathological neovascularization driven by vascular endothelial growth factor (VEGF) signaling pathway in retina. Current anti-VEGF therapy has shown some effective in a clinical trial, but is associated with the unintended effects on delayed eye growth and retinal vasculature development of preterm infants. Notably, cellular responses to hypoxia are characterized by robust increases in extracellular adenosine production and the markedly induced adenosine receptors, which provide a novel target for preferential control of pathological angiogenesis without affecting normal vascular development. Here, we review the experimental evidence in support of adenosine receptor-based therapeutic strategy for ROP, including the aberrant adenosine signaling in oxygen-induced retinopathy and the role of three adenosine receptor subtypes (A1R, A2AR, A2BR) in development and treatment of ROP using oxygen-induced retinopathy models. The clinical and initial animal evidence that implicate the therapeutic effect of caffeine (a non-selective adenosine receptor antagonist) in treatment of ROP are highlighted. Lastly, we discussed the translational potential as well therapeutic advantage of adenosine receptor- and caffeine-based therapy for ROR and possibly other proliferative retinopathy. PMID:28088487

  17. 2′,3′-cAMP, 3′-AMP, and 2′-AMP inhibit human aortic and coronary vascular smooth muscle cell proliferation via A2B receptors

    PubMed Central

    Ren, Jin; Gillespie, Delbert G.

    2011-01-01

    Rat vascular smooth muscle cells (VSMCs) from renal microvessels metabolize 2′,3′-cAMP to 2′-AMP and 3′-AMP, and these AMPs are converted to adenosine that inhibits microvascular VSMC proliferation via A2B receptors. The goal of this study was to test whether this mechanism also exists in VSMCs from conduit arteries and whether it is similarly expressed in human vs. rat VSMCs. Incubation of rat and human aortic VSMCs with 2′,3′-cAMP concentration-dependently increased levels of 2′-AMP and 3′-AMP in the medium, with a similar absolute increase in 2′-AMP vs. 3′-AMP. In contrast, in human coronary VSMCs, 2′,3′-cAMP increased 2′-AMP levels yet had little effect on 3′-AMP levels. In all cell types, 2′,3′-cAMP increased levels of adenosine, but not 5′-AMP, and 2′,3′-AMP inhibited cell proliferation. Antagonism of A2B receptors (MRS-1754), but not A1 (1,3-dipropyl-8-cyclopentylxanthine), A2A (SCH-58261), or A3 (VUF-5574) receptors, attenuated the antiproliferative effects of 2′,3′-cAMP. In all cell types, 2′-AMP, 3′-AMP, and 5′-AMP increased adenosine levels, and inhibition of ecto-5′-nucleotidase blocked this effect of 5′-AMP but not that of 2′-AMP nor 3′-AMP. Also, 2′-AMP, 3′-AMP, and 5′-AMP, like 2′,3′-cAMP, exerted antiproliferative effects that were abolished by antagonism of A2B receptors with MRS-1754. In conclusion, VSMCs from conduit arteries metabolize 2′,3′-cAMP to AMPs, which are metabolized to adenosine. In rat and human aortic VSMCs, both 2′-AMP and 3′-AMP are involved in this process, whereas, in human coronary VSMCs, 2′,3′-cAMP is mainly converted to 2′-AMP. Because adenosine inhibits VSMC proliferation via A2B receptors, local vascular production of 2′,3′-cAMP may protect conduit arteries from atherosclerosis. PMID:21622827

  18. Adenosine receptor agonists modulate visceral hyperalgesia in the rat.

    PubMed

    Sohn, Chong-Il; Park, Hyo Jin; Gebhart, G F

    2008-06-01

    Adenosine is an endogenous modulator of nociception. Its role in visceral nociception, particularly in visceral hyperalgesia, has not been studied. The aim of this study was to determine the effects of adenosine receptor agonists in a model of visceral hyperalgesia. The visceromotor response (VMR) in rats to colorectal distension (CRD; 80 mmHg, 20 seconds) was quantified by electromyographic recordings from the abdominal musculature. Three hours after the intracolonic administration of zymosan (25 mg/mL, 1 mL), VMRs to CRD were measured before and after either subcutaneous or intrathecal administration of an adenosine receptor agonist. Subcutaneous injection of 5'-N-ethylcarboxyamidoadenosine (NECA; an A1 and A2 receptor agonist), R(-)-N6-(2-phenylisopropyl)-adenosine (R-PIA; a selective A1 receptor agonist), or CGS-21680 hydrochloride (a selective A2a receptor agonist) dose-dependently (10-100 mg/kg) attenuated the VMR to CRD, although hindlimb weakness occurred at the higher doses tested. Intrathecal administration of NECA or R-PIA dose-dependently (0.1-1.0 microg/kg) decreased the VMR, whereas CGS-21680 hydrochloride was ineffective over the same concentration range. Higher intrathecal doses of the A1/A2 receptor agonist NECA produced motor weakness. Adenosine receptor agonists are antihyperalgesic, but also produce motor weakness at high doses. However, activation of the spinal A1 receptor significantly attenuates the VMR to CRD without producing motor weakness.

  19. Downregulation of adenosine and adenosine 1 receptor contributes to neuropathic pain in resiniferatoxin neuropathy.

    PubMed

    Kan, Hung-Wei; Chang, Chin-Hong; Lin, Chih-Lung; Lee, Yi-Chen; Hsieh, Sung-Tsang; Hsieh, Yu-Lin

    2018-04-16

    The neurochemical effects of adenosine signaling in small-fiber neuropathy leading to neuropathic pain are yet to be explored in a direct manner. This study examined this system at the level of ligand (via the ectonucleotidase activity of prostatic acid phosphatase, PAP) and adenosine A1 receptors (A1Rs) in resiniferatoxin (RTX) neuropathy, a peripheral neurodegenerative disorder which specifically affects nociceptive nerves expressing transient receptor potential vanilloid type 1 (TRPV1). We conducted immunohistochemistry on dorsal root ganglion neurons (DRG), high-performance liquid chromatography (HPLC) for functional assays, and pharmacological interventions to alter PAP and A1Rs in mice with RTX neuropathy. In DRG of RTX neuropathy, PAP(+) neurons were reduced compared with vehicle-treated mice (P = 0.002) . Functionally, PAP ectonucleotidase activity was consequently reduced (i.e., the content of adenosine in DRG, P = 0.012). PAP(+) neuronal density was correlated with the degree of mechanical allodynia, which was reversed by intrathecal lumbar puncture (i.t.) injection of recombinant PAP with a dose-dependent effect. Furthermore, A1Rs were downregulated (P = 0.002), and this downregulation was colocalized with the TRPV1 receptor (31.0% ± 2.8%). Mechanical allodynia was attenuated in a dose-dependent response by i.t. injection of the A1R ligand, adenosine; however, no analgesia was evident when an exogenous adenosine was blocked by A1R antagonist. This study demonstrated dual mechanisms of neuropathic pain in TRPV1-induced neuropathy, involving a reduced adenosine system at both the ligand (adenosine) and receptor (A1Rs) levels.

  20. 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. © 2016 Wiley Periodicals, Inc.

  1. Different efficacy of adenosine and NECA derivatives at the human A3 adenosine receptor: insight into the receptor activation switch.

    PubMed

    Dal Ben, Diego; Buccioni, Michela; Lambertucci, Catia; Kachler, Sonja; Falgner, Nico; Marucci, Gabriella; Thomas, Ajiroghene; Cristalli, Gloria; Volpini, Rosaria; Klotz, Karl-Norbert

    2014-01-15

    A3 Adenosine receptors are promising drug targets for a number of diseases and intense efforts are dedicated to develop selective agonists and antagonists of these receptors. A series of adenosine derivatives with 2-(ar)-alkynyl chains, with high affinity and different degrees of selectivity for human A3 adenosine receptors was tested for the ability to inhibit forskolin-stimulated adenylyl cyclase. All these derivatives are partial agonists at A3 adenosine receptors; their efficacy is not significantly modified by the introduction of small alkyl substituents in the N(6)-position. In contrast, the adenosine-5'-N-ethyluronamide (NECA) analogs of 2-(ar)-alkynyladenosine derivatives are full A3 agonists. Molecular modeling analyses were performed considering both the conformational behavior of the ligands and the impact of 2- and 5'-substituents on ligand-target interaction. The results suggest an explanation for the different agonistic behavior of adenosine and NECA derivatives, respectively. A sub-pocket of the binding site was analyzed as a crucial interaction domain for receptor activation. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells.

    PubMed

    Costa, M Adelina; Barbosa, A; Neto, E; Sá-e-Sousa, A; Freitas, R; Neves, J M; Magalhães-Cardoso, T; Ferreirinha, F; Correia-de-Sá, P

    2011-05-01

    Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation. Copyright © 2010 Wiley-Liss, Inc.

  3. Adenosine A2A receptor hyperexpression in patients with severe SIRS after cardiopulmonary bypass.

    PubMed

    Kerbaul, François; Bénard, Frédéric; Giorgi, Roch; Youlet, By; Carrega, Louis; Zouher, Ibrahim; Mercier, Laurence; Gérolami, Victoria; Bénas, Vincent; Blayac, Dorothée; Gariboldi, Vlad; Collart, Frédéric; Guieu, Régis

    2008-08-01

    Adenosine (ADO) is an endogenous nucleoside, which has been involved in blood pressure failure during severe systemic inflammatory response syndrome (severe SIRS) after cardiac surgery with cardiopulmonary bypass (CPB). Adenosine acts via its receptor subtypes, namely A1, A2A, A2B, or A3. Because A2A receptors are implicated in vascular tone, their expression might contribute to severe SIRS. We compared adenosine plasma levels (APLs) and A2A ADO receptor expression (ie, B, K, and mRNA amount) in patients with or without postoperative SIRS. : This was a prospective comparative observational study. Forty-four patients who underwent cardiac surgery involving CPB. Ten healthy subjects served as controls. Among the patients, 11 presented operative vasoplegia and postoperative SIRS (named complicated patients) and 33 were without vasoplegia or SIRS (named uncomplicated patients). Adenosine plasma levels, K, B, and mRNA amount (mean +/- SD) were measured on peripheral blood mononuclear cells. Adenosine plasma levels, B, and K were significantly higher in complicated patients than in uncomplicated patients (APLs: 2.7 +/- 1.0 vs 1.0 +/- 0.5 micromol l, P < 0.05; B: 210 +/- 43 vs 65 +/- 26 fmol/mg, P < 0.05; K: 35 +/- 10 vs 2 +/- 1 nM, P < 0.05). In uncomplicated patients, APLs remain higher than in controls (1 +/- 0.5 vs 0.6 +/- 0.25 micromol/L; P < 0.05). Mean arterial pressure was inversely correlated to APLs (R = -0.58; P < 0.001) and B (R = -0.64; P < 0.001) leading to an increased requirement of vasoactive drugs during the postoperative period in vasoplegic patients. High expression of A2A ADO receptor and high APLs may be a predictive factor of postoperative severe SIRS after CPB.

  4. Crosstalk between the equilibrative nucleoside transporter ENT2 and alveolar Adora2b adenosine receptors dampens acute lung injury

    PubMed Central

    Eckle, Tobias; Hughes, Kelly; Ehrentraut, Heidi; Brodsky, Kelley S.; Rosenberger, Peter; Choi, Doo-Sup; Ravid, Katya; Weng, Tingting; Xia, Yang; Blackburn, Michael R.; Eltzschig, Holger K.

    2013-01-01

    The signaling molecule adenosine has been implicated in attenuating acute lung injury (ALI). Adenosine signaling is terminated by its uptake through equilibrative nucleoside transporters (ENTs). We hypothesized that ENT-dependent adenosine uptake could be targeted to enhance adenosine-mediated lung protection. To address this hypothesis, we exposed mice to high-pressure mechanical ventilation to induce ALI. Initial studies demonstrated time-dependent repression of ENT1 and ENT2 transcript and protein levels during ALI. To examine the contention that ENT repression represents an endogenous adaptive response, we performed functional studies with the ENT inhibitor dipyridamole. Dipyridamole treatment (1 mg/kg; EC50=10 μM) was associated with significant increases in ALI survival time (277 vs. 395 min; P<0.05). Subsequent studies in gene-targeted mice for Ent1 or Ent2 revealed a selective phenotype in Ent2−/− mice, including attenuated pulmonary edema and improved gas exchange during ALI in conjunction with elevated adenosine levels in the bronchoalveolar fluid. Furthermore, studies in genetic models for adenosine receptors implicated the A2B adenosine receptor (Adora2b) in mediating ENT-dependent lung protection. Notably, dipyridamole-dependent attenuation of lung inflammation was abolished in mice with alveolar epithelial Adora2b gene deletion. Our newly identified crosstalk pathway between ENT2 and alveolar epithelial Adora2b in lung protection during ALI opens possibilities for combined therapies targeted to this protein set.—Eckle, T., Hughes, K., Ehrentraut, H., Brodsky, K. S., Rosenberger, P., Choi, D.-S., Ravid, K., Weng, T., Xia, Y., Blackburn, M. R., Eltzschig, H. K. Crosstalk between the equilibrative nucleoside transporter ENT2 and alveolar Adora2b adenosine receptors dampens acute lung injury. PMID:23603835

  5. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2014-03-25

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

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

    PubMed Central

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

    2014-01-01

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

  8. A2A adenosine receptor upregulation correlates with disease activity in patients with systemic lupus erythematosus.

    PubMed

    Bortoluzzi, Alessandra; Vincenzi, Fabrizio; Govoni, Marcello; Padovan, Melissa; Ravani, Annalisa; Borea, Pier Andrea; Varani, Katia

    2016-08-26

    Adenosine is a purine nucleoside implicated in the regulation of the innate and adaptive immune systems, acting through its interaction with four cell surface receptors: A1, A2A, A2B, and A3. There is intense interest in understanding how adenosine functions in health and during disease, but surprisingly little is known about the actual role of adenosine-mediated mechanisms in systemic lupus erythematosus (SLE). With this background, the aim of the present study was to test the hypothesis that dysregulation of A1, A2A, A2B, and A3 adenosine receptors (ARs) in lymphocytes of patients with SLE may be involved in the pathogenesis of the disease and to examine the correlations between the status of the ARs and the clinical parameters of SLE. ARs were analyzed by performing saturation-binding assays, as well as messenger RNA and Western blot analysis, with lymphocytes of patients with SLE in comparison with healthy subjects. We tested the effect of A2AAR agonists in the nuclear factor kB (NF-kB) pathway and on the release of interferon (IFN)-α; tumor necrosis factor (TNF)-α; and interleukin (IL)-2, IL-6, IL-1β, and IL-10. In lymphocytes obtained from 80 patients with SLE, A2AARs were upregulated compared with those of 80 age-matched healthy control subjects, while A1, A2B, and A3 ARs were unchanged. A2AAR density was inversely correlated with Systemic Lupus Erythematosus Disease Activity Index 2000 score disease activity through time evaluated according to disease course patterns, serositis, hypocomplementemia, and anti-double-stranded DNA positivity. A2AAR activation inhibited the NF-kB activation pathway and diminished inflammatory cytokines (IFN-α, TNF-α, IL-2, IL-6, IL-1β), but it potentiated the release of anti-inflammatory IL-10. These data suggest the involvement of A2AARs in the complex pathogenetic network of SLE, acting as a modulator of the inflammatory process. It could represent a compensatory pathway to better counteract disease activity. A2AAR

  9. Synthesis, biological evaluation, and molecular modeling of ribose-modified adenosine analogues as adenosine receptor agonists.

    PubMed

    Cappellacci, Loredana; Franchetti, Palmarisa; Pasqualini, Michela; Petrelli, Riccardo; Vita, Patrizia; Lavecchia, Antonio; Novellino, Ettore; Costa, Barbara; Martini, Claudia; Klotz, Karl-Norbert; Grifantini, Mario

    2005-03-10

    A number of 3'-C-methyl analogues of selective adenosine receptor agonists such as CPA, CHA, CCPA, 2'-Me-CCPA, NECA, and IB-MECA was synthesized to further investigate the subdomain of the receptor that binds the ribose moiety of the ligands. Affinity data at A(1), A(2A), and A(3) receptors in bovine brain membranes showed that the 3'-C-modification in adenosine resulted in a decrease of the affinity at all three receptor subtypes. When this modification was combined with N(6)-substitution with groups that induce high potency and selectivity at A(1) receptor, the affinity and selectivity were increased. However, all 3'-C-methyl derivatives proved to be very less active than the corresponding 2'-C-methyl analogues. The most active compound was found to be 3'-Me-CPA which displayed a K(i) value of 0.35 microM at A(1) receptor and a selectivity for A(1) vs A(2A) and A(3) receptors higher than 28-fold. 2'-Me-CCPA was confirmed to be the most selective, high affinity agonist so far known also at human A(1) receptor with a K(i) value of 3.3 nM and 2903- and 341-fold selective vs human A(2A) and A(3) receptors, respectively. In functional assay, 3'-Me-CPA, 3'-Me-CCPA, and 2-Cl-3'-Me-IB-MECA inhibited forskolin-stimulated adenylyl cyclase activity with IC(50) values ranging from 0.3 to 4.9 microM, acting as full agonists. A rhodopsin-based model of the bovine A(1)AR was built to rationalize the higher affinity and selectivity of 2'-C-methyl derivatives of N(6)-substituted-adenosine compared to that of 3'-C-methyl analogues. In the docking exploration, it was found that 2'-Me-CCPA was able to form a number of interactions with several polar residues in the transmembrane helices TM-3, TM-6, and TM-7 of bA(1)AR which were not preserved in the molecular dynamics simulation of 3'-Me-CCPA/bA(1)AR complex.

  10. Adenosine Receptors As Drug Targets for Treatment of Pulmonary Arterial Hypertension

    PubMed Central

    Alencar, Allan K. N.; Montes, Guilherme C.; Barreiro, Eliezer J.; Sudo, Roberto T.; Zapata-Sudo, Gisele

    2017-01-01

    Pulmonary arterial hypertension (PAH) is a clinical condition characterized by pulmonary arterial remodeling and vasoconstriction, which promote chronic vessel obstruction and elevation of pulmonary vascular resistance. Long-term right ventricular (RV) overload leads to RV dysfunction and failure, which are the main determinants of life expectancy in PAH subjects. Therapeutic options for PAH remain limited, despite the introduction of prostacyclin analogs, endothelin receptor antagonists, phosphodiesterase type 5 inhibitors, and soluble guanylyl cyclase stimulators within the last 15 years. Through addressing the pulmonary endothelial and smooth muscle cell dysfunctions associated with PAH, these interventions delay disease progression but do not offer a cure. Emerging approaches to improve treatment efficacy have focused on beneficial actions to both the pulmonary vasculature and myocardium, and several new targets have been investigated and validated in experimental PAH models. Herein, we review the effects of adenosine and adenosine receptors (A1, A2A, A2B, and A3) on the cardiovascular system, focusing on the A2A receptor as a pharmacological target. This receptor induces pulmonary vascular and heart protection in experimental models, specifically models of PAH. Targeting the A2A receptor could potentially serve as a novel and efficient approach for treating PAH and concomitant RV failure. A2A receptor activation induces pulmonary endothelial nitric oxide synthesis, smooth muscle cell hyperpolarization, and vasodilation, with important antiproliferative activities through the inhibition of collagen deposition and vessel wall remodeling in the pulmonary arterioles. The pleiotropic potential of A2A receptor activation is highlighted by its additional expression in the heart tissue, where it participates in the regulation of intracellular calcium handling and maintenance of heart chamber structure and function. In this way, the activation of A2A receptor could

  11. Adenosine Receptors As Drug Targets for Treatment of Pulmonary Arterial Hypertension.

    PubMed

    Alencar, Allan K N; Montes, Guilherme C; Barreiro, Eliezer J; Sudo, Roberto T; Zapata-Sudo, Gisele

    2017-01-01

    Pulmonary arterial hypertension (PAH) is a clinical condition characterized by pulmonary arterial remodeling and vasoconstriction, which promote chronic vessel obstruction and elevation of pulmonary vascular resistance. Long-term right ventricular (RV) overload leads to RV dysfunction and failure, which are the main determinants of life expectancy in PAH subjects. Therapeutic options for PAH remain limited, despite the introduction of prostacyclin analogs, endothelin receptor antagonists, phosphodiesterase type 5 inhibitors, and soluble guanylyl cyclase stimulators within the last 15 years. Through addressing the pulmonary endothelial and smooth muscle cell dysfunctions associated with PAH, these interventions delay disease progression but do not offer a cure. Emerging approaches to improve treatment efficacy have focused on beneficial actions to both the pulmonary vasculature and myocardium, and several new targets have been investigated and validated in experimental PAH models. Herein, we review the effects of adenosine and adenosine receptors (A 1 , A 2A , A 2B , and A 3 ) on the cardiovascular system, focusing on the A 2A receptor as a pharmacological target. This receptor induces pulmonary vascular and heart protection in experimental models, specifically models of PAH. Targeting the A 2A receptor could potentially serve as a novel and efficient approach for treating PAH and concomitant RV failure. A 2A receptor activation induces pulmonary endothelial nitric oxide synthesis, smooth muscle cell hyperpolarization, and vasodilation, with important antiproliferative activities through the inhibition of collagen deposition and vessel wall remodeling in the pulmonary arterioles. The pleiotropic potential of A 2A receptor activation is highlighted by its additional expression in the heart tissue, where it participates in the regulation of intracellular calcium handling and maintenance of heart chamber structure and function. In this way, the activation of A 2A

  12. Cordycepin Increases Nonrapid Eye Movement Sleep via Adenosine Receptors in Rats.

    PubMed

    Hu, Zhenzhen; Lee, Chung-Il; Shah, Vikash Kumar; Oh, Eun-Hye; Han, Jin-Yi; Bae, Jae-Ryong; Lee, Kinam; Chong, Myong-Soo; Hong, Jin Tae; Oh, Ki-Wan

    2013-01-01

    Cordycepin (3'-deoxyadenosine) is a naturally occurring adenosine analogue and one of the bioactive constituents isolated from Cordyceps militaris/Cordyceps sinensis, species of the fungal genus Cordyceps. It has traditionally been a prized Chinese folk medicine for the human well-being. Because of similarity of chemical structure of adenosine, cordycepin has been focused on the diverse effects of the central nervous systems (CNSs), like sleep regulation. Therefore, this study was undertaken to know whether cordycepin increases the natural sleep in rats, and its effect is mediated by adenosine receptors (ARs). Sleep was recorded using electroencephalogram (EEG) for 4 hours after oral administration of cordycepin in rats. Sleep architecture and EEG power spectra were analyzed. Cordycepin reduced sleep-wake cycles and increased nonrapid eye movement (NREM) sleep. Interestingly, cordycepin increased θ (theta) waves power density during NREM sleep. In addition, the protein levels of AR subtypes (A1, A2A, and A2B) were increased after the administration of cordycepin, especially in the rat hypothalamus which plays an important role in sleep regulation. Therefore, we suggest that cordycepin increases theta waves power density during NREM sleep via nonspecific AR in rats. In addition, this experiment can provide basic evidence that cordycepin may be helpful for sleep-disturbed subjects.

  13. Cordycepin Increases Nonrapid Eye Movement Sleep via Adenosine Receptors in Rats

    PubMed Central

    Hu, Zhenzhen; Lee, Chung-Il; Shah, Vikash Kumar; Oh, Eun-Hye; Han, Jin-Yi; Bae, Jae-Ryong; Lee, Kinam; Chong, Myong-Soo; Hong, Jin Tae; Oh, Ki-Wan

    2013-01-01

    Cordycepin (3′-deoxyadenosine) is a naturally occurring adenosine analogue and one of the bioactive constituents isolated from Cordyceps militaris/Cordyceps sinensis, species of the fungal genus Cordyceps. It has traditionally been a prized Chinese folk medicine for the human well-being. Because of similarity of chemical structure of adenosine, cordycepin has been focused on the diverse effects of the central nervous systems (CNSs), like sleep regulation. Therefore, this study was undertaken to know whether cordycepin increases the natural sleep in rats, and its effect is mediated by adenosine receptors (ARs). Sleep was recorded using electroencephalogram (EEG) for 4 hours after oral administration of cordycepin in rats. Sleep architecture and EEG power spectra were analyzed. Cordycepin reduced sleep-wake cycles and increased nonrapid eye movement (NREM) sleep. Interestingly, cordycepin increased θ (theta) waves power density during NREM sleep. In addition, the protein levels of AR subtypes (A1, A2A, and A2B) were increased after the administration of cordycepin, especially in the rat hypothalamus which plays an important role in sleep regulation. Therefore, we suggest that cordycepin increases theta waves power density during NREM sleep via nonspecific AR in rats. In addition, this experiment can provide basic evidence that cordycepin may be helpful for sleep-disturbed subjects. PMID:23710239

  14. Structural Probing and Molecular Modeling of the A3 Adenosine Receptor: A Focus on Agonist Binding

    PubMed Central

    Ciancetta, Antonella; Jacobson, Kenneth A.

    2017-01-01

    Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR) subtypes, termed A1, A2A, A2B and A3, which belong to the G protein-coupled receptor (GPCR) superfamily. The human A3AR (hA3AR) subtype is implicated in several cytoprotective functions. Therefore, hA3AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anticancer, and cardioprotective agents. Structure-based molecular modeling techniques have been applied over the years to rationalize the structure-activity relationships (SARs) of newly emerged A3AR ligands, guide the subsequent lead optimization, and interpret site-directed mutagenesis (SDM) data from a molecular perspective. In this review, we showcase selected modeling-based and guided strategies that were applied to elucidate the binding of agonists to the A3AR and discuss the challenges associated with an accurate prediction of the receptor extracellular vestibule through homology modeling from the available X-ray templates. PMID:28287473

  15. Structural Probing and Molecular Modeling of the A₃ Adenosine Receptor: A Focus on Agonist Binding.

    PubMed

    Ciancetta, Antonella; Jacobson, Kenneth A

    2017-03-11

    Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR) subtypes, termed A₁, A 2A , A 2B and A₃, which belong to the G protein-coupled receptor (GPCR) superfamily. The human A₃AR (hA₃AR) subtype is implicated in several cytoprotective functions. Therefore, hA₃AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anticancer, and cardioprotective agents. Structure-based molecular modeling techniques have been applied over the years to rationalize the structure-activity relationships (SARs) of newly emerged A₃AR ligands, guide the subsequent lead optimization, and interpret site-directed mutagenesis (SDM) data from a molecular perspective. In this review, we showcase selected modeling-based and guided strategies that were applied to elucidate the binding of agonists to the A₃AR and discuss the challenges associated with an accurate prediction of the receptor extracellular vestibule through homology modeling from the available X-ray templates.

  16. Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins

    PubMed Central

    Moreno, Estefanía; Canet, Júlia; Gracia, Eduard; Lluís, Carme; Mallol, Josefa; Canela, Enric I.; Cortés, Antoni; Casadó, Vicent

    2018-01-01

    Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits. PMID:29497379

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

    PubMed

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

    2014-01-09

    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.

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

    PubMed

    Chee, Hyun Keun; Oh, S June

    2013-12-01

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

  19. Functional Cross-Talk between Adenosine and Metabotropic Glutamate Receptors.

    PubMed

    Leon-Navarro, David Agustin; Albasanz, Jose Luis; Martin, Mairena

    2018-04-15

    G-protein coupled receptors are transmembrane proteins widely expressed in cells and their transduction pathways are mediated by controlling second messenger levels through different G-protein interactions. Many of these receptors have been described as involved in the physiopathology of neurodegenerative diseases and even considered as potential targets for the design of novel therapeutic strategies. Endogenous and synthetic allosteric and orthosteric selective ligands are able to modulate GPCRs at both gene and protein expression levels and can also modify their physiological function. GPCRs that coexist in the same cells can homo- and heteromerize therefore modulating their function. Adenosine receptors are GPCRs which stimulate or inhibit adenylyl cyclase activity through Gi/Gs protein and are involved in the control of neurotransmitter release as glutamate. In turn, metabotropic glutamate receptors are also GPCRs which inhibit adenylyl cyclase or stimulate phospholipase C activities through Gi or Gq proteins respectively. In recent years evidence of crosstalk mechanisms between different GPCRs have been described. The aim of the present review was to summarize the described mechanisms of interaction and crosstalking between adenosine and metabotropic glutamate receptors, mainly of group I, in both in vitro and in vivo systems, and their possible use for the design of novel ligands for the treatment of neurodegenerative diseases. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. 8-Azaxanthine derivatives as antagonists of adenosine receptors.

    PubMed

    Franchetti, P; Messini, L; Cappellacci, L; Grifantini, M; Lucacchini, A; Martini, C; Senatore, G

    1994-09-02

    A series of 1,3-dimethyl- and 1,3-dipropyl-8-azaxanthines, substituted at the N8 or N7 position with substituents which usually increase the affinity of the xanthines for the adenosine receptors, was synthesized and studied in radioligand binding experiments. The substitution of CH with N at the 8-position of both theophylline and caffeine dramatically reduced the affinity, as demonstrated by the fact that 8-azatheophylline and 8-azacaffeine were inert. The introduction of a methyl group at 8-position of 8-azatheophylline restored the antagonistic activity at A2 receptors, while a 8-cycloalkyl substituent increased the affinity for both receptor subtypes. A more favorable effect on affinity was produced by the substitution of the 7-methyl group in 8-azacaffeine with cycloalkyl groups. 7-Cyclopentyl-1,3-dimethyl-8-azaxanthine was 3 times more potent than caffeine at A1 receptors and 6 times less active at A2 receptors. On the contrary, the 7-cyclohexyl-1,3-dimethyl-8-azaxanthine was more potent than caffeine at A2 receptors. The substitution of 1- and 3-methyl groups with propyl in both 7- and 8-substituted 8-azatheophylline increased remarkably the affinity for A1 receptors. The 7-cyclopentyl-1,3-dipropyl-8-azaxanthine appears to be one of the most potent and selective among 7-alkyl-substituted xanthines at A1 receptors so far known. Because the 8-aza analogues of 8-substituted 1,3-dialkylxanthine were in any case less active than the corresponding xanthine derivatives, it was confirmed that the hydrogen atom at the 7-position of xanthines plays an important role in the binding to adenosine receptors.

  1. Expression of Receptors for Tetanus Toxin and Monoclonal Antibody A2B5 by Pancreatic Islet Cells

    NASA Astrophysics Data System (ADS)

    Eisenbarth, G. S.; Shimizu, K.; Bowring, M. A.; Wells, S.

    1982-08-01

    Studies of the reaction of antibody A2B5 and tetanus toxin with pancreatic islet cells, islet cell tumors, and other human amine precursor uptake and decarboxylation (APUD) tumors are described. By indirect immunofluorescence, antibody A2B5 and tetanus toxin were shown to specifically bind to the plasma membrane of human, rat, chicken, and mouse islet cells. The binding of antibody A2B5 to the cell surface of living islet cells has allowed isolation of these cells from a suspension of pancreatic cells by using a fluorescence-activated cell sorter. In studies designed to determine whether tetanus toxin and antibody A2B5 bound to the same surface antigen, A2B5 and tetanus toxin did not compete for binding to normal islet cells, a human islet cell tumor, or a rat islet cell tumor. In addition to binding to islet cell tumors, antibody A2B5 reacts with frozen sections, isolated cells, and cell lines of neural, neural crest, and APUD origin.

  2. Comparison of the Human A2A Adenosine Receptor Recognition by Adenosine and Inosine: New Insight from Supervised Molecular Dynamics Simulations.

    PubMed

    Deganutti, Giuseppe; Welihinda, Ajith; Moro, Stefano

    2017-08-22

    Adenosine deaminase converts adenosine into inosine. In contrast to adenosine, relatively little attention has been paid to the physiological roles of inosine. Nevertheless, recent studies have demonstrated that inosine has neuroprotective, cardioprotective, immunomodulatory, and antidepressive effects. Inosine was recently shown to be a less potent agonist than adenosine at the A 2A adenosine receptor. To better depict the differences in the mechanisms of receptor recognition between adenosine and inosine, we carried out supervised molecular dynamics (SuMD) simulations, and the results are analyzed herein. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Administration of exogenous adenosine triphosphate to ischemic skeletal muscle induces an energy-sparing effect: role of adenosine receptors.

    PubMed

    Maldonado, Claudio; Pushpakumar, Sathnur B; Perez-Abadia, Gustavo; Arumugam, Sengodagounder; Lane, Andrew N

    2013-05-01

    Ischemia-reperfusion injury is a devastating complication that occurs in allotransplantation and replantation of limbs. Over the years, several preservation strategies have been used to conserve the critical levels of intracellular adenosine triphosphate (ATP) during ischemia to sustain the ion gradients across the membranes and thus the tissue viability. The administration of exogenous ATP to ischemic tissues is known to provide beneficial effects during reperfusion, but it is unclear whether it provides protection during ischemia. The purpose of the present study was to determine the effect of ATP administration on high-energy phosphate levels in ischemic skeletal muscle and to examine the role of purinergic and adenosine receptors in mediating the response to exogenous ATP. The extensor digitorum longus muscles of Fischer rats were subjected to ischemia and treated with different concentrations of ATP with or without purinergic and adenosine receptor blockers. Phosphorus-31 nuclear magnetic resonance spectroscopy was used to measure the rate of decay of ATP, phosphocreatine (PCr), and the formation of adenosine monophosphate and acidification. Phosphorylated compounds were analyzed using a simple model of energy metabolism, and the PCr half-life was used as an index of internal depletion of ATP to distinguish between intracellular and extracellular ATP. PCr decay was rapid in all muscle groups and was followed by gradual ATP decay. The half-life of PCr was significantly longer in the ATP-treated muscles than in the vehicle controls and was maximally prolonged by treating with slow hydrolyzing adenosine 5'-O-(3-thio)triphosphate. Purinoceptor (P2X) blockade with ATP treatment significantly increased the half-life of PCr, and adenosine receptor blockers blunted the response. Administration of adenosine to ischemic muscles significantly increased the half-life of PCr compared with that in the vehicle controls. Exogenous ATP administration to ischemic skeletal

  4. Adenosine transiently modulates stimulated dopamine release in the caudate putamen via A1 receptors

    PubMed Central

    Ross, Ashley E.; Venton, B. Jill

    2014-01-01

    Adenosine modulates dopamine in the brain via A1 and A2A receptors, but that modulation has only been characterized on a slow time scale. Recent studies have characterized a rapid signaling mode of adenosine that suggests a possible rapid modulatory role. Here, fast-scan cyclic voltammetry was used to characterize the extent to which transient adenosine changes modulate stimulated dopamine release (5 pulses at 60 Hz) in rat caudate putamen brain slices. Exogenous adenosine was applied and dopamine concentration monitored. Adenosine only modulated dopamine when it was applied 2 or 5 s before stimulation. Longer time intervals and bath application of 5 µM adenosine did not decrease dopamine release. Mechanical stimulation of endogenous adenosine 2s before dopamine stimulation also decreased stimulated dopamine release by 41 ± 7 %, similar to the 54 ± 6 % decrease in dopamine after exogenous adenosine application. Dopamine inhibition by transient adenosine was recovered within 10 minutes. The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) blocked the dopamine modulation, whereas dopamine modulation was unaffected by the A2A receptor antagonist SCH 442416. Thus, transient adenosine changes can transiently modulate phasic dopamine release via A1 receptors. These data demonstrate that adenosine has a rapid, but transient, modulatory role in the brain. PMID:25219576

  5. Adenosine and inosine increase cutaneous vasopermeability by activating A3 receptors on mast cells

    PubMed Central

    Tilley, Stephen L.; Wagoner, Victoria A.; Salvatore, Christopher A.; Jacobson, Marlene A.; Koller, Beverly H.

    2000-01-01

    Adenosine has potent effects on both the cardiovascular and immune systems. Exposure of tissues to adenosine results in increased vascular permeability and extravasation of serum proteins. The mechanism by which adenosine brings about these physiological changes is poorly defined. Using mice deficient in the A3 adenosine receptor (A3AR), we show that increases in cutaneous vascular permeability observed after treatment with adenosine or its principal metabolite inosine are mediated through the A3AR. Adenosine fails to increase vascular permeability in mast cell–deficient mice, suggesting that this tissue response to adenosine is mast cell–dependent. Furthermore, this response is independent of activation of the high-affinity IgE receptor (FcεR1) by antigen, as adenosine is equally effective in mediating these changes in FcεR1 β-chain–deficient mice. Together these results support a model in which adenosine and inosine induce changes in vascular permeability indirectly by activating mast cells, which in turn release vasoactive substances. The demonstration in vivo that adenosine, acting through a specific receptor, can provoke degranulation of this important tissue-based effector cell, independent of antigen activation of the high-affinity IgE receptor, supports an important role for this nucleoside in modifying the inflammatory response. PMID:10675362

  6. Neuronal A1 receptors mediate increase in extracellular kynurenic acid after local intrastriatal adenosine infusion.

    PubMed

    Wu, Hui-Qiu; Fuxe, Kjell; Schwarcz, Robert

    2004-08-01

    The naturally occurring purine nucleoside adenosine has pronounced anticonvulsant and neuroprotective properties and plays a neuromodulatory role in the CNS. Kynurenic acid (KYNA) is an astrocyte-derived, endogenous neuroinhibitory compound, which shares several of adenosine's properties. In a first attempt to examine possible interactions between these two biologically active molecules, adenosine was focally applied into the striatum of freely moving rats by reverse microdialysis, and changes in extracellular KYNA were monitored over time. A 2-h infusion of adenosine increased KYNA levels in a dose-dependent manner, with 10 mm of adenosine causing a twofold elevation within 1 h. This effect was reversible and was effectively blocked by coinfusion of the specific A1 adenosine receptor antagonist 8-cyclopentyltheophylline (100 microm). In contrast, coinfusion of adenosine with MSX-3 (100 microm), an A2A receptor antagonist, did not affect the adenosine-induced increase in KYNA levels. Local striatal perfusion with the A1 receptor agonist N6-cyclopentyladenosine (100 microm) mimicked the effect of adenosine, whereas perfusion with the A2A receptor agonist CGS-21680 (100 microm) was ineffective. Finally, we tested the effect of adenosine (10 mm) on extracellular KYNA in striata that had been injected with quinolinate (60 nmol/1 microL) 7 days earlier. In this neuron-depleted tissue, perfusion with adenosine failed to affect extracellular KYNA levels. These data demonstrate that adenosine is capable of raising extracellular KYNA in the rat striatum by interacting with postsynaptic neuronal A1 receptors. This mechanism may result in a synergism between the neurobiological effects of adenosine and KYNA.

  7. Adenosine receptor antagonist and augmented vasodilation during hypoxic exercise

    PubMed Central

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

    2009-01-01

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

  8. Acetate supplementation modulates brain adenosine metabolizing enzymes and adenosine A₂A receptor levels in rats subjected to neuroinflammation.

    PubMed

    Smith, Mark D; Bhatt, Dhaval P; Geiger, Jonathan D; Rosenberger, Thad A

    2014-06-04

    Acetate supplementation reduces neuroglia activation and pro-inflammatory cytokine expression in rat models of neuroinflammation and Lyme neuroborreliosis. Because single-dose glyceryl triacetate (GTA) treatment increases brain phosphocreatine and reduces brain AMP levels, we postulate that GTA modulates adenosine metabolizing enzymes and receptors, which may be a possible mechanism to reduce neuroinflammation. To test this hypothesis, we quantified the ability of GTA to alter brain levels of ecto-5'-nucleotidase (CD73), adenosine kinase (AK), and adenosine A2A receptor using western blot analysis and CD73 activity by measuring the rate of AMP hydrolysis. Neuroinflammation was induced by continuous bacterial lipopolysaccharide (LPS) infusion in the fourth ventricle of the brain for 14 and 28 days. Three treatment strategies were employed, one and two where rats received prophylactic GTA through oral gavage with LPS infusion for 14 or 28 days. In the third treatment regimen, an interventional strategy was used where rats were subjected to 28 days of neuroinflammation, and GTA treatment was started on day 14 following the start of the LPS infusion. We found that rats subjected to neuroinflammation for 28 days had a 28% reduction in CD73 levels and a 43% increase in AK levels that was reversed with prophylactic acetate supplementation. CD73 activity in these rats was increased by 46% with the 28-day GTA treatment compared to the water-treated rats. Rats subjected to neuroinflammation for 14 days showed a 50% increase in levels of the adenosine A2A receptor, which was prevented with prophylactic acetate supplementation. Interventional GTA therapy, beginning on day 14 following the induction of neuroinflammation, resulted in a 67% increase in CD73 levels and a 155% increase in adenosine A2A receptor levels. These results support the hypothesis that acetate supplementation can modulate brain CD73, AK and adenosine A2A receptor levels, and possibly influence purinergic

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

    PubMed

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

    2014-10-01

    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 2-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. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Glutamate differently modulates excitatory and inhibitory adenosine receptors in neuronal and glial cells.

    PubMed

    Castillo, Carlos Alberto; León, David Agustín; Ballesteros-Yáñez, Inmaculada; Albasanz, José Luis; Martín, Mairena

    2010-08-01

    Adenosine is a neuromodulator which acts through adenosine receptors regulating functions such as inhibition of glutamate release. Adenosine A(1) and A(2A) receptor activations most often regulate opposing actions. Primary rat cortical neurons and rat C6 cells, an astrocytic derived cell line, were exposed to 100muM l-glutamate, and cell viability and transduction pathways mediated by both A(1) and A(2A) receptors were analyzed. Glutamate-induced excitotoxic damage was found only in cortical neurons, with C6 cells preserved. In C6 cells, adenosine A(1) and A(2A) receptors were increased and decreased, respectively. Consequently, A(1)-mediated adenylyl cyclase inhibition and A(2A)-mediated adenylyl cyclase stimulation were, respectively, increased and decreased after glutamate exposure. In cortical neurons, glutamate treatment increased both A(1) and A(2A) receptors. Moreover, adenylyl cyclase responsiveness to A(1) or A(2A) receptor agonists was heightened in these cells, in which pharmacological activation of AC induced cell death. Finally, activation of A(1) receptor or blockade of A(2A) receptor during glutamate treatment partially prevented the glutamate-induced cell death detected in cultured cortical neurons. Results show that adenosine receptors are regulated by glutamate, and that this regulation is dependent on the cell type, suggesting that adenosine receptors might be promising targets in the therapy against excitotoxic cell death.

  11. N6-Cycloalkyl- and N6-bicycloalkyl-C5'(C2')-modified adenosine derivatives as high-affinity and selective agonists at the human A1 adenosine receptor with antinociceptive effects in mice.

    PubMed

    Franchetti, Palmarisa; Cappellacci, Loredana; Vita, Patrizia; Petrelli, Riccardo; Lavecchia, Antonio; Kachler, Sonja; Klotz, Karl-Norbert; Marabese, Ida; Luongo, Livio; Maione, Sabatino; Grifantini, Mario

    2009-04-23

    To further investigate new potent and selective human A(1) adenosine receptor agonists, we have synthesized a series of 5'-chloro-5'-deoxy- and 5'-(2-fluorophenylthio)-5'-deoxy-N(6)-cycloalkyl(bicycloalkyl)-substituted adenosine and 2'-C-methyladenosine derivatives. These compounds were evaluated for affinity and efficacy at human A(1), A(2A), A(2B), and A(3) adenosine receptors. In the series of N(6)-cyclopentyl- and N(6)-(endo-norborn-2-yl)adenosine derivatives, 5'-chloro-5'-deoxy-CPA (1) and 5'-chloro-5'-deoxy-(+/-)-ENBA (3) displayed the highest affinity in the subnanomolar range and relevant selectivity for hA(1) vs the other human receptor subtypes. The higher affinity and selectivity of 5'-chloro-5'-deoxyribonucleoside derivatives 1 and 3 for hA(1) AR vs hA(3) AR compared to that of the parent 5'-hydroxy compounds CPA and (+/-)-ENBA was rationalized by a molecular modeling analysis. 5'-Chloro-5'-deoxy-(+/-)-ENBA, evaluated for analgesic activity in the formalin test in mice, was found to inhibit the first or the second phases of the nocifensive response induced by intrapaw injection of formalin at doses ranging between 1 and 2 mg/kg i.p.

  12. Modulation of adenosine A1 and A2A receptors in C6 glioma cells during hypoxia: involvement of endogenous adenosine.

    PubMed

    Castillo, Carlos A; León, David; Ruiz, María Angeles; Albasanz, José Luis; Martín, Mairena

    2008-06-01

    During hypoxia, extracellular adenosine levels are increased to prevent cell damage, playing a neuroprotective role mainly through adenosine A(1) receptors. The aim of the present study was to analyze the effect of hypoxia in both adenosine A(1) and A(2A) receptors endogenously expressed in C6 glioma cells. Two hours of hypoxia (5% O(2)) caused a significant decrease in adenosine A(1) receptors. The same effect was observed at 6 h and 24 h of hypoxia. However, adenosine A(2A) receptors were significantly increased at the same times. These effects were not due to hypoxia-induced alterations in cells number or viability. Changes in receptor density were not associated with variations in the rate of gene expression. Furthermore, hypoxia did not alter HIF-1alpha expression in C6 cells. However, HIF-3alpha, CREB and CREM were decreased. Adenosine A(1) and A(2A) receptor density in normoxic C6 cells treated with adenosine for 2, 6 and 24 h was similar to that observed in cells after oxygen deprivation. When C6 cells were subjected to hypoxia in the presence of adenosine deaminase, the density of receptors was not significantly modulated. Moreover, DPCPX, an A(1) receptor antagonist, blocked the effects of hypoxia on these receptors, while ZM241385, an A(2A) receptor antagonist, was unable to prevent these changes. These results suggest that moderate hypoxia modulates adenosine receptors and cAMP response elements in glial cells, through a mechanism in which endogenous adenosine and tonic A(1) receptor activation is involved.

  13. Profound hypothermia after adenosine kinase inhibition in A1AR-deficient mice suggests a receptor-independent effect of intracellular adenosine.

    PubMed

    Eisner, Christoph; Kim, SooMi; Grill, Alexandra; Qin, Yan; Hoerl, Marion; Briggs, Josephine; Castrop, Hayo; Thiel, Manfred; Schnermann, Jurgen

    2017-02-01

    Administration of the nucleoside adenosine has been shown to induce hypothermia in a number of species, an effect mediated predominantly by the adenosine 1 receptor (A1AR) subtype. The present experiments were performed to explore the possibility that the rise of intracellular adenosine levels expected to accompany adenosine administration may contribute to the hypothermic effect of adenosine independent of A1AR activation. Since phosphorylation of adenosine by adenosine kinase (ADK) is causal in the maintenance of low intracellular adenosine, we have examined the effect of ADK inhibition on core body temperature (CBT). Our data show that inhibition of ADK by A-134974 causes a long-lasting deep hypothermia in wild-type mice. Since there was an about 4-fold increase of adenosine plasma levels, experiments were repeated in A1AR-/- mice. ADK inhibition caused deep hypothermia despite the absence of A1AR, although the effect was significantly reduced compared to WT. Furthermore, the dose-dependent hypothermia caused by adenosine administration in WT mice was found to be reduced, but not abolished in A1AR-/- mice. To assess the possible role of A2AR and A3AR activation in our experimental setting, we compared the effects of the agonists CPA (A1AR), CGS21680 (A2AR), and IB-MECA (A3AR) on CBT. Hypothermia induced by CPA was much greater than that caused by CGS21680 or IB-MECA indicating that A1AR activation is the major receptor-dependent pathway for adenosine-induced hypothermia under our experimental conditions. Induction of deep hypothermia by inhibition of ADK, maintenance of this effect in A1AR-/- mice, and maintenance of adenosine-induced hypothermia in A1AR-deficient mice suggest that a receptor-independent action of adenosine requiring intact function of adenosine kinase contributes importantly to the hypothermia induced by adenosine.

  14. Endogenous expression of adenosine A1, A2 and A3 receptors in rat C6 glioma cells.

    PubMed

    Castillo, Carlos Alberto; Albasanz, José Luís; Fernández, Mercedes; Martín, Mairena

    2007-06-01

    Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [(3)H]DPCPX, selective A(1)R antagonist, revealed a single binding site with a K (D) = 9.4 +/- 1.4 nM and B (max) = 62.7 +/- 8.6 fmol/mg protein. Binding of [(3)H]DPCPX in intact cell revealed a K (D) = 17.7 +/- 1.3 nM and B (max )= 567.1 +/- 26.5 fmol/mg protein. On the other hand, [(3)H]ZM241385 binding experiments revealed a single binding site population of receptors with K (D) = 16.5 +/- 1.3 nM and B (max) = 358.9 +/- 52.4 fmol/mg protein in intact cells, and K (D) = 4.7 +/- 0.6 nM and B (max) = 74.3 +/- 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A(2A) receptor in C6 cells. A(1), A(2A), A(2B) and A(3 )adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Gialpha and Gsalpha proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A(1)R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A(1) and A(2) receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.

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

    PubMed

    Morton, R A; Davies, C H

    1997-07-01

    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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-06-01

    Inosine is an endogenous purine nucleoside that is produced by catabolism of adenosine. Adenosine has a short half-life (approximately 10s) and is rapidly deaminated to inosine, a stable metabolite with a half-life of approximately 15h. Resembling adenosine, inosine acting through adenosine receptors (ARs) exerts a wide range of anti-inflammatory and immunomodulatory effects in vivo. The immunomodulatory effects of inosine in vivo, at least in part, are mediated via the adenosine A2A receptor (A2AR), an observation that cannot be explained fully by in vitro pharmacological characterization of inosine at the A2AR. It is unclear whether the in vivo effects of inosine are due to inosine or a metabolite of inosine engaging the A2AR. Here, utilizing a combination of label-free, cell-based, and membrane-based functional assays in conjunction with an equilibrium agonist-binding assay we provide evidence for inosine engagement at the A2AR and subsequent activation of downstream signaling events. Inosine-mediated A2AR activation leads to cAMP production with an EC50 of 300.7μM and to extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation with an EC50 of 89.38μM. Our data demonstrate that inosine produces ERK1/2-biased signaling whereas adenosine produces cAMP-biased signaling at the A2AR, highlighting pharmacological differences between these two agonists. Given the in vivo stability of inosine, our data suggest an additional, previously unrecognized, mechanism that utilizes inosine to functionally amplify and prolong A2AR activation in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Targeting the adenosine 2A receptor enhances chimeric antigen receptor T cell efficacy

    PubMed Central

    Beavis, Paul A.; Henderson, Melissa A.; Giuffrida, Lauren; Mills, Jane K.; Sek, Kevin; Cross, Ryan S.; Davenport, Alexander J.; John, Liza B.; Mardiana, Sherly; Slaney, Clare Y.; Johnstone, Ricky W.; Trapani, Joseph A.; Stagg, John; Loi, Sherene; Kats, Lev; Gyorki, David; Kershaw, Michael H.; Darcy, Phillip K.

    2017-01-01

    Chimeric antigen receptor (CAR) T cells have been highly successful in treating hematological malignancies, including acute and chronic lymphoblastic leukemia. However, treatment of solid tumors using CAR T cells has been largely unsuccessful to date, partly because of tumor-induced immunosuppressive mechanisms, including adenosine production. Previous studies have shown that adenosine generated by tumor cells potently inhibits endogenous antitumor T cell responses through activation of adenosine 2A receptors (A2ARs). Herein, we have observed that CAR activation resulted in increased A2AR expression and suppression of both murine and human CAR T cells. This was reversible using either A2AR antagonists or genetic targeting of A2AR using shRNA. In 2 syngeneic HER2+ self-antigen tumor models, we found that either genetic or pharmacological targeting of the A2AR profoundly increased CAR T cell efficacy, particularly when combined with PD-1 blockade. Mechanistically, this was associated with increased cytokine production of CD8+ CAR T cells and increased activation of both CD8+ and CD4+ CAR T cells. Given the known clinical relevance of the CD73/adenosine pathway in several solid tumor types, and the initiation of phase I trials for A2AR antagonists in oncology, this approach has high translational potential to enhance CAR T cell efficacy in several cancer types. PMID:28165340

  19. Anti-inflammatory effects of inosine in allergic lung inflammation in mice: evidence for the participation of adenosine A2A and A 3 receptors.

    PubMed

    da Rocha Lapa, Fernanda; de Oliveira, Ana Paula Ligeiro; Accetturi, Beatriz Golega; de Oliveira Martins, Isabelli; Domingos, Helory Vanni; de Almeida Cabrini, Daniela; de Lima, Wothan Tavares; Santos, Adair Roberto Soares

    2013-09-01

    Inosine, a naturally occurring purine formed from the breakdown of adenosine, is associated with immunoregulatory effects. Evidence shows that inosine modulates lung inflammation and regulates cytokine generation. However, its role in controlling allergen-induced lung inflammation has yet to be identified. In this study, we aimed to investigate the role of inosine and adenosine receptors in a murine model of lung allergy induced by ovalbumin (OVA). Intraperitoneal administration of inosine (0.001-10 mg/kg, 30 min before OVA challenge) significantly reduced the number of leukocytes, macrophages, lymphocytes and eosinophils recovered in the bronchoalveolar lavage fluid of sensitized mice compared with controls. Interestingly, our results showed that pre-treatment with the selective A2A receptor antagonist (ZM241385), but not with the selective A2B receptor antagonist (alloxazine), reduced the inhibitory effects of inosine against macrophage count, suggesting that A2A receptors mediate monocyte recruitment into the lungs. In addition, the pre-treatment of mice with selective A3 antagonist (MRS3777) also prevented inosine effects against macrophages, lymphocytes and eosinophils. Histological analysis confirmed the effects of inosine and A2A adenosine receptors on cell recruitment and demonstrated that the treatment with ZM241385 and alloxazine reverted inosine effects against mast cell migration into the lungs. Accordingly, the treatment with inosine reduced lung elastance, an effect related to A2 receptors. Moreover, inosine reduced the levels of Th2-cytokines, interleukin-4 and interleukin-5, an effect that was not reversed by A2A or A2B selective antagonists. Our data show that inosine acting on A2A or A3 adenosine receptors can regulate OVA-induced allergic lung inflammation and also implicate inosine as an endogenous modulator of inflammatory processes observed in the lungs of asthmatic patients.

  20. Aged rat myocardium exhibits normal adenosine receptor-mediated bradycardia and coronary vasodilation but increased adenosine agonist-mediated cardioprotection.

    PubMed

    Kristo, Gentian; Yoshimura, Yukihiro; Keith, Byron J; Mentzer, Robert M; Lasley, Robert D

    2005-11-01

    The purpose of this study was to determine whether aged myocardium exhibits decreased responsiveness to adenosine A1 and A(2a) receptor activation. Studies were conducted in adult (4-6 months) and aged (24-26 months) Fischer 344 x Brown Norway hybrid (F344 x BN) rats. Effects of the adenosine A1/A(2a) agonist AMP579 were measured in isolated hearts and in rats submitted to in vivo regional myocardial ischemia. Aged isolated hearts exhibited lower spontaneous heart rates and higher coronary resistance, as well as normal A1- and A(2a)-mediated responses. There was no difference in control infarct size between adult and aged rats; however, AMP579 treatment resulted in a 50% greater infarct size reduction in aged rats (18 +/- 4% of risk area) compared to adult rats (37 +/- 3%). These findings suggest that adenosine A1 and A(2a) receptor-mediated effects are not diminished in normal aged myocardium, and that aged hearts exhibit increased adenosine agonist-induced infarct reduction.

  1. Adenosine A1 Receptors Promote Vasa Vasorum Endothelial Cell Barrier Integrity via Gi and Akt-Dependent Actin Cytoskeleton Remodeling

    PubMed Central

    Siddaramappa Umapathy, Nagavedi; Kaczmarek, Elzbieta; Fatteh, Nooreen; Burns, Nana; Lucas, Rudolf; Stenmark, Kurt R.; Verin, Alexander D.; Gerasimovskaya, Evgenia V.

    2013-01-01

    Background In a neonatal model of hypoxic pulmonary hypertension, a dramatic pulmonary artery adventitial thickening, accumulation of inflammatory cells in the adventitial compartment, and angiogenic expansion of the vasa vasorum microcirculatory network are observed. These pathophysiological responses suggest that rapidly proliferating vasa vasorum endothelial cells (VVEC) may exhibit increased permeability for circulating blood cells and macromolecules. However, the molecular mechanisms underlying these observations remain unexplored. Some reports implicated extracellular adenosine in the regulation of vascular permeability under hypoxic and inflammatory conditions. Thus, we aimed to determine the role of adenosine in barrier regulation of VVEC isolated from the pulmonary arteries of normoxic (VVEC-Co) or chronically hypoxic (VVEC-Hyp) neonatal calves. Principal Findings We demonstrate via a transendothelial electrical resistance measurement that exogenous adenosine significantly enhanced the barrier function in VVEC-Co and, to a lesser extent, in VVEC-Hyp. Our data from a quantitative reverse transcription polymerase chain reaction show that both VVEC-Co and VVEC-Hyp express all four adenosine receptors (A1, A2A, A2B, and A3), with the highest expression level of A1 receptors (A1Rs). However, A1R expression was significantly lower in VVEC-Hyp compared to VVEC-Co. By using an A1R-specific agonist/antagonist and siRNA, we demonstrate that A1Rs are mostly responsible for adenosine-induced enhancement in barrier function. Adenosine-induced barrier integrity enhancement was attenuated by pretreatment of VVEC with pertussis toxin and GSK690693 or LY294002, suggesting the involvement of Gi proteins and the PI3K-Akt pathway. Moreover, we reveal a critical role of actin cytoskeleton in VVEC barrier regulation by using specific inhibitors of actin and microtubule polymerization. Further, we show that adenosine pretreatment blocked the tumor necrosis factor alpha (TNF

  2. Monoclonal antibodies to adenosine receptor by an auto-anti-idiotypic approach

    SciTech Connect

    Ku, Hsing-Hsu.

    1988-01-01

    BALB/c mice were immunized with adenosine 6-aminocaproyl-BSA. Hybridoma cell lines that secreted anti-idiotypic antibodies were identified by their binding to rabbit anti-adenosine antibodies, but not to normal rabbit immunoglobulins. Two such monoclonal antibodies, AA18 and AA21, also inhibited the binding of ({sup 3}H)adenosine to the rabbit anti-adenosine antibodies. Therefore, both appeared to recognize idiotypic determinants on the rabbit anti-adenosine antibodies. The monoclonal antibodies AA18 and AA21 were established as being directed at adenosine receptors by the following criteria: (1) they bound to both rat and bovine brain membranes, and binding could be inhibited by CHA, an adenosine receptor agonist, (2)more » they inhibited the binding of ({sup 3}H)R-PIA, an adenosine receptor agonist, to rat brain membranes; and (3) they inhibited the adenylate cyclase of rat brain membranes. The monoclonal antibodies were used to screen cDNA libraries in lambda gt11.« less

  3. Acetate supplementation modulates brain adenosine metabolizing enzymes and adenosine A2A receptor levels in rats subjected to neuroinflammation

    PubMed Central

    2014-01-01

    Background Acetate supplementation reduces neuroglia activation and pro-inflammatory cytokine expression in rat models of neuroinflammation and Lyme neuroborreliosis. Because single-dose glyceryl triacetate (GTA) treatment increases brain phosphocreatine and reduces brain AMP levels, we postulate that GTA modulates adenosine metabolizing enzymes and receptors, which may be a possible mechanism to reduce neuroinflammation. Methods To test this hypothesis, we quantified the ability of GTA to alter brain levels of ecto-5’-nucleotidase (CD73), adenosine kinase (AK), and adenosine A2A receptor using western blot analysis and CD73 activity by measuring the rate of AMP hydrolysis. Neuroinflammation was induced by continuous bacterial lipopolysaccharide (LPS) infusion in the fourth ventricle of the brain for 14 and 28 days. Three treatment strategies were employed, one and two where rats received prophylactic GTA through oral gavage with LPS infusion for 14 or 28 days. In the third treatment regimen, an interventional strategy was used where rats were subjected to 28 days of neuroinflammation, and GTA treatment was started on day 14 following the start of the LPS infusion. Results We found that rats subjected to neuroinflammation for 28 days had a 28% reduction in CD73 levels and a 43% increase in AK levels that was reversed with prophylactic acetate supplementation. CD73 activity in these rats was increased by 46% with the 28-day GTA treatment compared to the water-treated rats. Rats subjected to neuroinflammation for 14 days showed a 50% increase in levels of the adenosine A2A receptor, which was prevented with prophylactic acetate supplementation. Interventional GTA therapy, beginning on day 14 following the induction of neuroinflammation, resulted in a 67% increase in CD73 levels and a 155% increase in adenosine A2A receptor levels. Conclusion These results support the hypothesis that acetate supplementation can modulate brain CD73, AK and adenosine A2A receptor

  4. Role of equilibrative adenosine transporters and adenosine receptors as modulators of the human placental endothelium in gestational diabetes mellitus.

    PubMed

    Pardo, F; Arroyo, P; Salomón, C; Westermeier, F; Salsoso, R; Sáez, T; Guzmán-Gutiérrez, E; Leiva, A; Sobrevia, L

    2013-12-01

    Gestational diabetes mellitus (GDM) is a diseases that alters human placenta macro and microvascular reactivity as a result of endothelial dysfunction. The human placenta is a highly vascularized organ which lacks innervation, so blood flux is governed by locally released vasoactive molecules, including the endogenous nucleoside adenosine and the free radical nitric oxide (NO). Altered adenosine metabolism and uptake by the endothelium leads to increased NO synthesis which then turns-off the expression of genes coding for a family of nucleoside membrane transporters belonging to equilibrative nucleoside transporters, particularly isoforms 1 (hENT1) and 2 (hENT2). This mechanism leads to increased extracellular adenosine and, as a consequence, activation of adenosine receptors to further sustain a tonic activation of NO synthesis. This is a phenomenon that seems operative in the placental macro and microvascular endothelium in GDM. We here summarize the findings available in the literature regarding these mechanisms in the human feto-placental circulation. This phenomenon is altered in the feto-placental vasculature, which could be crucial for understanding GDM deleterious effects in fetal growth and development. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Chalcone-based derivatives as new scaffolds for hA3 adenosine receptor antagonists.

    PubMed

    Vazquez-Rodriguez, Saleta; Matos, Maria João; Santana, Lourdes; Uriarte, Eugenio; Borges, Fernanda; Kachler, Sonja; Klotz, Karl-Norbert

    2013-05-01

    With the aim of finding new adenosine receptor (AR) ligands based on the chalcone scaffold, we report the synthesis of a new series of coumarin-chalcone hybrids and the pharmacological characterization of their actions at four subtypes of AR. The synthesized compounds 5-10 were characterized in radioligand binding (A1 , A2A and A3 ) and adenylyl cyclase activity assays (A2B ) to determine the affinity of the compounds for the four human AR (hAR) subtypes. Coumarin-chalcone hybrids were found to be ligands with a novel structure, not reported thus far, that showed varying affinity and selectivity for AR subtypes. The coumarin-chalcone hybrids in which ring B of the chalcone scaffold was a thiophene (compounds 5 and 9) were found to be the most potent compounds of the series. Compound 9, in which ring A of the chalcone moiety was the phenyl ring of the coumarin, showed similar activity against hA1 , hA2A and hA3 ARs, while compound 5, in which ring A of the chalcone was substituted by the benzopyrone ring of the coumarin moiety, showed similar activity only at the hA3 AR and, therefore, was deemed to be selective (Ki (dissociation constant) = 5160 nm). © 2013 The Authors. JPP © 2013 Royal Pharmaceutical Society.

  7. 5'-C-Ethyl-tetrazolyl-N(6)-substituted adenosine and 2-chloro-adenosine derivatives as highly potent dual acting A1 adenosine receptor agonists and A3 adenosine receptor antagonists.

    PubMed

    Petrelli, Riccardo; Torquati, Ilaria; Kachler, Sonja; Luongo, Livio; Maione, Sabatino; Franchetti, Palmarisa; Grifantini, Mario; Novellino, Ettore; Lavecchia, Antonio; Klotz, Karl-Norbert; Cappellacci, Loredana

    2015-03-12

    A series of N(6)-substituted-5'-C-(2-ethyl-2H-tetrazol-5-yl)-adenosine and 2-chloro-adenosine derivatives was synthesized as novel, highly potent dual acting hA1AR agonists and hA3AR antagonists, potentially useful in the treatment of glaucoma and other diseases. The best affinity and selectivity profiles were achieved by N(6)-substitution with a 2-fluoro-4-chloro-phenyl- or a methyl- group. Through an in silico receptor-driven approach, the molecular bases of the hA1- and hA3AR recognition and activation of this series of 5'-C-ethyl-tetrazolyl derivatives were explained.

  8. P2X7 receptor drives osteoclast fusion by increasing the extracellular adenosine concentration.

    PubMed

    Pellegatti, Patrizia; Falzoni, Simonetta; Donvito, Giovanna; Lemaire, Irma; Di Virgilio, Francesco

    2011-04-01

    Defects in bone homeostasis are a major health problem. Osteoclast differentiation and activation have a crucial role in bone remodeling in health and disease. Osteoclasts are bone-resorbing cells derived from mononuclear phagocyte progenitors. The key event in osteoclast formation is fusion of mononucleate precursors to form mature multinucleated osteclasts. Here we provide evidence of an absolute requirement for the P2X7 receptor, ATP release, and adenosine signaling in human osteoclast formation, as shown by the following findings: macrophage-colony stimulating factor/receptor activator for nuclear factor-κB ligand (M-CSF/RANKL)-stimulated fusion of human monocytes is fully prevented by an anti-P2X7 mAb, by specific P2X7 pharmacological antagonists, or by inhibition of CD39/NTPDase; fusion-competent monocytes release ATP via the P2X7 receptor; accelerated degradation of released ATP by addition of either apyrase or hexokinase strongly increases fusion; removal of extracellular adenosine by adenosine deaminase blocks, while addition of exogenous adenosine strongly potentiates, fusion; and pharmacologic stimulation of the adenosine A2A receptor increases, while selective A2A blockade inhibits, fusion. These results show that the purinergic axis plays a crucial and as yet undescribed role in osteoclast formation and reconcile previous evidence advocating a key role for either ATP or adenosine receptors in multinucleated giant cell formation.

  9. Adenosine A₂A and A₃ receptors are involved in the human endothelial progenitor cells migration.

    PubMed

    Fernandez, Paulina; Jara, Casandra; Aguilera, Valeria; Caviedes, Liska; Diaz, Francisca; Radojkovic, Claudia; Veas, Carlos; Lamperti, Liliana; Escudero, Carlos; Aguayo, Claudio

    2012-05-01

    Human endothelial progenitor cells (hEPC) are recruited to sites of neovascularization where they differentiate into endothelial cells. The signals/factors responsible for hEPC migration and adhesion to sites of injury are not well understood. Elevated levels of adenosine are known to increase mature endothelial cell migration in response to tissue injury. However, the understanding of the role of adenosine in the physiology of hEPC is very limited. Using quantitative polymerase chain reaction and western blot analyses, we detected the expression of the adenosine receptors A₂A, A₂B, and A₃ in hEPC. Stimulation of adenosine receptors using adenosine or the nonselective agonist adenosine-5'-N-ethylcarboxamide (NECA) increased hEPC migration in 1.4-fold and 2.1-fold (P < 0.01), respectively. Stimulation of hEPC using the A₂A-specific agonist CGS-21680 resembled the effect observed in migration when using adenosine or NECA. Consequently, NECA and CGS-21680-stimulated migration of hEPC were reverted using the A₂A receptor antagonist ZM-241385. NECA-stimulated migration was inhibited in dose-dependent manner using MRS-1523 (Ki of 147 ± 0.016 nM), MRS-1754 (Ki of 1900 ± 0.02 nM), or ZM-241385 (Ki of 0.2 ± 0.01 nM). In conclusion, adenosine stimulates hEPC migration by activating A₂A and A₃ but not A₂B receptors and provides evidence to support a role of adenosine in modulating angiogenic capacity of hEPC.

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

    PubMed Central

    Minic, Zeljka; O'Leary, Donal S.

    2015-01-01

    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. Modulation of Adenosine Receptors by [60]Fullerene Hydrosoluble Derivative in SK-N-MC Cells

    PubMed Central

    2011-01-01

    The most known fullerenes are spherical carbon compounds composed of 60 carbon atoms. C60 fullerenes have shown biochemical and biomedical properties in the last years such as as blockade of apoptosis and neuroprotection. The nucleoside adenosine has a neuroprotective role mainly due to inhibition of glutamate release, which is a neurotransmitter related to excitotoxicity and cell death. In the present work, we have determined the presence of adenosine receptors in SK-N-MC cells, a neuroepithelioma human cell line, and analyzed the effect of fullerenes in these receptors by using radioligand binding, immunoblotting, and quantitative real time PCR assays. Results demonstrated that SK-N-MC cells endogenously express adenosine receptors. Fullerene exposure of these cells did not affect cell viability measured by MTT reduction assay. However, adenosine A1 and A2A receptors were both increased in SK-N-MC cells after treatment. These results suggest for the first time the modulation of adenosine receptors after C60 fullerenes exposure. PMID:22816023

  12. Angiotensin II contributes to glomerular hyperfiltration in diabetic rats independently of adenosine type I receptors

    PubMed Central

    Patinha, Daniela; Fasching, Angelica; Pinho, Dora; Albino-Teixeira, António; Morato, Manuela

    2013-01-01

    Increased angiotensin II (ANG II) or adenosine can potentiate each other in the regulation of renal hemodynamics and tubular function. Diabetes is characterized by hyperfiltration, yet the roles of ANG II and adenosine receptors for controlling baseline renal blood flow (RBF) or tubular Na+ handling in diabetes is presently unknown. Accordingly, the changes in their functions were investigated in control and 2-wk streptozotocin-diabetic rats after intrarenal infusion of the ANG II AT1 receptor antagonist candesartan, the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), or their combination. Compared with controls, the baseline blood pressure, RBF, and renal vascular resistance (RVR) were similar in diabetics, whereas the glomerular filtration rate (GFR) and filtration fraction (FF) were increased. Candesartan, DPCPX, or the combination increased RBF and decreased RVR similarly in all groups. In controls, the GFR was increased by DPCPX, but in diabetics, it was decreased by candesartan. The FF was decreased by candesartan and DPCPX, independently. DPCPX caused the most pronounced increase in fractional Na+ excretion in both controls and diabetics, whereas candesartan or the combination only affected fractional Li+ excretion in diabetics. These results suggest that RBF, via a unifying mechanism, and tubular function are under strict tonic control of both ANG II and adenosine in both control and diabetic kidneys. Furthermore, increased vascular AT1 receptor activity is a contribution to diabetes-induced hyperfiltration independent of any effect of adenosine A1 receptors. PMID:23283998

  13. An update on adenosine A2A receptors as drug target in Parkinson's disease.

    PubMed

    Vallano, Antoni; Fernandez-Duenas, Victor; Pedros, Consuelo; Arnau, Josep Maria; Ciruela, Francisco

    2011-09-01

    Adenosine receptors are G protein-coupled receptors (GPCRs) that mediate the physiological functions of adenosine. In the central nervous system adenosine A(2A) receptors (A(2A)Rs) are highly enriched in striatopallidal neurons where they form functional oligomeric complexes with other GPCRs such us the dopamine D(2) receptor (D(2)R). Furthermore, it is assumed that the formation of balanced A(2A)R/D(2)R receptor oligomers are essential for correct striatal function as the allosteric receptor-receptor interactions established within the oligomer are needed for properly sensing adenosine and dopamine. Interestingly, A(2A)R activation reduces the affinity of striatal D(2)R for dopamine and the blockade of A(2A)R with specific antagonists facilitates function of the D(2)R. Thus, it may be postulated that A(2A)R antagonists are pro-dopaminergic agents. Therefore, A(2A)R antagonists will potentially reduce the effects associated with dopamine depletion in Parkinson's disease (PD). Accordingly, this class of compounds have recently attracted considerable attention as potential therapeutic agents for PD pharmacotherapy as they have shown potential effectiveness in counteracting motor dysfunctions and also displayed neuroprotective and anti-inflammatory effects in animal models of PD. Overall, we provide here an update of the current state of the art of these A(2A)R-based approaches that are under clinical study as agents devoted to alleviate PD symptoms.

  14. 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

  15. 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

  16. Adenosine receptors and muscarinic receptors cooperate in acetylcholine release modulation in the neuromuscular synapse.

    PubMed

    Santafe, M M; Priego, M; Obis, T; Garcia, N; Tomàs, M; Lanuza, M A; Tomàs, J

    2015-07-01

    Adenosine receptors (ARs) are present in the motor terminals at the mouse neuromuscular junction. ARs and the presynaptic muscarinic acetylcholine receptors (mAChRs) share the functional control of the neuromuscular junction. We analysed their mutual interaction in transmitter release modulation. In electrophysiological experiments with unaltered synaptic transmission (muscles paralysed by blocking the voltage-dependent sodium channel of the muscle cells with μ-conotoxin GIIIB), we found that: (i) a collaborative action between different AR subtypes reduced synaptic depression at a moderate activity level (40 Hz); (ii) at high activity levels (100 Hz), endogenous adenosine production in the synaptic cleft was sufficient to reduce depression through A1 -type receptors (A1 Rs) and A2 A-type receptors (A2 A Rs); (iii) when the non-metabolizable 2-chloroadenosine (CADO) agonist was used, both the quantal content and depression were reduced; (iv) the protective effect of CADO on depression was mediated by A1 Rs, whereas A2 A Rs seemed to modulate A1 Rs; (v) ARs and mAChRs absolutely depended upon each other for the modulation of evoked and spontaneous acetylcholine release in basal conditions and in experimental conditions with CADO stimulation; (vi) the purinergic and muscarinic mechanisms cooperated in the control of depression by sharing a common pathway although the purinergic control was more powerful than the muscarinic control; and (vii) the imbalance of the ARs created by using subtype-selective and non-selective inhibitory and stimulatory agents uncoupled protein kinase C from evoked transmitter release. In summary, ARs (A1 Rs, A2 A Rs) and mAChRs (M1 , M2 ) cooperated in the control of activity-dependent synaptic depression and may share a common protein kinase C pathway. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  17. Adenosine A2A receptor antagonists are potential antidepressants: evidence based on pharmacology and A2A receptor knockout mice

    PubMed Central

    Yacoubi, Malika El; Ledent, Catherine; Parmentier, Marc; Bertorelli, Rosalia; Ongini, Ennio; Costentin, Jean; Vaugeois, Jean-Marie

    2001-01-01

    Adenosine, an ubiquitous neuromodulator, and its analogues have been shown to produce ‘depressant' effects in animal models believed to be relevant to depressive disorders, while adenosine receptor antagonists have been found to reverse adenosine-mediated ‘depressant' effect. We have designed studies to assess whether adenosine A2A receptor antagonists, or genetic inactivation of the receptor would be effective in established screening procedures, such as tail suspension and forced swim tests, which are predictive of clinical antidepressant activity. Adenosine A2A receptor knockout mice were found to be less sensitive to ‘depressant' challenges than their wildtype littermates. Consistently, the adenosine A2A receptor blockers SCH 58261 (1 – 10 mg kg−1, i.p.) and KW 6002 (0.1 – 10 mg kg−1, p.o.) reduced the total immobility time in the tail suspension test. The efficacy of adenosine A2A receptor antagonists in reducing immobility time in the tail suspension test was confirmed and extended in two groups of mice. Specifically, SCH 58261 (1 – 10 mg kg−1) and ZM 241385 (15 – 60 mg kg−1) were effective in mice previously screened for having high immobility time, while SCH 58261 at 10 mg kg−1 reduced immobility of mice that were selectively bred for their spontaneous ‘helplessness' in this assay. Additional experiments were carried out using the forced swim test. SCH 58261 at 10 mg kg−1 reduced the immobility time by 61%, while KW 6002 decreased the total immobility time at the doses of 1 and 10 mg kg−1 by 75 and 79%, respectively. Administration of the dopamine D2 receptor antagonist haloperidol (50 – 200 μg kg−1 i.p.) prevented the antidepressant-like effects elicited by SCH 58261 (10 mg kg−1 i.p.) in forced swim test whereas it left unaltered its stimulant motor effects. In conclusion, these data support the hypothesis that A2A receptor antagonists prolong escape

  18. Progress towards novel adenosine receptor therapeutics gleaned from the recent patent literature.

    PubMed

    Press, Neil J; Fozard, John R

    2010-08-01

    The principle of treating disease with selective adenosine receptor ligands has been demonstrated with drugs on the market, while the lesser understood receptor subtypes are still being probed with new and drug-like pharmaceutical tools. The field of adenosine receptor research is, therefore, highly important as an emerging and proven point of intervention in disease. From 2008 to 2009, > 120 primary patent applications have claimed adenosine receptor ligands, which we analyze by applicant and target. Particularly significant disclosures are described in detail, paying particular attention to the biological data marshalled to support the case. The first published disclosure of new compounds, compound uses or drug targets is often in the patent literature, which can be difficult to trawl, interpret and verify as it is not subject to peer review. We have critically reviewed this area and share our conclusions regarding progress, trends and identification of early tool compounds or compounds of potential clinical significance ahead of peer-reviewed publication. Adenosine receptor research is a thriving field with continuing claims of exciting new compounds with high specificity and intriguing examples of new uses for such ligands.

  19. Could Adenosine Recognize its Receptors with a Stoichiometry Other than 1 : 1?

    PubMed

    Deganutti, Giuseppe; Salmaso, Veronica; Moro, Stefano

    2018-04-19

    One of the most largely accepted concepts in the G protein-coupled receptors (GPCRs) field is that the ligand, either agonist or antagonist, recognizes its receptor with a stoichiometry of 1 : 1. Recent experimental evidence, reporting ternary complexes formed by GPCR:orthosteric: allosteric ligands, has complicated the ligand-receptor 1 : 1 binding scenario. Molecular modeling simulations have been used to retrieve insights on the whole ligand-receptor recognition process, beyond information on the final bound state provided by experimental techniques. The simulation of adenosine binding pathways towards the A 2A adenosine receptor highlighted the presence of alternative binding sites (meta-binding sites) beside the canonical orthosteric one, mainly in proximity to the extracellular vestibule. In light of all these considerations, we investigated the possibility that a second molecule of adenosine engages its receptor when this is already in the holo form, generating a ternary complex with a stoichiometry of 2 : 1. Unexpectedly, supervised molecular dynamics (SuMD) simulations showed that the A 2A adenosine receptor could bind the second molecule of adenosine in one of the possible meta-binding sites as well as into its orthosteric site. The formation of this ternary complex, which favored the formation of the intracellular "ionic lock" between R102 (3.50) and E228 (6.30), could putatively be framed in the context of a negative allosteric regulation. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. 5'-Carbamoyl derivatives of 2'-C-methyl-purine nucleosides as selective A1 adenosine receptor agonists: affinity, efficacy, and selectivity for A1 receptor from different species.

    PubMed

    Cappellacci, Loredana; Franchetti, Palmarisa; Vita, Patrizia; Petrelli, Riccardo; Lavecchia, Antonio; Costa, Barbara; Spinetti, Francesca; Martini, Claudia; Klotz, Karl-Norbert; Grifantini, Mario

    2008-01-01

    A series of 5'-carbamoyl and 5'-thionocarbamoyl derivatives of 2'-C-methyl analogues of the A(1) adenosine receptor (A(1)AR) full agonists N(6)-cyclopentyladenosine (CPA), 2-chloro-N(6)-cyclopentyladenosine (CCPA), N(6)-[3-(R)-tetrahydrofuranyl]adenosine (tecadenoson), and 2-chloro analogue (2-Cl-tecadenoson) was synthesized and evaluated for their affinity for adenosine receptor subtypes from bovine, porcine, and human species. In the N(6)-cyclopentylamino series, the 5'-substituted derivatives showed a reduced affinity at the bovine A(1)AR compared to the parent compounds; however, the selectivity for A(1) versus A(2A) receptor was retained or increased. The corresponding N(6)-3-(R)-tetrahydrofuranylamino analogues displayed a very low affinity toward the bovine A(1)AR. The 5'-methylthionocarbamoyl derivative of 2'-Me-CCPA showed the best affinity at porcine A(1)AR with a K(i) value of 13 nM. At human AR subtypes tecadenoson derivatives showed 2.3- to 5-fold lower affinity at A(1)AR and very low affinity at the other subtypes (A(2A), A(2B), and A(3)) compared to the corresponding N(6)-cyclopentyl analogues. The 5'-carbamoyl and 5'-thionocarbamoyl derivatives of 2'-Me-CCPA 3, 4, 7 and tecadenoson derivative 12 were found to be partial A(1) agonists at the porcine receptor. Docking studies explained the lower affinity of N(6)-3-(R)-tetrahydrofuranyl-substituted compounds at bovine A(1)AR compared to that of N(6)-cyclopentyl analogues, showing that the oxygen of the tetrahydrofuranyl ring establishes unfavorable electrostatic interactions with the CO oxygen of Asn254. The low binding affinity of the 2'-C-methyl-N(6)-3-(R)-tetrahydrofuranyl adenosine analogues at human A(1)AR may be ascribed to the presence of unfavorable interactions between the hydrophilic tetrahydrofuranyl ring and the surrounding hydrophobic residues Leu250 (TM6) and Ile274 (TM7).

  1. 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 regionsmore » 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.« less

  2. Untangling dopamine-adenosine receptor-receptor assembly in experimental parkinsonism in rats

    PubMed Central

    Fernández-Dueñas, Víctor; Taura, Jaume J.; Cottet, Martin; Gómez-Soler, Maricel; López-Cano, Marc; Ledent, Catherine; Watanabe, Masahiko; Trinquet, Eric; Pin, Jean-Philippe; Luján, Rafael; Durroux, Thierry; Ciruela, Francisco

    2015-01-01

    Parkinson’s disease (PD) is a dopaminergic-related pathology in which functioning of the basal ganglia is altered. It has been postulated that a direct receptor-receptor interaction – i.e. of dopamine D2 receptor (D2R) with adenosine A2A receptor (A2AR) (forming D2R-A2AR oligomers) – finely regulates this brain area. Accordingly, elucidating whether the pathology prompts changes to these complexes could provide valuable information for the design of new PD therapies. Here, we first resolved a long-standing question concerning whether D2R-A2AR assembly occurs in native tissue: by means of different complementary experimental approaches (i.e. immunoelectron microscopy, proximity ligation assay and TR-FRET), we unambiguously identified native D2R-A2AR oligomers in rat striatum. Subsequently, we determined that, under pathological conditions (i.e. in a rat PD model), D2R-A2AR interaction was impaired. Collectively, these results provide definitive evidence for alteration of native D2R-A2AR oligomers in experimental parkinsonism, thus conferring the rationale for appropriate oligomer-based PD treatments. PMID:25398851

  3. Untangling dopamine-adenosine receptor-receptor assembly in experimental parkinsonism in rats.

    PubMed

    Fernández-Dueñas, Víctor; Taura, Jaume J; Cottet, Martin; Gómez-Soler, Maricel; López-Cano, Marc; Ledent, Catherine; Watanabe, Masahiko; Trinquet, Eric; Pin, Jean-Philippe; Luján, Rafael; Durroux, Thierry; Ciruela, Francisco

    2015-01-01

    Parkinson's disease (PD) is a dopaminergic-related pathology in which functioning of the basal ganglia is altered. It has been postulated that a direct receptor-receptor interaction - i.e. of dopamine D2 receptor (D2R) with adenosine A2A receptor (A2AR) (forming D2R-A2AR oligomers) - finely regulates this brain area. Accordingly, elucidating whether the pathology prompts changes to these complexes could provide valuable information for the design of new PD therapies. Here, we first resolved a long-standing question concerning whether D2R-A2AR assembly occurs in native tissue: by means of different complementary experimental approaches (i.e. immunoelectron microscopy, proximity ligation assay and TR-FRET), we unambiguously identified native D2R-A2AR oligomers in rat striatum. Subsequently, we determined that, under pathological conditions (i.e. in a rat PD model), D2R-A2AR interaction was impaired. Collectively, these results provide definitive evidence for alteration of native D2R-A2AR oligomers in experimental parkinsonism, thus conferring the rationale for appropriate oligomer-based PD treatments. © 2015. Published by The Company of Biologists Ltd.

  4. A ketogenic diet suppresses seizures in mice through adenosine A₁ receptors.

    PubMed

    Masino, Susan A; Li, Tianfu; Theofilas, Panos; Sandau, Ursula S; Ruskin, David N; Fredholm, Bertil B; Geiger, Jonathan D; Aronica, Eleonora; Boison, Detlev

    2011-07-01

    A ketogenic diet (KD) is a high-fat, low-carbohydrate metabolic regimen; its effectiveness in the treatment of refractory epilepsy suggests that the mechanisms underlying its anticonvulsive effects differ from those targeted by conventional antiepileptic drugs. Recently, KD and analogous metabolic strategies have shown therapeutic promise in other neurologic disorders, such as reducing brain injury, pain, and inflammation. Here, we have shown that KD can reduce seizures in mice by increasing activation of adenosine A1 receptors (A1Rs). When transgenic mice with spontaneous seizures caused by deficiency in adenosine metabolism or signaling were fed KD, seizures were nearly abolished if mice had intact A1Rs, were reduced if mice expressed reduced A1Rs, and were unaltered if mice lacked A1Rs. Seizures were restored by injecting either glucose (metabolic reversal) or an A1R antagonist (pharmacologic reversal). Western blot analysis demonstrated that the KD reduced adenosine kinase, the major adenosine-metabolizing enzyme. Importantly, hippocampal tissue resected from patients with medically intractable epilepsy demonstrated increased adenosine kinase. We therefore conclude that adenosine deficiency may be relevant to human epilepsy and that KD can reduce seizures by increasing A1R-mediated inhibition.

  5. Modulation of the dinucleotide receptor present in rat midbrain synaptosomes by adenosine and ATP

    PubMed Central

    Díaz-Hernández, Miguel; Pintor, Jesús; Miras-Portugal, M Teresa

    2000-01-01

    Diadenosine polyphosphates activate dinucleotide receptors in rat midbrain synaptic terminals. The agonist with highest affinity at this receptor, diadenosine pentaphosphate (Ap5A), elicits Ca2+ transients at concentrations ranging from 10−7 to 10−3 M with a single-phase curve and an EC50 value of 56.21±1.82 μM. Treatment of synaptosomal preparations with alkaline phosphatase (AP) changes the dose-response control curve into a biphasic one presenting two EC50 values of 6.47±1.25 nM and 11.16±0.83 μM respectively. The adenosine A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) reversed the biphasic concentration-response for Ap5A curve in the presence of AP, to a monophasic one with an EC50 value of 76.05±7.51 μM. The application of adenosine deaminase produced the same effect as DPCPX, the EC50 value for Ap5A, in the presence of AP being 18.62±4.03 μM. Activation of the adenosine A1 receptor by means of cyclohexyladenosine (CHA) shifted the dose response curve for Ap5A to the left, resulting in a monophasic curve with an EC50 of 5.01±0.02 pM. The destruction of extrasynaptosomal nucleotides by AP or the addition of pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), a broad P2 antagonist compound, enhance maximal effect of the Ap5A up to 55.6% on the dose response curve, thus suggesting a negative modulation by P2 receptors. In a summary, ATP and adenosine present at the extra-synaptosomal space, are relevant natural modulators of the dinucleotide receptor, via P2 and adenosine A1 receptors respectively. PMID:10807683

  6. Intrinsic A(1) adenosine receptor activation during ischemia or reperfusion improves recovery in mouse hearts.

    PubMed

    Peart, J; Headrick, J P

    2000-11-01

    We assessed the role of A(1) adenosine receptor (A(1)AR) activation by endogenous adenosine in the modulation of ischemic contracture and postischemic recovery in Langendorff-perfused mouse hearts subjected to 20 min of total ischemia and 30 min of reperfusion. In control hearts, the rate-pressure product (RPP) and first derivative of pressure development over time (+dP/dt) recovered to 57 +/- 3 and 58 +/- 3% of preischemia, respectively. Diastolic pressure remained elevated at 20 +/- 2 mmHg (compared with 3 +/- 1 mmHg preischemia). Interstitial adenosine, assessed by microdialysis, rose from approximately 0.3 to 1.9 microM during ischemia compared with approximately 15 microM in rat heart. Nonetheless, these levels will near maximally activate A(1)ARs on the basis of effects of exogenous adenosine and 2-chloroadenosine. Neither A(1)AR blockade with 200 nM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) during the ischemic period alone nor A(1)AR activation with 50 nM N(6)-cyclopentyladenosine altered rapidity or extent of ischemic contracture. However, ischemic DPCPX treatment significantly depressed postischemic recovery of RPP and +dP/dt (44 +/- 3 and 40 +/- 4% of preischemia, respectively). DPCPX treatment during the reperfusion period alone also reduced recovery of RPP and +dP/dt (to 44 +/- 2 and 47 +/- 2% of preischemia, respectively). These data indicate that 1) interstitial adenosine is lower in mouse versus rat myocardium during ischemia, 2) A(1)AR activation by endogenous adenosine or exogenous agonists does not modify ischemic contracture in murine myocardium, 3) A(1)AR activation by endogenous adenosine during ischemia attenuates postischemic stunning, and 4) A(1)AR activation by endogenous adenosine during the reperfusion period also improves postischemic contractile recovery.

  7. Inosine attenuates spontaneous activity in the rat neurogenic bladder through an A2B pathway

    PubMed Central

    Doyle, Claire; Cristofaro, Vivian; Sack, Bryan S.; Lukianov, Stefan N.; Schäfer, Mattias; Chung, Yeun Goo; Sullivan, Maryrose P.; Adam, Rosalyn M.

    2017-01-01

    Neurogenic detrusor overactivity (NDO) is among the most challenging complications of spinal cord injury (SCI). A recent report by us demonstrated an improvement in NDO in SCI rats following chronic systemic treatment with the purine nucleoside inosine. The objective of this study was to investigate the mechanism of action of inosine underlying improvement of NDO. Male Sprague-Dawley rats underwent complete spinal cord transection at T8. Inosine (1 mM) delivered intravesically to SCI rats during conscious cystometry significantly decreased the frequency of spontaneous non-voiding contractions. In isolated tissue assays, inosine (1 mM) significantly decreased the amplitude of spontaneous activity (SA) in SCI bladder muscle strips. This effect was prevented by a pan-adenosine receptor antagonist CGS15943, but not by A1 or A3 receptor antagonists. The A2A antagonist ZM241385 and A2B antagonist PSB603 prevented the effect of inosine. The effect of inosine was mimicked by the adenosine receptor agonist NECA and the A2B receptor agonist BAY60-6583. The inhibition of SA by inosine was not observed in the presence of the BK antagonist, iberiotoxin, but persisted in the presence of KATP and SK antagonists. These findings demonstrate that inosine acts via an A2B receptor-mediated pathway that impinges on specific potassium channel effectors. PMID:28294142

  8. Quantitative autoradiography of adenosine receptors in brains of chronic naltrexone-treated mice

    PubMed Central

    Bailey, Alexis; Hawkins, Rachel M; Hourani, Susanna M O; Kitchen, Ian

    2003-01-01

    Manipulation of μ opioid receptor expression either by chronic morphine treatment or by deletion of the gene encoding μ opioid receptors leads to changes in adenosine receptor expression. Chronic administration of the opioid receptor antagonist naltrexone leads to upregulation of μ receptor binding in the brain. To investigate if there are any compensatory alterations in adenosine systems in the brains of chronic naltrexone-treated mice, we carried out quantitative autoradiographic mapping of A1 and A2A adenosine receptors in the brains of mice treated for 1 week with naltrexone (8 mg−1 kg−1 day−1), administered subcutaneously via osmotic minipump. Adjacent coronal brain sections were cut from chronic saline- and naltrexone-treated mice for the determination of binding of [3H] D-Ala2-MePhe4-Gly-ol5 enkephalin ([3H] DAMGO), [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H] DPCPX) or [3H] 2-[p-(2-carbonylethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine ([3H] CGS21680) to μ, A1 and A2A receptors, respectively. A significant increase in μ and A1 receptor binding was detected in chronic naltrexone-treated brains. The changes in μ receptors were significant in several regions, but changes in A1 were relatively smaller but showed significant upregulation collectively. No significant change in A2A receptor binding was detected in chronic naltrexone-treated brains. The results show that blockade of opioid receptors causes upregulation of A1 receptors, but not A2A receptors, by as yet undefined mechanisms. PMID:12871838

  9. Potentiation of cytokine induction of group IIA phospholipase A(2) in rat mesangial cells by ATP and adenosine via the A2A adenosine receptor.

    PubMed

    Scholz-Pedretti, K; Pfeilschifter, J; Kaszkin, M

    2001-01-01

    1. In rat mesangial cells extracellular nucleotides were found to increase arachidonic acid release by a cytosolic phospholipase A(2) through the P2Y(2) purinergic receptor. 2. In this study we investigated the effects of ATP and UTP on interleukin-1ss (IL-1ss)-induced mRNA expression and activity of group IIA phospholipase A(2) (sPLA(2)-IIA) in rat mesangial cells. 3. Treatment of cells for 24 h with extracellular ATP potentiated IL-1ss-stimulated sPLA(2)-IIA induction, whereas UTP had no effect. 4. We obtained the following evidence that the P2Y(2) receptor is not involved in the potentiation of sPLA(2)-IIA induction: (i) ATP-gamma-S had no enhancing effect; (ii) suramin, a P(2) receptor antagonist, did not inhibit ATP-mediated potentiation; (iii) inhibition of degradation of extracellular nucleotides by the 5'-ectonucleotidase inhibitor AOPCP did not enhance sPLA(2)-IIA induction and (iv) adenosine deaminase treatment completely abolished the ATP-mediated potentiation of sPLA(2)-IIA induction. 5. In contrast, treatment of mesangial cells with adenosine or the A2A receptor agonist CGS 21680 mimicked the effects of ATP in enhancing IL-1ss-stimulated sPLA(2)-IIA induction, whereas the specific A2A receptor antagonist ZM 241385 completely abolished the potentiating effect of ATP or adenosine. 6. The protein kinase A inhibitor Rp-8-Br-cyclic AMPS dose-dependently inhibited the enhancing effect of ATP or adenosine indicating the participation of an adenosine receptor-mediated cyclic AMP-dependent signalling pathway. 7. These data indicate that ATP mediates proinflammatory long-term effects in rat mesangial cells via its degradation product adenosine through the A2A receptor resulting in potentiation of sPLA(2)-IIA induction.

  10. Glucose-induced intestinal vasodilation via adenosine A1 receptors requires nitric oxide but not K(+)(ATP) channels.

    PubMed

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

    2011-06-15

    Both nitric oxide (NO) and adenosine A1 receptor activation mediate microvascular vasodilation during intestinal glucose absorption. Our overall hypothesis is that adenosine triphosphate (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. Intravital videomicroscopy of the rat jejunum was used to record the vascular responses of inflow (termed 1A) arterioles, 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). 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. 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. Copyright

  11. C-nucleoside analogues of furanfurin as ligands to A1 adenosine receptors.

    PubMed

    Franchetti, P; Cappellacci, L; Marchetti, S; Martini, C; Costa, B; Varani, K; Borea, P A; Grifantini, M

    2000-09-01

    Furanfurin (2-beta-D-ribofuranosylfuran-4-carboxamide) derivatives and analogues were synthesized and their affinity for adenosine receptors was determined. The agonistic behavior of furanfurin against A1 receptors is preserved only when the furan ring is substituted with isosteric pentatomic ring systems such as oxazole, thiazole or thiophene, and the carboxamide group is unsubstituted. Replacement of the hydrogen atoms of the carboxamide group with alkyl, cycloalkyl or arylalkyl groups generates compounds endowed with moderate antagonistic activity.

  12. The A3 adenosine receptor attenuates the calcium rise triggered by NMDA receptors in retinal ganglion cells.

    PubMed

    Zhang, Mei; Hu, Huiling; Zhang, Xiulan; Lu, Wennan; Lim, Jason; Eysteinsson, Thor; Jacobson, Kenneth A; Laties, Alan M; Mitchell, Claire H

    2010-01-01

    The A(3) adenosine receptor is emerging as an important regulator of neuronal signaling, and in some situations receptor stimulation can limit excitability. As the NMDA receptor frequently contributes to neuronal excitability, this study examined whether A(3) receptor activation could alter the calcium rise accompanying NMDA receptor stimulation. Calcium levels were determined from fura-2 imaging of isolated rat retinal ganglion cells as these neurons possess both receptor types. Brief application of glutamate or NMDA led to repeatable and reversible elevations of intracellular calcium. The A(3) agonist Cl-IB-MECA reduced the response to both glutamate and NMDA. While adenosine mimicked the effect of Cl-IB-MECA, the A(3) receptor antagonist MRS 1191 impeded the block by adenosine, implicating a role for the A(3) receptor in response to the natural agonist. The A(1) receptor antagonist DPCPX provided additional inhibition, implying a contribution from both A(1) and A(3) adenosine receptors. The novel A(3) agonist MRS 3558 (1'S,2'R,3'S,4'R,5'S)-4-(2-chloro-6-(3-chlorobenzylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo [3.1.0] hexane-1-carboxamide and mixed A(1)/A(3) agonist MRS 3630 (1'S,2'R,3'S,4'R,5'S)-4-(2-chloro-6-(cyclopentylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo [3.1.0] hexane-1-carboxamide also inhibited the calcium rise induced by NMDA. Low levels of MRS 3558 were particularly effective, with an IC(50) of 400 pM. In all cases, A(3) receptor stimulation inhibited only 30-50% of the calcium rise. In summary, stimulation of the A(3) adenosine receptor by either endogenous or synthesized agonists can limit the calcium rise accompanying NMDA receptor activation. It remains to be determined if partial block of the calcium rise by A(3) agonists can modify downstream responses to NMDA receptor stimulation. 2009 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2016-09-01

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

  14. Activation of nicotinic ACh receptors with alpha4 subunits induces adenosine release at the rat carotid body.

    PubMed

    Conde, Sílvia V; Monteiro, Emília C

    2006-04-01

    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, DHbetaE and alpha-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 DHbetaE>mecamylamine>D-tubocurarine>alpha-bungarotoxin. The effect of the endogenous agonist, ACh, which was mimicked by nicotine, was antagonized by DHbetaE, 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 alpha4 and beta2 receptor subunits.

  15. Adenosine A1 Receptor-Mediated Attenuation of Reciprocal Dendro-Dendritic Inhibition in the Mouse Olfactory Bulb

    PubMed Central

    Schulz, Kristina; Rotermund, Natalie; Grzelka, Katarzyna; Benz, Jan; Lohr, Christian; Hirnet, Daniela

    2018-01-01

    It is well described that A1 adenosine receptors inhibit synaptic transmission at excitatory synapses in the brain, but the effect of adenosine on reciprocal synapses has not been studied so far. In the olfactory bulb, the majority of synapses are reciprocal dendro-dendritic synapses mediating recurrent inhibition. We studied the effect of A1 receptor activation on recurrent dendro-dendritic inhibition in mitral cells using whole-cell patch-clamp recordings. Adenosine reduced dendro-dendritic inhibition in wild-type, but not in A1 receptor knock-out mice. Both NMDA receptor-mediated and AMPA receptor-mediated dendro-dendritic inhibition were attenuated by adenosine, indicating that reciprocal synapses between mitral cells and granule cells as well as parvalbumin interneurons were targeted by A1 receptors. Adenosine reduced glutamatergic self-excitation and inhibited N-type and P/Q-type calcium currents, but not L-type calcium currents in mitral cells. Attenuated glutamate release, due to A1 receptor-mediated calcium channel inhibition, resulted in impaired dendro-dendritic inhibition. In behavioral tests we tested the ability of wild-type and A1 receptor knock-out mice to find a hidden piece of food. Knock-out mice were significantly faster in locating the food. Our results indicate that A1 adenosine receptors attenuates dendro-dendritic reciprocal inhibition and suggest that they affect odor information processing. PMID:29379418

  16. Involvement of A1 adenosine receptors in osmotic volume regulation of retinal glial cells in mice

    PubMed Central

    Wurm, Antje; Lipp, Stephan; Pannicke, Thomas; Linnertz, Regina; Färber, Katrin; Wiedemann, Peter; Reichenbach, Andreas

    2009-01-01

    Purpose Osmotic swelling of Müller glial cells has been suggested to contribute to retinal edema. We determined the role of adenosine signaling in the inhibition of Müller cell swelling in the murine retina. Methods The size of Müller cell somata was recorded before and during perfusion of retinal sections and isolated Müller cells with a hypoosmolar solution. Retinal tissues were freshly isolated from wild-type mice and mice deficient in A1 adenosine receptors (A1AR−/−), or cultured as whole-mounts for three days. The potassium conductance of Müller cells was recorded in isolated cells, and retinal slices were immunostained against Kir4.1. Results Hypotonic exposure for 4 min induced a swelling of Müller cell bodies in retinal slices from A1AR−/− mice but not wild-type mice. Pharmacological inhibition of A1 receptors or of the ecto-5′-nucleotidase induced hypoosmotic swelling of Müller cells from wild-type mice. Exogenous adenosine prevented the swelling of Müller cells from wild-type but not A1AR−/− mice. The antiinflammatory corticosteroid, triamcinolone acetonide, inhibited the swelling of Müller cells from wild-type mice; this effect was blocked by an antagonist of A1 receptors. The potassium conductance of Müller cells and the Kir4.1 immunolabeling of retinal slices were not different between A1AR−/− and wild-type mice, both in freshly isolated tissues and retinal organ cultures. Conclusions The data suggest that autocrine activation of A1 receptors by extracellularly generated adenosine mediates the volume homeostasis of Müller cells in the murine retina. The swelling-inhibitory effect of triamcinolone is mediated by enhancement of endogenous adenosine signaling. PMID:19756184

  17. Role of adenosine signaling in penile erection and erectile disorders.

    PubMed

    Phatarpekar, Prasad V; Wen, Jiaming; Xia, Yang

    2010-11-01

    Penile erection is a hemodynamic process, which results from increased flow and retention of blood in the penile organ due to the relaxation of smooth muscle cells. Adenosine, a physiological vasorelaxant, has been shown to be a modulator of penile erection. To summarize the research on the role of adenosine signaling in normal penile erection and erectile disorders. Evidence in the literature on the association between adenosine signaling and normal and abnormal penile erection, i.e., erectile dysfunction (ED) and priapism. The article reviews the literature on the role of endogenous and exogenous adenosine in normal penile erection, as well as in erectile disorders namely, ED and priapism. Adenosine has been shown to relax corpus cavernosum from various species including human in both in vivo and in vitro studies. Neuromodulatory role of adenosine in corpus cavernosum has also been demonstrated. Impaired adenosine signaling through A(2B) receptor causes partial resistance of corpus cavernosum, from men with organic ED, to adenosine-mediated relaxation. Increased level of adenosine has been shown to be a causative factor for priapism. Overall, the research reviewed here suggests a general role of exogenous and endogenous adenosine signaling in normal penile erection. From this perspective, it is not surprising that impaired adenosine signaling is associated with ED, and excessive adenosine signaling is associated with priapism. Adenosine signaling represents a potentially important diagnostic and therapeutic target for the treatment of ED and priapism. © 2009 International Society for Sexual Medicine.

  18. Role of Adenosine Signaling in Penile Erection and Erectile Disorders

    PubMed Central

    Phatarpekar, Prasad V.; Wen, Jiaming; Xia, Yang

    2010-01-01

    Introduction Penile erection is a hemodynamic process, which results from increased flow and retention of blood in the penile organ due to the relaxation of smooth muscle cells. Adenosine, a physiological vasorelaxant, has been shown to be a modulator of penile erection. Aim To summarize the research on the role of adenosine signaling in normal penile erection and erectile disorders. Main Outcome Measures Evidence in the literature on the association between adenosine signaling and normal and abnormal penile erection, i.e., erectile dysfunction (ED) and priapism. Methods The article reviews the literature on the role of endogenous and exogenous adenosine in normal penile erection, as well as in erectile disorders namely, ED and priapism. Results Adenosine has been shown to relax corpus cavernosum from various species including human in both in vivo and in vitro studies. Neuromodulatory role of adenosine in corpus cavernosum has also been demonstrated. Impaired adenosine signaling through A2B receptor causes partial resistance of corpus cavernosum, from men with organic ED, to adenosine-mediated relaxation. Increased level of adenosine has been shown to be a causative factor for priapism. Conclusion Overall, the research reviewed here suggests a general role of exogenous and endogenous adenosine signaling in normal penile erection. From this perspective, it is not surprising that impaired adenosine signaling is associated with ED, and excessive adenosine signaling is associated with priapism. Adenosine signaling represents a potentially important diagnostic and therapeutic target for the treatment of ED and priapism. PMID:19889148

  19. Pharmacological characterisation and inhibitory effects of (2R,3R,4S,5R)-2-(6-amino-2-{[(1S)-2-hydroxy-1-(phenylmethyl)ethyl]amino}-9H-purin-9-yl)-5-(2-ethyl-2H-tetrazol-5-yl)tetrahydro-3,4-furandiol, a novel ligand that demonstrates both adenosine A(2A) receptor agonist and adenosine A(3) receptor antagonist activity.

    PubMed

    Bevan, Nicola; Butchers, Peter R; Cousins, Rick; Coates, Jill; Edgar, Emma V; Morrison, Val; Sheehan, Michael J; Reeves, Julian; Wilson, David J

    2007-06-14

    The pharmacological properties of the novel ligand, (2R,3R,4S,5R)-2-(6-amino-2-{[(1S)-2-hydroxy-1-(phenylmethyl)ethyl]amino}-9H-purin-9-yl)-5-(2-ethyl-2H-tetrazol-5-yl)tetrahydro-3,4-furandiol (I), at the human adenosine receptors were investigated using Chinese hamster ovary cell lines recombinantly expressing these receptors. Functional studies were performed using a cyclic AMP-coupled reporter gene system. Binding studies were performed using membranes from these cells. The effects of ligand (I) were also determined on functional responses of human neutrophils and eosinophils. Ligand (I) had a high affinity for the adenosine A(2A) receptor (pKi 7.8+/-0.2) and was a potent agonist at this receptor (pEC(50) 9.0+/-0.2). Ligand (I) had a similar affinity for the adenosine A(3) receptor (pKi 7.8+/-0.1) but displayed no agonist activity, acting instead as a competitive antagonist (pA(2) 8.3+/-0.04). Ligand (I) had lower affinity for adenosine A(1) and A(2B) receptors (pKireceptors (pEC(50) 7.1 at both receptors). Ligand (I) was a potent inhibitor of the generation of reactive oxygen species from human neutrophils and eosinophils (pEC(50) 9.7+/-0.1 and 9.4+/-0.2 respectively). The inhibitory effect of ligand (I) on the release of reactive oxygen species from neutrophils was antagonised competitively by the adenosine A(2A) receptor antagonist 9-chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS15943) with a pA(2) value (10.03+/-0.44) consistent with an effect on adenosine A(2A) receptors. Ligand (I) also inhibited the release of granule proteins from neutrophils and eosinophils (pEC(50) 8.7 and 8.9 respectively), albeit less potently than as an inhibitor of reactive oxygen species generation. In summary, ligand (I) is a potent and selective agonist for the adenosine A(2A) receptor and a competitive antagonist at the adenosine A(3) receptor. Ligand (I) has potent anti-inflammatory effects on human

  20. The adenosine a2a receptor inhibits matrix-induced inflammation in a novel fashion.

    PubMed

    Scheibner, Kara A; Boodoo, Sada; Collins, Samuel; Black, Katharine E; Chan-Li, Yee; Zarek, Paul; Powell, Jonathan D; Horton, Maureen R

    2009-03-01

    Endogenous mediators within the inflammatory milieu play a critical role in directing the scope, duration, and resolution of inflammation. High-molecular-weight extracellular matrix hyaluronan (HA) helps to maintain homeostasis. During inflammation, hyaluronan is broken down into fragments that induce chemokines and cytokines, thereby augmenting the inflammatory response. Tissue-derived adenosine, released during inflammation, inhibits inflammation via the anti-inflammatory A2 adenosine receptor (A2aR). We demonstrate that adenosine modulates HA-induced gene expression via the A2aR. A2aR stimulation inhibits HA fragment-induced pro-fibrotic genes TNF-alpha, keratinocyte chemoattractant (KC), macrophage inflammatory protein (MIP)-2, and MIP-1alpha while simultaneously synergizing with hyaluronan fragments to up-regulate the TH1 cytokine IL-12. Interestingly, A2aR stimulation mediates these affects via the novel cAMP-activated guanine nucleotide exchange factor EPAC. In addition, A2aR-null mice are more susceptible to bleomycin-induced lung injury, consistent with a role for endogenous adenosine in inhibiting the inflammation that may lead to fibrosis. Indeed, the bleomycin treated A2aR-null mice demonstrate increased lung inflammation, HA accumulation, and histologic damage. Overall, our data elucidate the opposing roles of tissue-derived HA fragments and adenosine in regulating noninfectious lung inflammation and support the pursuit of A2aR agonists as a means of pharmacologically inhibiting inflammation that may lead to fibrosis.

  1. Prostacyclin regulates spinal nociceptive processing through cyclic adenosine monophosphate-induced translocation of glutamate receptors.

    PubMed

    Schuh, Claus Dieter; Brenneis, Christian; Zhang, Dong Dong; Angioni, Carlo; Schreiber, Yannick; Ferreiros-Bouzas, Nerea; Pierre, Sandra; Henke, Marina; Linke, Bona; Nüsing, Rolf; Scholich, Klaus; Geisslinger, Gerd

    2014-02-01

    Prostacyclin (PGI2) is known to be an important mediator of peripheral pain sensation (nociception) whereas little is known about its role in central sensitization. The levels of the stable PGI2-metabolite 6-keto-prostaglandin F1α (6-keto-PGF1α) and of prostaglandin E2 (PGE2) were measured in the dorsal horn with the use of mass spectrometry after peripheral inflammation. Expression of the prostanoid receptors was determined by immunohistology. Effects of prostacyclin receptor (IP) activation on spinal neurons were investigated with biochemical assays (cyclic adenosine monophosphate-, glutamate release-measurement, Western blot analysis) in embryonic cultures and adult spinal cord. The specific IP antagonist Cay10441 was applied intrathecally after zymosan-induced mechanical hyperalgesia in vivo. Peripheral inflammation caused a significant increase of the stable PGI2 metabolite 6-keto-PGF1α in the dorsal horn of wild-type mice (n = 5). IP was located on spinal neurons and did not colocalize with the prostaglandin E2 receptors EP2 or EP4. The selective IP-agonist cicaprost increased cyclic adenosine monophosphate synthesis in spinal cultures from wild-type but not from IP-deficient mice (n = 5-10). The combination of fluorescence-resonance-energy transfer-based cyclic adenosine monophosphate imaging and calcium imaging showed a cicaprost-induced cyclic adenosine monophosphate synthesis in spinal cord neurons (n = 5-6). Fittingly, IP activation increased glutamate release from acute spinal cord sections of adult mice (n = 13-58). Cicaprost, but not agonists for EP2 and EP4, induced protein kinase A-dependent phosphorylation of the GluR1 subunit and its translocation to the membrane. Accordingly, intrathecal administration of the IP receptor antagonist Cay10441 had an antinociceptive effect (n = 8-11). Spinal prostacyclin synthesis during early inflammation causes the recruitment of GluR1 receptors to membrane fractions, thereby augmenting the onset of central

  2. Ribose-modified nucleosides as ligands for adenosine receptors: synthesis, conformational analysis, and biological evaluation of 1'-C-methyl adenosine analogues.

    PubMed

    Cappellacci, Loredana; Barboni, Grazia; Palmieri, Micaela; Pasqualini, Michela; Grifantini, Mario; Costa, Barbara; Martini, Claudia; Franchetti, Palmarisa

    2002-03-14

    1'-C-Methyl analogues of adenosine and selective adenosine A(1) receptor agonists, such as N-[(1R)-1-methyl-2-phenylethyl]adenosine ((R)-PIA) and N(6)-cyclopentyladenosine, were synthesized to further investigate the subdomain that binds the ribose moiety. Binding affinities of these new compounds at A(1) and A(2A) receptors in rat brain membranes and at A(3) in rat testis membranes were determined and compared. It was found that the 1'-C-methyl modification in adenosine resulted in a decrease of affinity, particularly at A(1) and A(2A) receptors. When this modification was combined with N(6) substitutions with groups that induce high potency and selectivity at A(1) receptors, the high affinity was in part restored and the selectivity was increased. The most potent compound proved to be the 1'-C-methyl analogue of (R)-PIA with a K(i) of 23 nM for the displacement of [(3)H]CHA binding from rat brain A(1) receptors and a > 435-fold selectivity over A(2A) receptors. In functional assays, these compounds inhibited forskolin-stimulated adenylate cyclase with IC(50) values ranging from 0.065 to 3.4 microM, acting as full agonists. Conformational analysis based on vicinal protonminus signproton J-coupling constants and molecular mechanics calculations using the MM2 force field proved that the methyl group on C1' in adenosine has a pronounced impact on the furanose conformation by driving its conformational equilibrium toward the north, gamma+, syn form.

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

    PubMed Central

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

    1994-01-01

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

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

    PubMed

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

    1994-12-01

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

  5. Interactions between Calmodulin, Adenosine A2A, and Dopamine D2 Receptors*

    PubMed Central

    Navarro, Gemma; Aymerich, Marisol S.; Marcellino, Daniel; Cortés, Antoni; Casadó, Vicent; Mallol, Josefa; Canela, Enric I.; Agnati, Luigi; Woods, Amina S.; Fuxe, Kjell; Lluís, Carmen; Lanciego, Jose Luis; Ferré, Sergi; Franco, Rafael

    2009-01-01

    The Ca2+-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors, including the dopamine D2 receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D2 receptor, within an Arg-rich epitope that is also involved in the binding to Gi/o proteins and to the adenosine A2A receptor, with the formation of A2A-D2 receptor heteromers. In the present work, by using proteomics and bioluminescence resonance energy transfer (BRET) techniques, we provide evidence for the binding of CaM to the A2A receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A2A-D2 receptor oligomerization. BRET competition experiments indicated that, in the A2A-D2 receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A2A receptor. Furthermore, Ca2+ was found to induce conformational changes in the CaM-A2A-D2 receptor oligomer and to selectively modulate A2A and D2 receptor-mediated MAPK signaling in the A2A-D2 receptor heteromer. These results may have implications for basal ganglia disorders, since A2A-D2 receptor heteromers are being considered as a target for anti-parkinsonian agents. PMID:19632986

  6. Excessive penile norepinephrine level underlies impaired erectile function in adenosine A1 receptor deficient mice.

    PubMed

    Ning, Chen; Qi, Lin; Wen, Jiaming; Zhang, Yujin; Zhang, Weiru; Wang, Wei; Blackburn, Michael; Kellems, Rodney; Xia, Yang

    2012-10-01

    Penile erection is a complex neurovascular physiological event controlled by multiple factors and signaling pathways. A considerable amount of evidence indicates that adenosine plays a significant role in cavernosal smooth muscle relaxation. However, the specific role of adenosine and its receptors in erectile physiology and pathology is not fully understood. To determine the role of the adenosine A1 receptor (ADORA1) in penile erection. Adenosine A1 receptor deficient (Adora1-/-) mice and aged-matched wild-type (WT) mice were utilized. We evaluated the in vivo erectile function by measuring the intracavernosal pressure (ICP) in response to cavernous nerve stimulation (CNS). Enzyme-linked immunosorbent assay was used to measure the norepinephrine (NE) plasma concentration in the corpus cavernosum and systemic circulation. We also evaluated the myosin light chain phosphorylation (p-MLC) in penile tissue pre- and post-CNS. The main outcome measurement of this research was the evaluation of in vivo erectile response to CNS by measuring the ICP in Adora1-/- mice and WT mice and to identify the localization and specific neuron types of ADORA1 expression by dual immunostaining and immunofluorescence co-localization. In vivo, both the ratio of CNS-induced Maximum ICP to mean arterial pressure and CNS-induced slope in Adora1-/- mice were significantly lower than WT mice. At the cellular level in penile tissue, we determined that ADORA1 was highly abundant in neuronal cells. During penile erection, Adora1-/- mice exhibited a higher level of NE plasma concentration in the penis than WT mice. And WT mice had a significantly greater reduction in p-MLC compared to Adora1-/- mice. Our results show that ADORA1 is enriched on neuron cells where it functions to control NE release. Activation of this receptor during penile erection results in reduced NE release and reduced cavernosal smooth muscle contraction, therefore facilitating penile erection. © 2012 International Society for

  7. Caffeine, Adenosine Receptors and Estrogen in Toxin Models of Parkinson’s Disease

    DTIC Science & Technology

    2010-10-30

    van Calker, D., Muller, M., Hamprecht, B., 1979. Adenosine regulates via two different types of receptors, the accumulation of cyclic AMP in cultured...calcium/calmodulin­ dependent protein kinase type II/IV; cAMP, cyclic AMP ; CREB, cAMP response element-binding protein; Kþ, potassium channel; DARPP-32...patients. Neurology, 52, 1916. Kulisevsky, J., Barbanoj, M., Gironell, A., Antonijoan, R., Casas , M., & Pascual-Sedano, B. (2002). A double-blind

  8. 2-n-Butyl-9-methyl-8-[1,2,3]triazol-2-yl-9H-purin-6-ylamine and analogues as A2A adenosine receptor antagonists. Design, synthesis, and pharmacological characterization.

    PubMed

    Minetti, Patrizia; Tinti, Maria Ornella; Carminati, Paolo; Castorina, Massimo; Di Cesare, Maria Assunta; Di Serio, Stefano; Gallo, Grazia; Ghirardi, Orlando; Giorgi, Fabrizio; Giorgi, Luca; Piersanti, Giovanni; Bartoccini, Francesca; Tarzia, Giorgio

    2005-11-03

    Two types of adenosine receptor ligands were designed, i.e., 9H-purine and 1H-imidazo[4,5-c]pyridines, to obtain selective A(2A) antagonists, and we report here their synthesis and binding affinities for the four adenosine receptor subtypes A(1), A(2A), A(2B) and A(3). The design was carried out on the basis of the molecular modeling of a number of potent adenosine receptor antagonists described in the literature. Three compounds (25b-d) showed an interesting affinity and selectivity for the A(2A) subtype. One of them, i.e., ST1535 (2-n-butyl-9-methyl-8-[1,2,3]triazol-2-yl-9H-purin-6-ylamine, 25b) (K(i) A(2A) = 6.6 nM, K(i) A(1)/A(2A) = 12; K(i) A(2B)/A(2A) = 58; K(i) A(3)/A(2A) > 160), was selected for in vivo study and shown to induce a dose-related increase in locomotor activity, suggestive of an A(2A) antagonist type of activity.

  9. Mast cell degranulation following adenosine A3 receptor activation in rats.

    PubMed

    Fozard, J R; Pfannkuche, H J; Schuurman, H J

    1996-03-18

    The present studies were carried out to provide further evidence for the hypothesis that the hypotensive response to adenosine A3 receptor activation in the anaesthetized rat involves mediator release from mast cells. Male Sprague-Dawley rats were anaesthetized and given just supramaximal hypotensive doses of either the non-selective A3 receptor agonist, N6(-2)-(4-aminophenyl)ethyladenosine (APNEA: 100 micrograms/kg, preceded by the A1/A2 receptor antagonist, 8-p-(sulphophenyl)theophylline, to "isolate' the A3 receptor-mediated component of the response), the mast cell degranulating agent, compound 48/80 (300 micrograms/kg) or the vehicle for APNEA, intravenously. Blood was withdrawn from a carotid artery between 2 and 10 min after the injection and plasma and serum histamine concentrations measured. Samples of connective tissue (surrounding the abdominal musculature), thymus, mesenteric lymph node, kidney, skin and diaphragm were removed for histological analysis. The plasma and serum histamine concentrations were markedly and significantly higher in the APNEA- or compound 48/80-treated animals compared to vehicle-treated controls. Consistent with this, a substantially greater proportion of mast cells was seen to be undergoing degranulation in all tissues removed from animals treated with APNEA or compound 48/80 compared to those from rats treated with vehicle. Thus, adenosine A3 receptor activation results in rapid mast cell degranulation in the anaesthetized rat. The data provide further evidence of a key role for the mast cell in adenosine A3 receptor-mediated hypotension in this species.

  10. 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

  11. The A1 adenosine receptor as a new player in microglia physiology.

    PubMed

    Luongo, L; Guida, F; Imperatore, R; Napolitano, F; Gatta, L; Cristino, L; Giordano, C; Siniscalco, D; Di Marzo, V; Bellini, G; Petrelli, R; Cappellacci, L; Usiello, A; de Novellis, V; Rossi, F; Maione, S

    2014-01-01

    The purinergic system is highly involved in the regulation of microglial physiological processes. In addition to the accepted roles for the P2 X4,7 and P2 Y12 receptors activated by adenosine triphosphate (ATP) and adenosine diphosphate, respectively, recent evidence suggests a role for the adenosine A2A receptor in microglial cytoskeletal rearrangements. However, the expression and function of adenosine A1 receptor (A1AR) in microglia is still unclear. Several reports have demonstrated possible expression of A1AR in microglia, but a new study has refuted such evidence. In this study, we investigated the presence and function of A1AR in microglia using biomolecular techniques, live microscopy, live calcium imaging, and in vivo electrophysiological approaches. The aim of this study was to clarify the expression of A1AR in microglia and to highlight its possible roles. We found that microglia express A1AR and that it is highly upregulated upon ATP treatment. Moreover, we observed that selective stimulation of A1AR inhibits the morphological activation of microglia, possibly by suppressing the Ca(2+) influx induced by ATP treatment. Finally, we recorded the spontaneous and evoked activity of spinal nociceptive-specific neuron before and after application of resting or ATP-treated microglia, with or without preincubation with a selective A1AR agonist. We found that the microglial cells, pretreated with the A1AR agonist, exhibit lower capability to facilitate the nociceptive neurons, as compared with the cells treated with ATP alone. Copyright © 2013 Wiley Periodicals, Inc.

  12. Adenosine A(2A) receptors are necessary and sufficient to trigger memory impairment in adult mice.

    PubMed

    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

    2015-08-01

    Caffeine (a non-selective adenosine receptor antagonist) prevents memory deficits in aging and Alzheimer's disease, an effect mimicked by adenosine A2 A 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. We determined whether A2 A receptors are necessary for the emergence of memory impairments induced by scopolamine and whether A2 A 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. 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 A2 A 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 A2 A 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. These results show that A2 A 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. © 2015 The British Pharmacological Society.

  13. 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

  14. Effects of adenosine receptor agonist on the rocuroniuminduced neuromuscular block and sugammadex-induced recovery.

    PubMed

    Kim, Yong Beom; Lee, Sangseok; Choi, Hey Ran; In, Junyong; Chang, Young Jin; Kim, Ha Jung; Ro, Young Jin; Yang, Hong-Seuk

    2018-04-25

    Several types of receptors are found at neuromuscular presynaptic membranes. Presynaptic inhibitory A1 and facilitatory A2A receptors mediate different modulatory functions on acetylcholine release. This study investigated whether adenosine A1 receptor agonist contributes to the first twitch tension (T1) of train-of-four (TOF) stimulation depression and TOF fade during rocuronium-induced neuromuscular blockade, and sugammadex-induced recovery. Phrenic nerve-diaphragm tissues were obtained from 30 adult Sprague-Dawley rats. Each tissue specimen was randomly allocated to either control group or 2-chloroadenosine (CADO, 10 μM) group. One hour of reaction time was allowed before initiating main experimental data collection. Loading and boost doses of rocuronium were sequentially administered until > 95% depression of the T1 was achieved. After confirming that there was no T1 twitch tension response, 15 min of resting time was allowed, after which sugammadex was administered. Recovery profiles (T1, TOF ratio [TOFR], and recovery index) were collected for 1 h and compared between groups. There were statistically significant differences on amount of rocuronium (actually used during experiment), TOFR changes during concentration-response of rocuronium (P = 0.04), and recovery profiles (P < 0.01) of CADO group comparing with the control group. However, at the initial phase of this experiment, dose-response of rocuronium in each group demonstrated no statistically significant differences (P = 0.12). The adenosine A1 receptor agonist (CADO) influenced the TOFR and the recovery profile. After activating adenosine receptor, sugammadex-induced recovery from rocuronium-induced neuromuscular block was delayed.

  15. Adenosine A2A Receptor Deletion Blocks the Beneficial Effects of Lactobacillus reuteri in Regulatory T-Deficient Scurfy Mice

    PubMed Central

    He, Baokun; Hoang, Thomas K.; Tran, Dat Q.; Rhoads, Jon Marc; Liu, Yuying

    2017-01-01

    The lack of a functional Foxp3 transcription factor and regulatory T (Treg) cells causes lethal, CD4+ T cell-driven autoimmune diseases in scurfy (SF) mice and humans. Recent studies have shown that adenosine A2A receptor activation limits inflammation and tissue damage, thereby playing an anti-inflammatory role. However, the role of the adenosine A2A receptor in the development of disease in SF mice remains unclear. Using a genetic approach, we found that adenosine A2A receptor deletion in SF mice (SF⋅A2A-/-) does not affect early life events, the development of a lymphoproliferative disorder, or hyper-production of pro-inflammatory cytokines seen in the Treg-deficiency state. As shown previously, Lactobacillus reuteri DSM 17938 treatment prolonged survival and reduced multiorgan inflammation in SF mice. In marked contrast, A2A receptor deletion completely blocked these beneficial effects of L. reuteri in SF mice. Altogether, these results suggest that although absence of the adenosine A2A receptor does not affect the development of disease in SF mice, it plays a critical role in the immunomodulation by L. reuteri in Treg-deficiency disease. The adenosine A2A receptor and its activation may have a role in treating other Treg dysfunction-mediated autoimmune diseases. PMID:29270168

  16. Reduced adenosine A2a receptor-mediated efferent arteriolar vasodilation contributes to diabetes-induced glomerular hyperfiltration.

    PubMed

    Persson, Patrik; Hansell, Peter; Palm, Fredrik

    2015-01-01

    Diabetes is associated with increased risk for development of kidney disease, and an increased glomerular filtration rate is an early indication of altered kidney function. Here we determine whether reduced adenosine A2a receptor-mediated vasodilation of the efferent arteriole contributes to the increased glomerular filtration rate in diabetes. The glomerular filtration rate, renal blood flow, and proximal tubular stop flow pressure were investigated in control and streptozotocin-diabetic rats during baseline and after administration of the adenosine A2a receptor antagonist ZM241385 or the adenosine A2a receptor agonist CGS21680. The diabetes-induced glomerular hyperfiltration was reduced by 24% following A2a receptor stimulation but was unaffected by A2a receptor inhibition. Contrarily, glomerular filtration rate in controls increased by 22% after A2a receptor inhibition and was unaffected by A2a stimulation. The increased glomerular filtration rate after A2a receptor inhibition in controls and decreased glomerular filtration rate after A2a receptor activation in diabetics were caused by increased and decreased stop flow pressure, respectively. None of the interventions affected renal blood flow. Thus, the normal adenosine A2a receptor-mediated tonic vasodilation of efferent arterioles is abolished in the diabetic kidney. This causes increased efferent arteriolar resistance resulting in increased filtration fraction and hyperfiltration.

  17. Caffeine acts via A1 adenosine receptors to disrupt embryonic cardiac function.

    PubMed

    Buscariollo, Daniela L; Breuer, Gregory A; Wendler, Christopher C; Rivkees, Scott A

    2011-01-01

    Evidence suggests that adenosine acts via cardiac A1 adenosine receptors (A1ARs) to protect embryos against hypoxia. During embryogenesis, A1ARs are the dominant regulator of heart rate, and A1AR activation reduces heart rate. Adenosine action is inhibited by caffeine, which is widely consumed during pregnancy. In this study, we tested the hypothesis that caffeine influences developing embryos by altering cardiac function. Effects of caffeine and adenosine receptor-selective antagonists on heart rate were studied in vitro using whole murine embryos at E9.5 and isolated hearts at E12.5. Embryos were examined in room air (21% O(2)) or hypoxic (2% O(2)) conditions. Hypoxia decreased heart rates of E9.5 embryos by 15.8% and in E12.5 isolated hearts by 27.1%. In room air, caffeine (200 µM) had no effect on E9.5 heart rates; however, caffeine increased heart rates at E12.5 by 37.7%. Caffeine abolished hypoxia-mediated bradycardia at E9.5 and blunted hypoxia-mediated bradycardia at E12.5. Real-time PCR analysis of RNA from isolated E9.5 and E12.5 hearts showed that A1AR and A2aAR genes were expressed at both ages. Treatment with adenosine receptor-selective antagonists revealed that SCH-58261 (A2aAR-specific antagonist) had no affects on heart function, whereas DPCPX (A1AR-specific antagonist) had effects similar to caffeine treatment at E9.5 and E12.5. At E12.5, embryonic hearts lacking A1AR expression (A1AR-/-) had elevated heart rates compared to A1AR+/- littermates, A1AR-/- heart rates failed to decrease to levels comparable to those of controls. Caffeine did not significantly affect heart rates of A1AR-/- embryos. These data show that caffeine alters embryonic cardiac function and disrupts the normal cardiac response to hypoxia through blockade of A1AR action. Our results raise concern for caffeine exposure during embryogenesis, particularly in pregnancies with increased risk of embryonic hypoxia.

  18. Caffeine Acts via A1 Adenosine Receptors to Disrupt Embryonic Cardiac Function

    PubMed Central

    Buscariollo, Daniela L.; Breuer, Gregory A.; Wendler, Christopher C.; Rivkees, Scott A.

    2011-01-01

    Background Evidence suggests that adenosine acts via cardiac A1 adenosine receptors (A1ARs) to protect embryos against hypoxia. During embryogenesis, A1ARs are the dominant regulator of heart rate, and A1AR activation reduces heart rate. Adenosine action is inhibited by caffeine, which is widely consumed during pregnancy. In this study, we tested the hypothesis that caffeine influences developing embryos by altering cardiac function. Methodology/Principal Findings Effects of caffeine and adenosine receptor-selective antagonists on heart rate were studied in vitro using whole murine embryos at E9.5 and isolated hearts at E12.5. Embryos were examined in room air (21% O2) or hypoxic (2% O2) conditions. Hypoxia decreased heart rates of E9.5 embryos by 15.8% and in E12.5 isolated hearts by 27.1%. In room air, caffeine (200 µM) had no effect on E9.5 heart rates; however, caffeine increased heart rates at E12.5 by 37.7%. Caffeine abolished hypoxia-mediated bradycardia at E9.5 and blunted hypoxia-mediated bradycardia at E12.5. Real-time PCR analysis of RNA from isolated E9.5 and E12.5 hearts showed that A1AR and A2aAR genes were expressed at both ages. Treatment with adenosine receptor-selective antagonists revealed that SCH-58261 (A2aAR-specific antagonist) had no affects on heart function, whereas DPCPX (A1AR-specific antagonist) had effects similar to caffeine treatment at E9.5 and E12.5. At E12.5, embryonic hearts lacking A1AR expression (A1AR−/−) had elevated heart rates compared to A1AR+/− littermates, A1AR−/− heart rates failed to decrease to levels comparable to those of controls. Caffeine did not significantly affect heart rates of A1AR−/− embryos. Conclusions/Significance These data show that caffeine alters embryonic cardiac function and disrupts the normal cardiac response to hypoxia through blockade of A1AR action. Our results raise concern for caffeine exposure during embryogenesis, particularly in pregnancies with increased risk of embryonic

  19. Ecto-5′-Nucleotidase (CD73)-Mediated Formation of Adenosine Is Critical for the Striatal Adenosine A2A Receptor Functions

    PubMed Central

    Augusto, Elisabete; Matos, Marco; Sévigny, Jean; El-Tayeb, Ali; Bynoe, Margaret S.; Müller, Christa E.

    2013-01-01

    Adenosine is a neuromodulator acting through inhibitory A1 receptors (A1Rs) and facilitatory A2ARs, which have similar affinities for adenosine. It has been shown that the activity of intracellular adenosine kinase preferentially controls the activation of A1Rs, but the source of the adenosine activating A2ARs is unknown. We now show that ecto-5′-nucleotidase (CD73), the major enzyme able to convert extracellular AMP into adenosine, colocalizes with A2ARs in the basal ganglia. In addition to astrocytes, striatal CD73 is prominently localized to postsynaptic sites. Notably, CD73 coimmunoprecipitated with A2ARs and proximity ligation assays confirmed the close proximity of CD73 and A2ARs in the striatum. Accordingly, the cAMP formation in synaptosomes as well as the hypolocomotion induced by a novel A2AR prodrug that requires CD73 metabolization to activate A2ARs were observed in wild-type mice, but not in CD73 knock-out (KO) mice or A2AR KO mice. Moreover, CD73 KO mice displayed increased working memory performance and a blunted amphetamine-induced sensitization, mimicking the phenotype of global or forebrain-A2AR KO mice, as well as upon pharmacological A2AR blockade. These results show that CD73-mediated formation of extracellular adenosine is responsible for the activation of striatal A2AR function. This study points to CD73 as a new target that can fine-tune A2AR activity, and a novel therapeutic target to manipulate A2AR-mediated control of striatal function and neurodegeneration. PMID:23843511

  20. Optimization of Adenosine 5′-Carboxamide Derivatives as Adenosine Receptor Agonists Using Structure-Based Ligand Design and Fragment Screening

    PubMed Central

    Tosh, Dilip K.; Phan, Khai; Gao, Zhan-Guo; Gakh, Andrei A.; Xu, Fei; Deflorian, Francesca; Abagyan, Ruben; Stevens, Raymond C.; Jacobson, Kenneth A.; Katritch, Vsevolod

    2012-01-01

    Structures of G protein-coupled receptors (GPCRs) have a proven utility in the discovery of new antagonists and inverse agonists modulating signaling of this important family of clinical targets. Applicability of active-state GPCR structures to virtual screening and rational optimization of agonists, however, remains to be assessed. In this study of adenosine 5′ derivatives, we evaluated the performance of an agonist-bound A2A adenosine receptor (AR) structure in retrieval of known agonists and then employed the structure to screen for new fragments optimally fitting the corresponding subpocket. Biochemical and functional assays demonstrate high affinity of new derivatives that include polar heterocycles. The binding models also explain modest selectivity gain for some substituents toward the closely related A1AR subtype and the modified agonist efficacy of some of these ligands. The study suggests further applicability of in silico fragment screening to rational lead optimization in GPCRs. PMID:22486652

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

    PubMed

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

    1989-09-01

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

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

    PubMed Central

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

    2012-01-01

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

  3. Adenosine A(1) receptors in mouse pontine reticular formation depress breathing, increase anesthesia recovery time, and decrease acetylcholine release.

    PubMed

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

    2013-02-01

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

  4. Roles of the Adenosine Receptor and CD73 in the Regulatory Effect of γδ T Cells

    PubMed Central

    Liang, Dongchun; Zuo, Aijun; Shao, Hui; Chen, Mingjiazi; Kaplan, Henry J.; Sun, Deming

    2014-01-01

    The adenosine A2A receptor (A2AR), the main functional adenosine receptor on murine T cells, plays a unique role in the attenuation of inflammation and tissue damage in vivo. Here, we showed that, of the immune cell types tested, activated γδ T cells expressed the highest levels of A2AR mRNA and that A2AR ligation inhibited αβ T cell activation, but enhanced γδ T cell activation. We also showed that the inhibitory effect of an adenosine receptor agonist on autoreactive T cells was prevented by addition of a low percentage of activated γδ T cells. Furthermore, compared to resting cells, activated γδ T cells expressed significantly lower levels of CD73, an enzyme involved in the generation of extracellular adenosine. Exogenous AMP had a significant inhibitory effect on autoreactive T cell responses, but only in the presence of CD73+ γδ T cells, and this effect was abolished by a CD73 inhibitor. Our results show that expression of increased amounts of A2AR allows γδ T cells to bind adenosine and thereby attenuate its suppressive effect, while decreased expression of CD73 results in less generation of adenosine in the inflammatory site. Together, these events allow activated γδ T cells to acquire increased proinflammatory activity, leading to augmented autoimmune responses. PMID:25268760

  5. Kinetic Aspects of the Interaction between Ligand and G Protein-Coupled Receptor: The Case of the Adenosine Receptors.

    PubMed

    Guo, Dong; Heitman, Laura H; IJzerman, Adriaan P

    2017-01-11

    Ligand-receptor binding kinetics is an emerging topic in the drug research community. Over the past years, medicinal chemistry approaches from a kinetic perspective have been increasingly applied to G protein-coupled receptors including the adenosine receptors (AR), which are involved in a plethora of physiological and pathological conditions. The study of ligand-AR binding kinetics offers room for detailed structure-kinetics relationships next to more traditional structure-activity relationships. Their combination may facilitate the triage of candidate compounds in hit-to-lead campaigns. Furthermore, kinetic studies also help in understanding AR allosterism. Allosteric modulation may yield a change in the activity and conformation of a receptor resulting from the binding of a compound at a site distinct from where the endogenous agonist adenosine binds. Hence, in this Review, we summarize available data and evidence for the binding kinetics of orthosteric and allosteric AR ligands. We hope this Review will raise awareness to consider the kinetic aspects of drug-target interactions on both ARs and other drug targets.

  6. 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.

  7. Adenosine receptors as markers of brain iron deficiency: Implications for Restless Legs Syndrome.

    PubMed

    Quiroz, César; Gulyani, Seema; Ruiqian, Wan; Bonaventura, Jordi; Cutler, Roy; Pearson, Virginia; Allen, Richard P; Earley, Christopher J; Mattson, Mark P; Ferré, Sergi

    2016-12-01

    Deficits of sensorimotor integration with periodic limb movements during sleep (PLMS) and hyperarousal and sleep disturbances in Restless Legs Syndrome (RLS) constitute two pathophysiologically distinct but interrelated clinical phenomena, which seem to depend mostly on alterations in dopaminergic and glutamatergic neurotransmission, respectively. Brain iron deficiency is considered as a main pathogenetic mechanism in RLS. Rodents with brain iron deficiency represent a valuable pathophysiological model of RLS, although they do not display motor disturbances. Nevertheless, they develop the main neurochemical dopaminergic changes found in RLS, such as decrease in striatal dopamine D 2 receptor density. On the other hand, brain iron deficient mice exhibit the characteristic pattern of hyperarousal in RLS, providing a tool to find the link between brain iron deficiency and sleep disturbances in RLS. The present study provides evidence for a role of the endogenous sleep-promoting factor adenosine. Three different experimental preparations, long-term (22 weeks) severe or moderate iron-deficient (ID) diets (3- or 7-ppm iron diet) in mice and short-term (3 weeks) severe ID diet (3-ppm iron diet) in rats, demonstrated a significant downregulation (Western blotting in mouse and radioligand binding saturation experiments in rat brain tissue) of adenosine A 1 receptors (A1R) in the cortex and striatum, concomitant to striatal D2R downregulation. On the other hand, the previously reported upregulation of adenosine A 2A receptors (A2AR) was only observed with severe ID in both mice and rats. The results suggest a key role for A1R downregulation in the PLMS and hyperarousal in RLS. Published by Elsevier Ltd.

  8. Adenosine receptors as markers of brain iron deficiency: Implications for Restless Legs Syndrome

    PubMed Central

    Quiroz, César; Gulyani, Seema; Ruiqian, Wan; Bonaventura, Jordi; Cutler, Roy; Pearson, Virginia; Allen, Richard P.; Earley, Christopher J.; Mattson, Mark P.; Ferré, Sergi

    2016-01-01

    Deficits of sensorimotor integration with periodic limb movements during sleep (PLMS) and hyperarousal and sleep disturbances in Restless Legs Syndrome (RLS) constitute two pathophysiologically distinct but interrelated clinical phenomena, which seem to depend mostly on alterations in dopaminergic and glutamatergic neurotransmission, respectively. Brain iron deficiency is considered as a main pathogenetic mechanism in RLS. Rodents with brain iron deficiency represent a valuable pathophysiological model of RLS, although they do not display motor disturbances. Nevertheless, they develop the main neurochemical dopaminergic changes found in RLS, such as decrease in striatal dopamine D2 receptor density. On the other hand, brain iron deficient mice exhibit the characteristic pattern of hyperarousal in RLS, providing a tool to find the link between brain iron deficiency and sleep disturbances in RLS. The present study provides evidence for a role of the endogenous sleep-promoting factor adenosine. Three different experimental preparations, long-term (22 weeks) severe or moderate iron-deficient (ID) diets (3- or 7-ppm iron diet) in mice and short-term (3 weeks) severe ID diet (3-ppm iron diet) in rats, demonstrated a significant downregulation (Western blotting in mouse and radioligand binding saturation experiments in rat brain tissue) of adenosine A1 receptors (A1R) in the cortex and striatum, concomitant to striatal D2R downregulation. On the other hand, the previously reported upregulation of adenosine A2A receptors (A2AR) was only observed with severe ID in both mice and rats. The results suggest a key role for A1R downregulation in the PLMS and hyperarousal in RLS. PMID:27600688

  9. Pharmacochaperoning of the A1 adenosine receptor is contingent on the endoplasmic reticulum.

    PubMed

    Málaga-Diéguez, Laura; Yang, Qiong; Bauer, Jan; Pankevych, Halyna; Freissmuth, Michael; Nanoff, Christian

    2010-06-01

    Exchanging each of the conserved aromatic residues of the NPxxY(x)(5,6)F sequence (at the boundary of helices 7 and 8) generated variants of the A(1) adenosine receptor that were retained within the cell. The mutations disconnected a link between alpha-helix 7 and cytosolic helix 8, likely destabilizing the structure of the proximal carboxyl terminus. The mutant receptors were rescued by incubation of cells with a pharmacochaperone, a membrane-permeable ligand that homosterically binds to the receptor; pharmacochaperoning restored the density of functional receptors at the plasma membrane. The following observations support the assumption that retention and the site of pharmacochaperone action were within bounds of the endoplasmic reticulum (ER): 1) the retained receptor colocalized with an ER marker; 2) pharmacochaperoning initiated receptor transfer to Golgi stacks; and 3) the inhibitor of glycoprotein synthesis tunicamycin suppressed receptor chaperoning. Our data are consistent with the hypothesis that pharmacochaperoning stabilizes the structure of late folding intermediates and lifts a block on maturation, allowing the receptors to exit from the ER. We suggest that the ER-associated 40-kDa heat shock protein family member D(1) receptor interacting protein 78 (DRiP78; M(r), approximately 78,000) represents a model executor of quality control. Overexpressed DRiP78 interacted physically with the A(1) receptor, inhibited export to the plasma membrane, and in this action was selective for the mutants relative to the wild-type receptor. Both agonist and antagonist were effective chaperone ligands. Thus, occupancy of the binding pocket corrected the mutation-induced disorder, indicating a mutual impingement of the transmembrane domain and the proximal carboxyl terminus in establishing the stable receptor fold.

  10. The effects of caffeine on sleep in Drosophila require PKA activity, but not the adenosine receptor.

    PubMed

    Wu, Mark N; Ho, Karen; Crocker, Amanda; Yue, Zhifeng; Koh, Kyunghee; Sehgal, Amita

    2009-09-02

    Caffeine is one of the most widely consumed stimulants in the world and has been proposed to promote wakefulness by antagonizing function of the adenosine A2A receptor. Here, we show that chronic administration of caffeine reduces and fragments sleep in Drosophila and also lengthens circadian period. To identify the mechanisms underlying these effects of caffeine, we first generated mutants of the only known adenosine receptor in flies (dAdoR), which by sequence is most similar to the mammalian A2A receptor. Mutants lacking dAdoR have normal amounts of baseline sleep, as well as normal homeostatic responses to sleep deprivation. Surprisingly, these mutants respond normally to caffeine. On the other hand, the effects of caffeine on sleep and circadian rhythms are mimicked by a potent phosphodiesterase inhibitor, IBMX (3-isobutyl-1-methylxanthine). Using in vivo fluorescence resonance energy transfer imaging, we find that caffeine induces widespread increase in cAMP levels throughout the brain. Finally, the effects of caffeine on sleep are blocked in flies that have reduced neuronal PKA activity. We suggest that chronic administration of caffeine promotes wakefulness in Drosophila, at least in part, by inhibiting cAMP phosphodiesterase activity.

  11. The Effects of Caffeine on Sleep in Drosophila Require PKA Activity, but not the Adenosine Receptor

    PubMed Central

    Wu, Mark N.; Ho, Karen; Crocker, Amanda; Yue, Zhifeng; Koh, Kyunghee; Sehgal, Amita

    2009-01-01

    Caffeine is one of the most widely consumed stimulants in the world and has been proposed to promote wakefulness by antagonizing function of the adenosine A2A receptor. Here, we show that chronic administration of caffeine reduces and fragments sleep in Drosophila and also lengthens circadian period. To identify the mechanisms underlying these effects of caffeine, we first generated mutants of the only known adenosine receptor in flies (dAdoR), which by sequence is most similar to the mammalian A2A receptor. Mutants lacking dAdoR have normal amounts of baseline sleep, as well as normal homeostatic responses to sleep deprivation. Surprisingly, these mutants respond normally to caffeine. On the other hand, the effects of caffeine on sleep and circadian rhythms are mimicked by a potent phosphodiesterase inhibitor, IBMX (3-isobutyl-1-methylxanthine). Using in vivo FRET imaging, we find that caffeine induces widespread increase in cAMP levels throughout the brain. Finally, the effects of caffeine on sleep are blocked in flies that have reduced neuronal PKA activity. We suggest that chronic administration of caffeine promotes wakefulness in Drosophila, at least in part, by inhibiting cAMP phosphodiesterase activity. PMID:19726661

  12. Caffeine May Reduce Perceived Sweet Taste in Humans, Supporting Evidence That Adenosine Receptors Modulate Taste.

    PubMed

    Choo, Ezen; Picket, Benjamin; Dando, Robin

    2017-09-01

    Multiple recent reports have detailed the presence of adenosine receptors in sweet sensitive taste cells of mice. These receptors are activated by endogenous adenosine in the plasma to enhance sweet signals within the taste bud, before reporting to the primary afferent. As we commonly consume caffeine, a powerful antagonist for such receptors, in our daily lives, an intriguing question we sought to answer was whether the caffeine we habitually consume in coffee can inhibit the perception of sweet taste in humans. 107 panelists were randomly assigned to 2 groups, sampling decaffeinated coffee supplemented with either 200 mg of caffeine, about the level found in a strong cup of coffee, or an equally bitter concentration of quinine. Participants subsequently performed sensory testing, with the session repeated in the alternative condition in a second session on a separate day. Panelists rated both the sweetened coffee itself and subsequent sucrose solutions as less sweet in the caffeine condition, despite the treatment having no effect on bitter, sour, salty, or umami perception. Panelists were also unable to discern whether they had consumed the caffeinated or noncaffeinated coffee, with ratings of alertness increased equally, but no significant improvement in reaction times, highlighting coffee's powerful placebo effect. This work validates earlier observations in rodents in a human population. © 2017 Institute of Food Technologists®.

  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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Adenosine receptor-mediated coronary vascular protection in post-ischemic mouse heart.

    PubMed

    Zatta, Amanda J; Matherne, G Paul; Headrick, John P

    2006-04-18

    This study evaluated the ability of A1 and A3 adenosine receptor (AR) agonism, and A1, A2A, A2B and A3AR antagonism (revealing "intrinsic" responses), to modify post-ischemic coronary dysfunction in mouse heart. Vascular function was assessed before and after 20 min global ischemia and 30-45 min reperfusion in Langendorff perfused C57/Bl6 mouse hearts. Ischemic insult impaired coronary sensitivity to the endothelial-dependent dilators ADP (pEC50=6.8+/-0.1 vs. 7.6+/-0.1, non-ischemic) and acetylcholine (pEC50=6.1+/-0.1 vs. 7.3+/-0.1 in non-ischemic), and for the mixed endothelial-dependent/independent dilator 2-chloroadenosine (pEC50=7.5+/-0.1 vs. 8.4+/-0.1, non-ischemic). Endothelium-independent dilation in response to nitroprusside was unaltered (pEC50=7.0+/-0.1 vs. 7.1+/-0.1 in non-ischemic). Pre-treatment with a selective A1AR agonist (50 nM CHA) failed to modify coronary dysfunction, whereas A1AR antagonism (200 nM DPCPX) worsened the effects of I/R (2-chloroadenosine pEC50=6.9+/-0.1). Conversely, A3AR agonism (100 nM Cl-IB-MECA) did reduce effects of I/R (pEC50s=8.0+/-0.1 and 7.3+/-0.1 for 2-chloroadenosine and ADP, respectively), whereas antagonism (100 nM MRS1220) was without effect. While A2AAR agonism could not be assessed (due to pronounced vasodilatation), A2AAR antagonism (100 nM SCH58261) was found to exert no effect, and antagonism of A2BARs (50 nM MRS1754) was also ineffective. The protective actions of A3AR agonism were also manifest as improved reactive hyperemic responses. Interestingly, post-ischemic coronary dysfunction was also limited by: Na+-H+ exchange (NHE) inhibition with 10 or 50 microM BIIB-513 (2-chloroadenosine pEC50s=7.8+/-0.1, either dose), an effect not additive with A3AR agonism; Ca2+ antagonism with 0.3 microM verapamil (2-chloroadenosine pEC50=7.9+/-0.1); and Ca2+ desensitization with 5 mM BDM (2-chloroadenosine pEC50=7.8+/-0.1). In contrast, endothelin antagonism (200 nM PD142893) and anti-oxidant therapy (300 microM MPG+150 U

  15. Adenosine A2 receptor activation ameliorates mitochondrial oxidative stress upon reperfusion through the posttranslational modification of NDUFV2 subunit of complex I in the heart.

    PubMed

    Xu, Jingman; Bian, Xiyun; Liu, Yuan; Hong, Lan; Teng, Tianming; Sun, Yuemin; Xu, Zhelong

    2017-05-01

    While it is well known that adenosine receptor activation protects the heart from ischemia/reperfusion injury, the precise mitochondrial mechanism responsible for the action remains unknown. This study probed the mitochondrial events associated with the cardioprotective effect of 5'-(N-ethylcarboxamido) adenosine (NECA), an adenosine A 2 receptor agonist. Isolated rat hearts were subjected to 30min ischemia followed by 10min of reperfusion, whereas H9c2 cells experienced 20min ischemia and 10min reperfusion. NECA prevented mitochondrial structural damage, decreases in respiratory control ratio (RCR), and collapse of mitochondrial membrane potential (ΔΨm). Both the adenosine A 2A receptor antagonist SCH58261 and A 2B receptor antagonist MRS1706 inhibited the action of NECA. NECA reduced mitochondrial proteins carbonylation, H 2 O 2 , and superoxide generation at reperfusion, but did not change superoxide dismutase (SOD) activity. In support, the protective effects of NECA and Peg-SOD on ΔΨm upon reperfusion were additive, implying that NECA's protection is attributable to the reduced superoxide generation but not to the enhancement of the superoxide-scavenging capacity. NECA increased the mitochondrial Src tyrosine kinase activity and suppressed complex I activity at reperfusion in a Src-dependent manner. NECA also reduced mitochondrial superoxide through Src tyrosine kinase. Studies with liquid chromatography-mass spectrometer (LC-MS) identified Tyr118 of the NDUFV2 subunit of complex 1 as a likely site of the tyrosine phosphorylation. Furthermore, the complex I activity of cells transfected with the Y118F mutant was increased, suggesting that this site might be a negative regulator of complex I activity. In support, NECA failed to suppress complex I activity at reperfusion in cells transfected with the Y118F mutant of NDUFV2. In conclusion, NECA prevents mitochondrial oxidative stress by decreasing mitochondrial superoxide generation through inhibition of

  16. The importance of the adenosine A(2A) receptor-dopamine D(2) receptor interaction in drug addiction.

    PubMed

    Filip, M; Zaniewska, M; Frankowska, M; Wydra, K; Fuxe, K

    2012-01-01

    Drug addiction is a serious brain disorder with somatic, psychological, psychiatric, socio-economic and legal implications in the developed world. Illegal (e.g., psychostimulants, opioids, cannabinoids) and legal (alcohol, nicotine) drugs of abuse create a complex behavioral pattern composed of drug intake, withdrawal, seeking and relapse. One of the hallmarks of drugs that are abused by humans is that they have different mechanisms of action to increase dopamine (DA) neurotransmission within the mesolimbic circuitry of the brain and indirectly activate DA receptors. Among the DA receptors, D(2) receptors are linked to drug abuse and addiction because their function has been proven to be correlated with drug reinforcement and relapses. The recognition that D(2) receptors exist not only as homomers but also can form heteromers, such as with the adenosine (A)(2A) receptor, that are pharmacologically and functionally distinct from their constituent receptors, has significantly expanded the range of potential drug targets and provided new avenues for drug design in the search for novel drug addiction therapies. The aim of this review is to bring current focus on A(2A) receptors, their physiology and pharmacology in the central nervous system, and to discuss the therapeutic relevance of these receptors to drug addiction. We concentrate on the contribution of A(2A) receptors to the effects of different classes of drugs of abuse examined in preclinical behavioral experiments carried out with pharmacological and genetic tools. The consequences of chronic drug treatment on A(2A) receptor-assigned functions in preclinical studies are also presented. Finally, the neurochemical mechanism of the interaction between A(2A) receptors and drugs of abuse in the context of the heteromeric A(2A)-D(2) receptor complex is discussed. Taken together, a significant amount of experimental analyses provide evidence that targeting A(2A) receptors may offer innovative translational strategies

  17. 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

  18. 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

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

    PubMed

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

    2016-02-01

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

  20. Differential effects of the adenosine A1 receptor agonist adenosine amine congener on renal, femoral and carotid vascular conductance in preterm fetal sheep.

    PubMed

    Booth, Lindsea C; Tummers, Leonie; Jensen, Ellen C; Barrett, Carolyn J; Malpas, Simon C; Gunn, Alistair J; Bennet, Laura

    2008-11-01

    1. Adenosine A(1) receptor activation is critical for endogenous neuroprotection from hypoxia-ischaemia, raising the possibility that treatment with A(1) receptor agonists may be an effective physiological protection strategy for vulnerable preterm infants. However, the A(1) receptor can mediate unwanted systemic effects, including vasoconstriction of the afferent glomerular arteriole. There is limited information on whether this occurs at doses that improve cerebral perfusion in the immature brain. 2. Therefore, in the present study, we examined whether infusion of the selective A(1) receptor agonist adenosine amine congener (ADAC) is associated with reduced renal perfusion in chronically instrumented preterm (0.7 gestation) fetal sheep. In the present study, ADAC was given in successive doses of 2.5, 5.0 and 15.0 microg, 45 min apart. 3. Treatment with ADAC was associated with a marked reduction in renal vascular conductance (and blood flow), whereas carotid conductance was increased and there was no significant effect on femoral conductance. In contrast with the stable effects of increasing ADAC dose on vascular conductance, there was a significant dose-related fall in fetal heart rate and blood pressure. 4. In conclusion, these short-term data support the concern that A(1) receptor agonist infusion can selectively impair renal perfusion, even at low doses.

  1. Dipyridamole enhances ischaemia-induced reactive hyperaemia by increased adenosine receptor stimulation

    PubMed Central

    Meijer, P; Wouters, C W; van den Broek, P H H; Scheffer, G J; Riksen, N P; Smits, P; Rongen, G A

    2008-01-01

    Background and purpose: Dipyridamole enhances post-occlusive reactive hyperaemia (PORH) in the human forearm vascular bed. We hypothesize that this effect is completely mediated by increased adenosine receptor stimulation. To test this hypothesis, the effect of caffeine (an adenosine receptor antagonist) on dipyridamole-induced augmentation of PORH was explored. Experimental approach: The forearm vasodilator responses to three increasing periods of forearm ischaemia (2, 5 and 13 min) were determined during placebo infusion. Forty minutes after the last reperfusion period, this procedure was repeated during intra-arterial infusion of dipyridamole (7.4 nmol min−1 per 100 ml forearm). At least 2 weeks later, this whole procedure was repeated, but now in the presence of caffeine (90 μg min−1 per 100 ml volume). Key results: After 2, 5 and 13 min of ischaemia, the average forearm blood flow increased to 5.6±0.7, 9.7±1.3 and 34.5±2.1 ml min−1 per 100 ml. After infusion of dipyridamole into the brachial artery, these numbers were significantly increased to 7.7±0.8, 12.5±1.5 and 41.6±3.1 ml min−1 per 100 ml. This response was abolished by the concomitant infusion of caffeine (6.6±0.5, 10.2±0.6, 35.1±2.2 (caffeine) versus 7.4±0.4, 10.5±0.6, 33.7±2.2 ml min−1per 100 ml (caffeine/dipyridamole)). Conclusions and implications: Caffeine prevented the augmenting effect of dipyridamole on PORH. This indicates that dipyridamole-induced augmentation of PORH is mediated via increased adenosine receptor stimulation as a result of elevated extracellular formation of adenosine during ischaemia. PMID:18264130

  2. Allosteric interactions across native adenosine-A3 receptor homodimers: quantification using single-cell ligand-binding kinetics

    PubMed Central

    May, Lauren T.; Bridge, Lloyd J.; Stoddart, Leigh A.; Briddon, Stephen J.; Hill, Stephen J.

    2011-01-01

    A growing awareness indicates that many G-protein-coupled receptors (GPCRs) exist as homodimers, but the extent of the cooperativity across the dimer interface has been largely unexplored. Here, measurement of the dissociation kinetics of a fluorescent agonist (ABA-X-BY630) from the human A1 or A3 adenosine receptors expressed in CHO-K1 cells has provided evidence for highly cooperative interactions between protomers of the A3-receptor dimer in single living cells. In the absence of competitive ligands, the dissociation rate constants of ABA-X-BY630 from A1 and A3 receptors were 1.45 ± 0.05 and 0.57 ± 0.07 min−1, respectively. At the A3 receptor, this could be markedly increased by both orthosteric agonists and antagonists [15-, 9-, and 19-fold for xanthine amine congener (XAC), 5′-(N-ethyl carboxamido)adenosine (NECA), and adenosine, respectively] and reduced by coexpression of a nonbinding (N250A) A3-receptor mutant. The changes in ABA-X-BY630 dissociation were much lower at the A1 receptor (1.5-, 1.4-, and 1.5-fold). Analysis of the pEC50 values of XAC, NECA, and adenosine for the ABA-X-BY630-occupied A3-receptor dimer yielded values of 6.0 ± 0.1, 5.9 ± 0.1, and 5.2 ± 0.1, respectively. This study provides new insight into the spatial and temporal specificity of drug action that can be provided by allosteric modulation across a GPCR homodimeric interface.—May, L. T., Bridge, L. J., Stoddart, L. A., Briddon, S. J., Hill, S. J. Allosteric interactions across native adenosine-A3 receptor homodimers: quantification using single-cell ligand-binding kinetics. PMID:21715680

  3. 2'-C-Methyl analogues of selective adenosine receptor agonists: synthesis and binding studies.

    PubMed

    Franchetti, P; Cappellacci, L; Marchetti, S; Trincavelli, L; Martini, C; Mazzoni, M R; Lucacchini, A; Grifantini, M

    1998-05-07

    2'-C-Methyl analogues of selective adenosine receptor agonists such as (R)-PIA, CPA, CCPA, NECA, and IB-MECA were synthesized in order to further investigate the subdomain that binds the ribose moiety. Binding affinities of these new compounds at A1 and A2A receptors in bovine brain membranes and at A3 in rat testis membranes were determined and compared. It was found that the 2'-C-methyl modification resulted in a decrease of the affinity, particularly at A2A and A3 receptors. When such modification was combined with N6-substitutions with groups which induce high potency and selectivity at A1 receptors, the high affinity was retained and the selectivity was increased. Thus, 2-chloro-2'-C-methyl-N6-cyclopentyladenosine (2'-Me-CCPA), which displayed a Ki value of 1.8 nM at A1 receptors, was selective for A1 vs A2A and A3 receptors by 2166- and 2777-fold, respectively, resulting in one of the most potent and A1-selective agonists so far known. In functional assay, this compound inhibited forskolin-stimulated adenylyl cyclase activity with an IC50 value of 13.1 nM, acting as a full agonist.

  4. Caffeine Suppresses the Activation of Hepatic Stellate Cells cAMP-Independently by Antagonizing Adenosine Receptors.

    PubMed

    Yamaguchi, Momoka; Saito, Shin-Ya; Nishiyama, Ryota; Nakamura, Misuzu; Todoroki, Kenichiro; Toyo'oka, Toshimasa; Ishikawa, Tomohisa

    2017-01-01

    During liver injury, hepatic stellate cells (HSCs) are activated by various cytokines and transdifferentiated into myofibroblast-like activated HSCs, which produce collagen, a major source of liver fibrosis. Therefore, the suppression of HSC activation is regarded as a therapeutic target for liver fibrosis. Several epidemiological reports have revealed that caffeine intake decreases the risk of liver disease. In this study, therefore, we investigated the effect of caffeine on the activation of primary HSCs isolated from mice. Caffeine suppressed the activation of HSC in a concentration-dependent manner. BAPTA-AM, an intracellular Ca 2+ chelator, had no effect on the caffeine-induced suppression of HSC activation. None of the isoform-selective inhibitors of phosphodiesterase1 to 5 affected changes in the morphology of HSC during activation, whereas CGS-15943, an adenosine receptor antagonist, inhibited them. Caffeine had no effect on intracellular cAMP level or on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2. In contrast, caffeine significantly decreased the phosphorylation of Akt1. These results suggest that caffeine inhibits HSC activation by antagonizing adenosine receptors, leading to Akt1 signaling activation.

  5. Reduced response to the formalin test and lowered spinal NMDA glutamate receptor binding in adenosine A2A receptor knockout mice.

    PubMed

    Hussey, Martin J; Clarke, Geoffrey D; Ledent, Catherine; Hourani, Susanna M O; Kitchen, Ian

    2007-06-01

    Adenosine is a neuromodulator with complex effects on pain pathways. Mice lacking the adenosine A2A receptor are hypoalgesic, and have altered analgesic responses to receptor-selective opioid agonists. These and other findings suggest a role for the adenosine A2A receptor in sensitizing afferent fibres projecting to the spinal cord. To test this hypothesis formalin (20 microl, 5%) was injected into the paw and nociceptive responses were measured in wildtype and adenosine A2A receptor knockout mice. There was a significant reduction in nociception associated with sensory nerve activation in the knockout mice as measured by time spent biting/licking the formalin-injected paw and number of flinches seen during the first phase, but only the number of flinches was reduced during the second inflammatory phase. In addition, the selective adenosine A2A antagonist SCH58261 (3 and 10 mg/kg) also antagonised both phases of the formalin test. We also labelled NMDA glutamate and NK1 receptors in spinal cord sections as an indirect measure of nociceptive transmission from peripheral sites to the spinal cord. [3H]-Substance P binding to NK1 receptors was unaltered but there was a substantial reduction in binding of [3H]-MK801 to NMDA glutamate receptors in all regions of the spinal cord from knockout mice. The decrease in NMDA glutamate receptor binding may reflect reduced peripheral sensory input to the spinal cord during development and could relate to the hypoalgesia in this genotype. These results support a key role for the adenosine A2A receptor in peripheral nociceptive pathways.

  6. The wakefulness promoting drug Modafinil causes adenosine receptor-mediated upregulation of receptor activator of nuclear factor κB ligand in osteoblasts: Negative impact of the drug on peak bone accrual in rats.

    PubMed

    China, Shyamsundar Pal; Pal, Subhashis; Chattopadhyay, Sourav; Porwal, Konica; Mittal, Monika; Sanyal, Sabyasachi; Chattopadhyay, Naibedya

    2018-04-09

    Modafinil is primarily prescribed for treatment of narcolepsy and other sleep-associated disorders. However, its off-prescription use as a cognition enhancer increased considerably, specially among youths. Given its increasing use in young adults the effect of modafinil on peak bone accrual is an important issue but has never been investigated. Modafinil treatment to young male rats caused trabecular and cortical bone loss in tibia and femur, and reduction in biomechanical strength. Co-treatment of modafinil with alendronate (a drug that suppresses bone resorption) reversed the trabecular bone loss but failed to prevent cortical loss. Modafinil increased serum type 1 pro-collagen N-terminal protein (PINP) and collagen type 1 cross-linked C-telopeptide (CTX-1) indicating a high turnover bone loss. The drug also increased receptor activator of nuclear factor κB ligand (RANKL) to osteoprotegerin (OPG) ratio in serum which likely resulted in increased osteoclast number per bone surface. Furthermore, conditioned medium from modafinil treated osteoblasts increased the expression of osteoclastogenic genes in bone marrow-derived macrophages and the effect was blocked by RANKL neutralizing antibody. In primary osteoblasts, modafinil stimulated cAMP production and using pharmacological approach, we showed that modafinil signalled via adenosine receptors (A 2A R and A 2B R) which resulted in increased RANKL expression. ZM-241,385 (an A 2A R inhibitor) and MRS 1754 (an A 2B R inhibitor) suppressed modafinil-induced upregulation of RANKL/OPG ratio in the calvarium of new born rat pups. Our data suggests that by activating osteoblast adenosine receptors modafinil increases the production of osteoclastogenic cytokine, RANKL that in turn results in high turnover bone loss in young rats. Copyright © 2018. Published by Elsevier Inc.

  7. Adenosine A1 receptor-dependent antinociception induced by inosine in mice: pharmacological, genetic and biochemical aspects.

    PubMed

    Nascimento, Francisney Pinto; Macedo-Júnior, Sérgio José; Pamplona, Fabrício Alano; Luiz-Cerutti, Murilo; Córdova, Marina Machado; Constantino, Leandra; Tasca, Carla Inês; Dutra, Rafael Cypriano; Calixto, João B; Reid, Allison; Sawynok, Jana; Santos, Adair Roberto Soares

    2015-01-01

    Inosine is an endogenous nucleoside that has anti-inflammatory and antinociceptive properties. Inosine is a metabolite of adenosine, and some of its actions suggest the involvement of adenosine A1 receptors (A1Rs). The purpose of this study was to better understand mechanisms of inosine-induced antinociception by investigating the role of A1Rs and purine metabolism inhibitors. Inosine antinociception was evaluated using the formalin test in mice. An A1R-selective antagonist (DPCPX), A1R knockout mice (gene deletion) and mice with A1R reduced expression (antisense oligonucleotides) were used to assess the role of A1Rs in the antinociceptive action of inosine. Binding assays were performed to compare the affinity of inosine and adenosine for A1Rs. Finally, the role of adenosine and inosine breakdown was assessed using deoxycoformycin (DCF) and forodesine (FDS) as enzymatic inhibitors of adenosine deaminase and purine nucleoside phosphorylase, respectively. Inosine induced antinociception in the formalin test when given by systemic, spinal and peripheral routes. Systemically, inosine exhibited a potency similar to adenosine, and its effects were inhibited by DPCPX. Inosine did not induce antinociception in A1R knockout mice or in mice with reduced A1R expression. In binding studies, inosine bound to A1Rs with an affinity similar to adenosine. DCF had no effect on inosine actions. FDS augmented the antinociceptive effect of a low systemic dose of inosine and, at a higher dose, induced antinociception by itself. Collectively, these data indicate that inosine is an agonist for A1Rs with antinociceptive properties and a potency similar to adenosine and can be considered another endogenous ligand for this receptor.

  8. Pharmacological evidence for different populations of postsynaptic adenosine A2A receptors in the rat striatum

    PubMed Central

    Orrú, Marco; Quiroz, César; Guitart, Xavier; Ferré, Sergi

    2011-01-01

    Adenosine A2A receptors (A2ARs) are highly concentrated in the striatum. Two pharmacological different functional populations of A2ARs have been recently described based on their different affinities for the A2AR antagonist SCH-442416. This compound has high affinity for A2ARs not forming heteromers or forming heteromers with adenosine A1 receptors (A1Rs) while showing very low affinity for A2ARs forming heteromers with dopamine D2 receptors (D2Rs). It has been widely described that striatal A1R-A2AR heteromers are preferentially localized presynaptically in the glutamatergic terminals that contact GABAergic dynorphinergic neurons, and that A2AR-D2R heteromers are localized postsynaptically in GABAergic enkephalinergic neurons. In the present study we provide evidence suggesting that SCH-442416 also targets postsynaptic A2AR not forming heteromers with D2R, which are involved in the motor depressant effects induced by D2R antagonists. SCH-442416 counteracted motor depression in rats induced by the D2R antagonist raclopride at a dose that did not produce motor activation or that blocked motor depression induced by an A2AR agonist. Furthermore, we re-evaluated the recently suggested key role of cannabinoid CB1 receptors (CB1Rs) in the effects of A2AR antagonists acting at postsynaptic A2ARs. By recording locomotor activity and monitoring striatal glutamate release induced by cortical electrical stimulation in rats after administration of A2AR and CB1R antagonists, we did not find evidence for any significant role of endocannabinoids in the post- or presynaptic effects of A2AR antagonists. The present results further suggest the existence of at least two functionally and pharmacologically different populations of striatal postsynaptic A2ARs. PMID:21752341

  9. Novel Alexa Fluor-488 labeled antagonist of the A(2A) adenosine receptor: Application to a fluorescence polarization-based receptor binding assay.

    PubMed

    Kecskés, Miklós; Kumar, T Santhosh; Yoo, Lena; Gao, Zhan-Guo; Jacobson, Kenneth A

    2010-08-15

    Fluorescence polarization (FP) assay has many advantages over the traditional radioreceptor binding studies. We developed an A(2A) adenosine receptor (AR) FP assay using a newly synthesized fluorescent antagonist of the A(2A)AR (MRS5346), a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivative conjugated to the fluorescent dye Alexa Fluor-488. MRS5346 displayed a K(i) value of 111+/-16nM in radioligand binding using [(3)H]CGS21680 and membranes prepared from HEK293 cells stably expressing the human A(2A)AR. In a cyclic AMP functional assay, MRS5346 was shown to be an A(2A)AR antagonist. MRS5346 did not show any effect on A(1) and A(3) ARs in binding or the A(2B)AR in a cyclic AMP assay at 10microM. Its suitability as a fluorescent tracer was indicated in an initial observation of an FP signal following A(2A)AR binding. The FP signal was optimal with 20nM MRS5346 and 150microg protein/mL HEK293 membranes. The association and dissociation kinetic parameters were readily determined using this FP assay. The K(d) value of MRS5346 calculated from kinetic parameters was 16.5+/-4.7nM. In FP competition binding experiments using MRS5346 as a tracer, K(i) values of known AR agonists and antagonists consistently agreed with K(i) values from radioligand binding. Thus, this FP assay, which eliminates using radioisotopes, appears to be appropriate for both routine receptor binding and high-throughput screening with respect to speed of analysis, displaceable signal and precision. The approach used in the present study could be generally applicable to other GPCRs. Published by Elsevier Inc.

  10. Predicting Subtype Selectivity for Adenosine Receptor Ligands with Three-Dimensional Biologically Relevant Spectrum (BRS-3D)

    NASA Astrophysics Data System (ADS)

    He, Song-Bing; Ben Hu; Kuang, Zheng-Kun; Wang, Dong; Kong, De-Xin

    2016-11-01

    Adenosine receptors (ARs) are potential therapeutic targets for Parkinson’s disease, diabetes, pain, stroke and cancers. Prediction of subtype selectivity is therefore important from both therapeutic and mechanistic perspectives. In this paper, we introduced a shape similarity profile as molecular descriptor, namely three-dimensional biologically relevant spectrum (BRS-3D), for AR selectivity prediction. Pairwise regression and discrimination models were built with the support vector machine methods. The average determination coefficient (r2) of the regression models was 0.664 (for test sets). The 2B-3 (A2B vs A3) model performed best with q2 = 0.769 for training sets (10-fold cross-validation), and r2 = 0.766, RMSE = 0.828 for test sets. The models’ robustness and stability were validated with 100 times resampling and 500 times Y-randomization. We compared the performance of BRS-3D with 3D descriptors calculated by MOE. BRS-3D performed as good as, or better than, MOE 3D descriptors. The performances of the discrimination models were also encouraging, with average accuracy (ACC) 0.912 and MCC 0.792 (test set). The 2A-3 (A2A vs A3) selectivity discrimination model (ACC = 0.882 and MCC = 0.715 for test set) outperformed an earlier reported one (ACC = 0.784). These results demonstrated that, through multiple conformation encoding, BRS-3D can be used as an effective molecular descriptor for AR subtype selectivity prediction.

  11. Effects of the adenosine A1 receptor inhibitor FK 838 on proximal tubular fluid output in rats.

    PubMed

    Bak, Martin; Thomsen, Klaus

    2004-05-01

    Adenosine A(1) receptor blockade has been suggested as a treatment in conditions with sodium and fluid retention because it increases urinary Na(+) excretion and increases proximal tubular fluid output. In the present study, we examine the time course for the renal responses to adenosine A(1) receptor blockade in order to investigate whether the effects may be prolonged and not just temporary. The acute effects of the adenosine A(1) receptor inhibitor FK 838 on segmental tubular Na(+) handling were examined by a renal clearance technique in conscious chronically instrumented rats. Lithium clearance (C(Li)) was used as a clearance marker of proximal tubular fluid output. Acute adenosine A(1) receptor inhibition did not affect the glomerular filtration rate (GFR) significantly. In contrast, the inhibition led to significant increases in C(Li) (from 290+/-28 to 431+/-28 microl/min/100 g), fractional Li(+) excretion (FE(Li)) (from 33+/-2 to 47+/-3%) and fractional Na(+) excretion (FE(Na)) (from 0.44+/-0.07 to 2.03+/-0.42%). Sodium excretion, expressed as a fraction of proximal tubular fluid output (C(Na)/C(Li)), rose from 1.3+/-0.2 to 4.2+/-0.4%, suggesting that the natriuretic effect was supported by inhibition of distal nephron Na(+) reabsorption. All values returned to baseline values during the clearance study and thereby indicated that neither proximal tubular fluid output nor urinary sodium excretion remained elevated for a prolonged time. It is concluded that in conscious unstressed rats, acute adenosine A(1) receptor inhibition by FK 838 led to a significant natriuresis that was caused by inhibition of proximal tubular Na(+) reabsorption, possibly with a contribution from inhibition of distal nephron Na(+) reabsorption. The increased proximal tubular fluid output and the increased urinary Na(+) excretion returned to baseline values during the clearance study, indicating that none of these effects of adenosine A(1) blockade were long lasting.

  12. CD73-generated adenosine promotes osteoblast differentiation

    PubMed Central

    Takedachi, Masahide; Oohara, Hiroyuki; Smith, Brenda J.; Iyama, Mitsuyoshi; Kobashi, Mariko; Maeda, Kenichiro; Long, Courtney L.; Humphrey, Mary B.; Stoecker, Barbara J.; Toyosawa, Satoru; Thompson, Linda F.; Murakami, Shinya

    2011-01-01

    CD73 is a GPI-anchored cell surface protein with ecto-5′-nucleotidase enzyme activity that plays a crucial role in adenosine production. While the roles of adenosine receptors (AR) on osteoblasts and osteoclasts have been unveiled to some extent, the roles of CD73 and CD73-generated adenosine in bone tissue are largely unknown. To address this issue, we first analyzed the bone phenotype of CD73-deficient (cd73−/−) mice. The mutant male mice showed osteopenia, with significant decreases of osteoblastic markers. Levels of osteoclastic markers were, however, comparable to those of wild type mice. A series of in vitro studies revealed that CD73 deficiency resulted in impairment in osteoblast differentiation but not in the number of osteoblast progenitors. In addition, over expression of CD73 on MC3T3-E1 cells resulted in enhanced osteoblastic differentiation. Moreover, MC3T3-E1 cells expressed adenosine A2A receptors (A2AAR) and A2B receptors (A2BAR) and expression of these receptors increased with osteoblastic differentiation. Enhanced expression of osteocalcin (OC) and bone sialoprotein (BSP) observed in MC3T3-E1 cells over expressing CD73 were suppressed by treatment with an A2BAR antagonist but not with an A2AAR antagonist. Collectively, our results indicate that CD73 generated adenosine positively regulates osteoblast differentiation via A2BAR signaling. PMID:21882189

  13. Effects of A1-adenosine receptor antagonists on purinergic transmission in the guinea-pig vas deferens in vitro

    PubMed Central

    Hardy, Todd A; Brock, James A

    1999-01-01

    Intracellularly recorded excitatory junction potentials (e.j.ps) were used to study the effects of adenosine receptor antagonists on neurotransmitter release from postganglionic sympathetic nerve terminals in the guinea-pig vas deferens in vitro.The A1 adenosine receptor antagonists, 8-phenyltheophylline (10 μM) and 8-cyclopentyl-1,3-dipropylxanthine (0.1 μM), increased the amplitude of e.j.ps evoked during trains of 20 stimuli at 1 Hz in the presence, but not in the absence, of the α2-adrenoceptor antagonist, yohimbine (1 μM) or the non-selective α-adrenoceptor antagonist, phentolamine (1 μM).Adenosine (100 μM) reduced the amplitude of e.j.ps, both in the presence and in the absence of phentolamine (1 μM). This inhibitory effect of adenosine is most likely caused by a reduction in transmitter release as there was no detectable change in spontaneous e.j.p. amplitudes.In the presence of phentolamine, application of the adenosine uptake inhibitor, S-(p-nitrobenzyl)-6-thioinosine (0.1 μM), had no effect on e.j.p. amplitudes.The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (100 μM), significantly increased the amplitudes of all e.j.ps evoked during trains of 20 stimuli at 1 Hz, both in the presence and in the absence of phentolamine (1 μM).These results suggest that endogenous adenosine modulates neurotransmitter release by an action at prejunctional A1 adenosine receptors only when α2-adrenoceptors are blocked. PMID:10372818

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

    SciTech Connect

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

    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 adenosinemore » 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.« less

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

    PubMed

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

    2015-07-07

    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.

  16. 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

  17. Persistent reduction of cocaine seeking by pharmacological manipulation of adenosine A1 and A 2A receptors during extinction training in rats.

    PubMed

    O'Neill, Casey E; Hobson, Benjamin D; Levis, Sophia C; Bachtell, Ryan K

    2014-08-01

    Adenosine receptor stimulation and blockade have been shown to modulate a variety of cocaine-related behaviors. These studies identify the direct effects of adenosine receptor stimulation on cocaine seeking during extinction training and the persistent effects on subsequent reinstatement to cocaine seeking. Rats self-administered cocaine on a fixed ratio one schedule in daily sessions over 3 weeks. Following a 1-week withdrawal, the direct effects of adenosine receptor modulation were tested by administering the adenosine A1 receptor agonist, N(6)-cyclopentyladenosine (CPA, 0.03 and 0.1 mg/kg), the adenosine A2A agonist, CGS 21680 (0.03 and 0.1 mg/kg), the presynaptic adenosine A2A receptor antagonist, SCH 442416 (0.3, 1, and 3 mg/kg), or vehicle prior to each of six daily extinction sessions. The persistent effects of adenosine receptor modulation during extinction training were subsequently tested on reinstatement to cocaine seeking induced by cues, cocaine, and the dopamine D2 receptor agonist, quinpirole. All doses of CPA and CGS 21680 impaired initial extinction responding; however, only CPA treatment during extinction produced persistent impairment in subsequent cocaine- and quinpirole-induced seeking. Dissociating CPA treatment from extinction did not alter extinction responding or subsequent reinstatement. Administration of SCH 442416 had no direct effects on extinction responding but produced dose-dependent persistent impairment of cocaine- and quinpirole-induced seeking. These findings demonstrate that adenosine A1 or A2A receptor stimulation directly impair extinction responding. Interestingly, adenosine A1 receptor stimulation or presynaptic adenosine A2A receptor blockade during extinction produces lasting changes in relapse susceptibility.

  18. 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.

  19. Antinociception by systemically-administered acetaminophen (paracetamol) involves spinal serotonin 5-HT7 and adenosine A1 receptors, as well as peripheral adenosine A1 receptors.

    PubMed

    Liu, Jean; Reid, Allison R; Sawynok, Jana

    2013-03-01

    Acetaminophen (paracetamol) is a widely used analgesic, but its sites and mechanisms of action remain incompletely understood. Recent studies have separately implicated spinal adenosine A(1) receptors (A(1)Rs) and serotonin 5-HT(7) receptors (5-HT(7)Rs) in the antinociceptive effects of systemically administered acetaminophen. In the present study, we determined whether these two actions are linked by delivering a selective 5-HT(7)R antagonist to the spinal cord of mice and examining nociception using the formalin 2% model. In normal and A(1)R wild type mice, antinociception by systemic (i.p.) acetaminophen 300mg/kg was reduced by intrathecal (i.t.) delivery of the selective 5-HT(7)R antagonist SB269970 3μg. In mice lacking A(1)Rs, i.t. SB269970 did not reverse antinociception by systemic acetaminophen, indicating a link between spinal 5-HT(7)R and A(1)R mechanisms. We also explored potential roles of peripheral A(1)Rs in antinociception by acetaminophen administered both locally and systemically. In normal mice, intraplantar (i.pl.) acetaminophen 200μg produced antinociception in the formalin test, and this was blocked by co-administration of the selective A(1)R antagonist DPCPX 4.5μg. Acetaminophen administered into the contralateral hindpaw had no effect, indicating a local peripheral action. When acetaminophen was administered systemically, its antinociceptive effect was reversed by i.pl. DPCPX in normal mice; this was also observed in A(1)R wild type mice, but not in those lacking A(1)Rs. In summary, we demonstrate a link between spinal 5-HT(7)Rs and A(1)Rs in the spinal cord relevant to antinociception by systemic acetaminophen. Furthermore, we implicate peripheral A(1)Rs in the antinociceptive effects of locally- and systemically-administered acetaminophen. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  20. Insulin restores L-arginine transport requiring adenosine receptors activation in umbilical vein endothelium from late-onset preeclampsia.

    PubMed

    Salsoso, R; Guzmán-Gutiérrez, E; Sáez, T; Bugueño, K; Ramírez, M A; Farías, M; Pardo, F; Leiva, A; Sanhueza, C; Mate, A; Vázquez, C; Sobrevia, L

    2015-03-01

    Preeclampsia is associated with impaired placental vasodilation and reduced endothelial nitric oxide synthase (eNOS) activity in the foetoplacental circulation. Adenosine and insulin stimulate vasodilation in endothelial cells, and this activity is mediated by adenosine receptor activation in uncomplicated pregnancies; however, this activity has yet to be examined in preeclampsia. Early onset preeclampsia is associated with severe placental vasculature alterations that lead to altered foetus growth and development, but whether late-onset preeclampsia (LOPE) alters foetoplacental vascular function is unknown. Vascular reactivity to insulin (0.1-1000 nmol/L, 5 min) and adenosine (1 mmol/L, 5 min) was measured in KCl-preconstricted human umbilical vein rings from normal and LOPE pregnancies using a wire myograph. The protein levels of human cationic amino acid transporter 1 (hCAT-1), adenosine receptor subtypes, total and Ser¹¹⁷⁷- or Thr⁴⁹⁵-phosphorylated eNOS were detected via Western blot, and L-arginine transport (0-1000 μmol/L L-arginine, 3 μCi/mL L-[³H]arginine, 20 s, 37 °C) was measured in the presence or absence of insulin and adenosine receptor agonists or antagonists in human umbilical vein endothelial cells (HUVECs) from normal and LOPE pregnancies. LOPE increased the maximal L-arginine transport capacity and hCAT-1 and eNOS expression and activity compared with normal conditions. The A(2A) adenosine receptor (A(2A)AR) antagonist ZM-241385 blocked these effects of LOPE. Insulin-mediated umbilical vein ring relaxation was lower in LOPE pregnancies than in normal pregnancies and was restored using the A(2A)AR antagonist. The reduced foetoplacental vascular response to insulin may result from A(2A)AR activation in LOPE pregnancies. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. 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

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

    PubMed

    Kim, Youngsoo; Elmenhorst, David; Weisshaupt, Angela; Wedekind, Franziska; Kroll, Tina; McCarley, Robert W; Strecker, Robert E; Bauer, Andreas

    2015-10-01

    Although chronic sleep restriction frequently produces long-lasting behavioural and physiological impairments in humans, the underlying neural mechanisms are unknown. Here we used a rat model of chronic sleep restriction to investigate the role of brain adenosine and noradrenaline systems, known to regulate sleep and wakefulness, respectively. The density of adenosine A1 and A2a receptors and β-adrenergic receptors before, during and following 5 days of sleep restriction was assessed with autoradiography. Rats (n = 48) were sleep-deprived for 18 h day(-1) for 5 consecutive days (SR1-SR5), followed by 3 unrestricted recovery sleep days (R1-R3). Brains were collected at the beginning of the light period, which was immediately after the end of sleep deprivation on sleep restriction days. Chronic sleep restriction increased adenosine A1 receptor density significantly in nine of the 13 brain areas analysed with elevations also observed on R3 (+18 to +32%). In contrast, chronic sleep restriction reduced adenosine A2a receptor density significantly in one of the three brain areas analysed (olfactory tubercle which declined 26-31% from SR1 to R1). A decrease in β-adrenergic receptors density was seen in substantia innominata and ventral pallidum which remained reduced on R3, but no changes were found in the anterior cingulate cortex. These data suggest that chronic sleep restriction can induce long-term changes in the brain adenosine and noradrenaline receptors, which may underlie the long-lasting neurocognitive impairments observed in chronic sleep restriction. © 2015 European Sleep Research Society.

  3. Nucleus Accumbens Adenosine A2A Receptors Regulate Exertion of Effort by Acting on the Ventral Striatopallidal Pathway

    PubMed Central

    Mingote, Susana; Font, Laura; Farrar, Andrew M.; Vontell, Regina; Worden, Lila T.; Stopper, Colin M.; Port, Russell G.; Sink, Kelly S.; Bunce, Jamie G.; Chrobak, James J.; Salamone, John D.

    2009-01-01

    Goal-directed actions are sensitive to work-related response costs, and dopamine in nucleus accumbens is thought to modulate the exertion of effort in motivated behavior. Dopamine-rich striatal areas such as nucleus accumbens also contain high numbers of adenosine A2A receptors, and, for that reason, the behavioral and neurochemical effects of the adenosine A2A receptor agonist CGS 21680 [2-p-(2-carboxyethyl) phenethylamino-5′-N-ethylcarboxamidoadenosine] were investigated. Stimulation of accumbens adenosine A2A receptors disrupted performance of an instrumental task with high work demands (i.e., an interval lever-pressing schedule with a ratio requirement attached) but had little effect on a task with a lower work requirement. Immunohistochemical studies revealed that accumbens neurons that project to the ventral pallidum showed adenosine A2A receptors immunoreactivity. Moreover, activation of accumbens A2A receptors by local injections of CGS 21680 increased extracellular GABA levels in the ventral pallidum. Combined contralateral injections of CGS 21680 into the accumbens and the GABAA agonist muscimol into ventral pallidum (i.e., “disconnection” methods) also impaired response output, indicating that these structures are part of a common neural circuitry regulating the exertion of effort. Thus, accumbens adenosine A2A receptors appear to regulate behavioral activation and effort-related processes by modulating the activity of the ventral striatopallidal pathway. Research on the effort-related functions of these forebrain systems may lead to a greater understanding of pathological features of motivation, such as psychomotor slowing, anergia, and fatigue in depression. PMID:18768698

  4. AMP-guided tumour-specific nanoparticle delivery via adenosine A1 receptor.

    PubMed

    Dai, Tongcheng; Li, Na; Han, Fajun; Zhang, Hua; Zhang, Yuanxing; Liu, Qin

    2016-03-01

    Active targeting-ligands have been increasingly used to functionalize nanoparticles for tumour-specific clinical applications. Here we utilize nucleotide adenosine 5'-monophosphate (AMP) as a novel ligand to functionalize polymer-based fluorescent nanoparticles (NPs) for tumour-targeted imaging. We demonstrate that AMP-conjugated NPs (NPs-AMP) efficiently bind to and are following internalized into colon cancer cell CW-2 and breast cancer cell MDA-MB-468 in vitro. RNA interference and inhibitor assays reveal that the targeting effects mainly rely on the specific binding of AMP to adenosine A1 receptor (A1R), which is greatly up-regulated in cancer cells than in matched normal cells. More importantly, NPs-AMP specifically accumulate in the tumour site of colon and breast tumour xenografts and are further internalized into the tumour cells in vivo via tail vein injection, confirming that the high in vitro specificity of AMP can be successfully translated into the in vivo efficacy. Furthermore, NPs-AMP exhibit an active tumour-targeting behaviour in various colon and breast cancer cells, which is positively related to the up-regulation level of A1R in cancer cells, suggesting that AMP potentially suits for more extensive A1R-overexpressing cancer models. This work establishes AMP to be a novel tumour-targeting ligand and provides a promising strategy for future diagnostic or therapeutic applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Adenosine A2A Receptor Signaling in the Immunopathogenesis of Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Rajasundaram, Skanda

    2018-01-01

    Our increasing appreciation of adenosine as an endogenous signaling molecule that terminates inflammation has generated excitement regarding the potential to target adenosine receptors (ARs) in the treatment of multiple sclerosis (MS), a disease of chronic neuroinflammation. Of the four G protein-coupled ARs, A2ARs are the principal mediator of adenosine’s anti-inflammatory effects and accordingly, there is a growing body of evidence surrounding the role of A2ARs in experimental autoimmune encephalomyelitis (EAE), the dominant animal model of MS. Such evidence points to a complex, often paradoxical role for A2ARs in the immunopathogenesis of EAE, where they have the ability to both exacerbate and alleviate disease severity. This review seeks to interpret these paradoxical findings and evaluate the therapeutic promise of A2ARs. In essence, the complexities of A2AR signaling arise from two properties. Firstly, A2AR signaling downregulates the inflammatory potential of TH lymphocytes whilst simultaneously facilitating the recruitment of these cells into the CNS. Secondly, A2AR expression by myeloid cells – infiltrating macrophages and CNS-resident microglia – has the capacity to promote both tissue injury and repair in chronic neuroinflammation. Consequently, the therapeutic potential of targeting A2ARs is greatly undermined by the risk of collateral tissue damage in the periphery and/or CNS. PMID:29559972

  6. The plant hormone zeatin riboside inhibits T lymphocyte activity via adenosine A2A receptor activation.

    PubMed

    Lappas, Courtney M

    2015-01-01

    Cytokinins are plant hormones that play an integral role in multiple aspects of plant growth and development. The biological functions of cytokinins in mammalian systems are, however, largely uncharacterized. The naturally occurring cytokinin zeatin riboside has recently been demonstrated to activate the mammalian adenosine A(2A) receptor, which is broadly expressed by various cell types including immune system cells, with the activation of the A(2A)R playing a role in the regulation of cells involved in both innate and adaptive immunity. We show for the first time that zeatin riboside modulates mammalian immune system activity via an A(2A)R-dependent mechanism. Specifically, zeatin riboside treatment induces the production of cyclic adenosine monophosphate (cAMP) by T lymphocytes and inhibits the production by CD3(+)CD4(+) T cells of interferon (IFN)-γ, IL-2, tumor-necrosis factor (TNF)-α, IL-4 and IL-13, and the production by CD3(+)CD8(+) T cells of IFN-γ, IL-2 and TNF-α. Additionally, the upregulation of CD25, CD69 and CD40L by activated T lymphocytes is modulated by zeatin riboside. Zeatin riboside treatment also potently inhibits thioglycollate-induced peritoneal leukocytosis. The immunomodulatory activities of zeatin riboside are blocked by co-treatment with the selective A(2A)R antagonist ZM241385. These data suggest that zeatin riboside possesses therapeutic potential as a mammalian immunomodulatory agent.

  7. An Allosteric Modulator of the Adenosine A1 Receptor Improves Cardiac Function Following Ischaemia in Murine Isolated Hearts

    PubMed Central

    Butcher, Anna; Scammells, Peter J.; White, Paul J.; Devine, Shane M.; Rose’Meyer, Roselyn B.

    2013-01-01

    The effect of an allosteric modulator of the adenosine A1 receptors was investigated using an ischaemia-reperfusion protocol in murine isolated hearts. Isolated hearts were perfused with Kreb-Henseleit solution gassed with carbogen gas (95% O2 and 5% CO2) in Langendorff mode and electrically paced at 480 bpm. Following 20 min equilibration and 20 min global normothermic ischaemia, the allosteric modulator VCP333 (1 μM) or the adenosine A1 receptor partial agonist VCP102 (10 μM) were infused after 5 min of reperfusion for 15 min. Upon termination of the drug treatment, reperfusion continued for a further 40 min. At the end of 60 min reperfusion, treatment with VCP333 or VCP102 improved the recovery of the left ventricular developed pressure when compared to control group responses (p < 0.05). Neither compound affected end diastolic pressure, coronary flow rates or dP/dtmax values when compared to control tissues during reperfusion (p > 0.05). The infusion of VCP102 or VCP333 during reperfusion reduced cardiac troponin I efflux to 6.7% and 25% respectively of control heart efflux (p < 0.05). This data indicates that the allosteric modulator of the adenosine A1 receptor (VCP333) has similar characteristics to the adenosine receptor partial agonist VCP102 as it improves cardiac function and reduces myocardial cell death following an ischaemic episode. PMID:24276124

  8. Structure-Based Design, Synthesis, and In Vivo Antinociceptive Effects of Selective A1Adenosine Receptor Agonists.

    PubMed

    Petrelli, Riccardo; Scortichini, Mirko; Belardo, Carmela; Boccella, Serena; Luongo, Livio; Capone, Fabio; Kachler, Sonja; Vita, Patrizia; Del Bello, Fabio; Maione, Sabatino; Lavecchia, Antonio; Klotz, Karl-Norbert; Cappellacci, Loredana

    2018-01-11

    Our previous work discovered that combining the appropriate 5'- and N 6 -substitution in adenosine derivatives leads to the highly selective human A 1 adenosine receptor (hA 1 AR) agonists or highly potent dual hA 1 AR agonists and hA 3 AR antagonists. In order to explore novel dual adenosine receptor ligands, a series of N 6 -substituted-5'-pyrazolyl-adenosine and 2-chloro-adenosine derivatives were synthesized and assayed in vitro at all ARs. The N 6 -(±)-endo-norbornyl derivative 12 was the most potent and selective at A 1 AR and effective as an analgesic in formalin test in mice, but none of the 5'-pyrazolyl series compounds showed a dual behavior at hA 1 and hA 3 AR. Molecular modeling studies rationalized the structure-activity relationships and the selectivity profiles of the new series of A 1 AR agonists. Interestingly, an unexpected inverted binding mode of the N 6 -tetrahydrofuranyl derivative 14 was hypothesized to explain its low affinity at A 1 AR.

  9. 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

  10. Role of Microglia Adenosine A2A Receptors in Retinal and Brain Neurodegenerative Diseases

    PubMed Central

    Santiago, Ana R.; Baptista, Filipa I.; Santos, Paulo F.; Cristóvão, Gonçalo; Ambrósio, António F.; Cunha, Rodrigo A.; Gomes, Catarina A.

    2014-01-01

    Neuroinflammation mediated by microglial cells in the brain has been commonly associated with neurodegenerative diseases. Whether this microglia-mediated neuroinflammation is cause or consequence of neurodegeneration is still a matter of controversy. However, it is unequivocal that chronic neuroinflammation plays a role in disease progression and halting that process represents a potential therapeutic strategy. The neuromodulator adenosine emerges as a promising targeting candidate based on its ability to regulate microglial proliferation, chemotaxis, and reactivity through the activation of its G protein coupled A2A receptor (A2AR). This is in striking agreement with the ability of A2AR blockade to control several brain diseases. Retinal degenerative diseases have been also associated with microglia-mediated neuroinflammation, but the role of A2AR has been scarcely explored. This review aims to compare inflammatory features of Parkinson's and Alzheimer's diseases with glaucoma and diabetic retinopathy, discussing the therapeutic potential of A2AR in these degenerative conditions. PMID:25132733

  11. CHOLESTEROL 27-HYDROXYLASE BUT NOT APOLIPOPROTEIN apoE CONTRIBUTES TO A2A ADENOSINE RECEPTOR STIMULATED REVERSE CHOLESTEROL TRANSPORT

    PubMed Central

    Bingham, Taiese Crystal; Parathath, Saj; Tian, Heather; Reiss, Allison; Chan, Edwin; Fisher, Edward A.; Cronstein, Bruce N.

    2011-01-01

    Movement of free cholesterol between the cellular compartment and acceptor is governed by cholesterol gradients that are determined by several enzymes and reverse cholesterol transport proteins. We have previously demonstrated that adenosine A2A receptors inhibit foam cell formation and stimulate production of cholesterol 27-hydroxylase (CYP27A1), an enzyme involved in the conversion of cholesterol to oxysterols. We therefore asked whether the effect of adenosine A2A receptors on foam cell formation in vitro are mediated by CYP27A1 or apoE, a carrier for cholesterol in the serum. We found that specific lentiviral siRNA infection markedly reduced apoE or 27-hydroxylase mRNA in THP-1 cells. Despite diminished apoE expression (p< 0.0002, IFNγ CGS vs. IFNγ alone, n= 4) CGS-21680, an adenosine A2A receptor agonist, inhibits foam cell formation. In contrast, CGS-21680 had no effect on reducing foam cell formation in CYP27A1 KD cells (4±2% p<0.5113 inhibition vs. IFNγ alone n= 4). Previously we reported the A2A agonist CGS-21680 increases apoAI-mediated cholesterol efflux nearly 2-fold in wildtype macrophages. Adenosine receptor activation had no effect on cholesterol efflux in CYP27A1 KD cells but reduced efflux in apoE KD cells. These results demonstrate that adenosine A2A receptor occupancy diminishes foam cell formation by increasing expression and function of CYP27A1. PMID:21258856

  12. Cholesterol 27-hydroxylase but not apolipoprotein apoE contributes to A2A adenosine receptor stimulated reverse cholesterol transport.

    PubMed

    Bingham, Taiese Crystal; Parathath, Saj; Tian, Heather; Reiss, Allison; Chan, Edwin; Fisher, Edward A; Cronstein, Bruce N

    2012-02-01

    Movement of free cholesterol between the cellular compartment and acceptor is governed by cholesterol gradients that are determined by several enzymes and reverse cholesterol transport proteins. We have previously demonstrated that adenosine A(2A) receptors inhibit foam cell formation and stimulate production of cholesterol 27-hydroxylase (CYP27A1), an enzyme involved in the conversion of cholesterol to oxysterols. We therefore asked whether the effect of adenosine A(2A) receptors on foam cell formation in vitro is mediated by CYP27A1 or apoE, a carrier for cholesterol in the serum. We found that specific lentiviral siRNA infection markedly reduced apoE or 27-hydroxylase mRNA in THP-1 cells. Despite diminished apoE expression (p < 0.0002, interferon-gamma (IFNγ) CGS vs. IFNγ alone, n=4), CGS-21680, an adenosine A(2A) receptor agonist, inhibits foam cell formation. In contrast, CGS-21680 had no effect on reducing foam cell formation in CYP27A1 KD cells (4 ± 2%; p<0.5113, inhibition vs. IFNγ alone, n=4). Previously, we reported the A(2A) agonist CGS-21680 increases apoAI-mediated cholesterol efflux nearly twofold in wild-type macrophages. Adenosine receptor activation had no effect on cholesterol efflux in CYP27A1 KD cells but reduced efflux in apoE KD cells. These results demonstrate that adenosine A(2A) receptor occupancy diminishes foam cell formation by increasing expression and function of CYP27A1.

  13. Predicted Structures of Agonist and Antagonist Bound Complexes of Adenosine A3 Receptor

    PubMed Central

    Kim, Soo-Kyung; Riley, Lindsay; Abrol, Ravinder; Jacobson, Kenneth A.; Goddard, William A.

    2011-01-01

    We used the GEnSeMBLE Monte Carlo method to predict ensemble of the 20 best packings (helix rotations and tilts) based on the neutral total energy (E) from a vast number (10 trillion) of potential packings for each of the 4 subtypes of the adenosine G protein-coupled receptors (GPCRs), which are involved in many cytoprotective functions. We then used the DarwinDock Monte Carlo methods to predict the binding pose for the human A3 adenosine receptor (hAA3R) for subtype selective agonists and antagonists. We find that all four A3 agonists stabilize the 15th lowest conformation of apo-hAA3R while also binding strongly to the 1st and 3rd. In contrast the four A3 antagonists stabilize the 2nd or 3rd lowest conformation. These results show that different ligands can stabilize different GPCR conformations, which will likely affect function, complicating the design of functionally unique ligands. Interestingly all agonists lead to a trans χ1 angle for W6.48 that experiments on other GPCRs associate with G-protein activation while all 20 apo-AA3R conformations have a W6.48 gauche+ χ1 angle associated experimentally with inactive GPCRs for other systems. Thus docking calculations have identified critical ligand-GPCR structures involved with activation. We find that the predicted binding site for selective agonist Cl-IB-MECA to the predicted structure of hAA3R shows favorable interactions to three subtype variable residues, I2536.58, V169EL2, and Q167EL2, while the predicted structure for hAA2AR shows weakened to the corresponding amino acids: T2566.58, E169EL2, and L167EL2, explaining the observed subtype selectivity. PMID:21488099

  14. New 2-heterocyclyl-imidazo[2,1-i]purin-5-one derivatives as potent and selective human A3 adenosine receptor antagonists.

    PubMed

    Baraldi, Pier Giovanni; Preti, Delia; Zaid, Abdel Naser; Saponaro, Giulia; Tabrizi, Mojgan Aghazadeh; Baraldi, Stefania; Romagnoli, Romeo; Moorman, Allan R; Varani, Katia; Cosconati, Sandro; Di Maro, Salvatore; Marinelli, Luciana; Novellino, Ettore; Borea, Pier Andrea

    2011-07-28

    A series of 4-allyl/benzyl-7,8-dihydro-8-methyl/ethyl-2-[(substituted)isoxazol/pyrazol-3/5-yl]-1H-imidazo[2,1-i]purin-5(4H)-ones has been synthesized and evaluated in radioligand binding assays to determine their affinities at the human A(1), A(2A), and A(3) adenosine receptors. Efficacy at the hA(2B) AR and antagonism of selected ligands at the hA(3) AR were also assessed through cAMP experiments. All of the synthesized molecules exhibited high affinity at the hA(3) AR (K(i) values ranging from 1.46 to 44.8 nM), as well as remarkable selectivity versus A(1), A(2A), and A(2B) AR subtypes. Compound (R)-4-allyl-8-ethyl-7,8-dihydro-2-(3-methoxy-1-methyl-1H-pyrazol-5-yl)-1H-imidazo[2,1-i]purin-5(4H)-one (R-33) was found to be the most potent and selective ligand of the series (K(i) hA(3) = 1.46 nM, K(i) hA(2A)/K(i) hA(3) > 3425; IC(50) hA(2B)/K(i) hA(3) > 3425; K(i) hA(1)/K(i) hA(3) = 1729). Molecular modeling studies were helpful in rationalizing the available structure-activity relationships along with the selectivity profiles of the new series of ligands.

  15. A1 adenosine receptor allosteric enhancer PD-81723 protects against renal ischemia-reperfusion injury.

    PubMed

    Park, Sang Won; Kim, Joo Yun; Ham, Ahrom; Brown, Kevin M; Kim, Mihwa; D'Agati, Vivette D; Lee, H Thomas

    2012-09-01

    Activation of A(1) adenosine receptors (ARs) protects against renal ischemia-reperfusion (I/R) injury by reducing necrosis, apoptosis, and inflammation. However, extrarenal side effects (bradycardia, hypotension, and sedation) may limit A(1)AR agonist therapy for ischemic acute kidney injury. Here, we hypothesized that an allosteric enhancer for A(1)AR (PD-81723) protects against renal I/R injury without the undesirable side effects of systemic A(1)AR activation by potentiating the cytoprotective effects of renal adenosine generated locally by ischemia. Pretreatment with PD-81723 produced dose-dependent protection against renal I/R injury in A(1)AR wild-type mice but not in A(1)AR-deficient mice. Significant reductions in renal tubular necrosis, neutrophil infiltration, and inflammation as well as tubular apoptosis were observed in A(1)AR wild-type mice treated with PD-81723. Furthermore, PD-81723 decreased apoptotic cell death in human proximal tubule (HK-2) cells in culture, which was attenuated by a specific A(1)AR antagonist (8-cyclopentyl-1,3-dipropylxanthine). Mechanistically, PD-81723 induced sphingosine kinase (SK)1 mRNA and protein expression in HK-2 cells and in the mouse kidney. Supporting a critical role of SK1 in A(1)AR allosteric enhancer-mediated renal protection against renal I/R injury, PD-81723 failed to protect SK1-deficient mice against renal I/R injury. Finally, proximal tubule sphingosine-1-phosphate type 1 receptors (S1P(1)Rs) are critical for PD-81723-induced renal protection, as mice selectively deficient in renal proximal tubule S1P(1)Rs (S1P(1)R(flox/flox) PEPCK(Cre/-) mice) were not protected against renal I/R injury with PD-81723 treatment. Taken together, our experiments demonstrate potent renal protection with PD-81723 against I/R injury by reducing necrosis, inflammation, and apoptosis through the induction of renal tubular SK1 and activation of proximal tubule S1P(1)Rs. Our findings imply that selectively enhancing A(1)AR activation

  16. Adenosine-Induced Atrial Fibrillation: Localized Reentrant Drivers in Lateral Right Atria due to Heterogeneous Expression of Adenosine A1 Receptors and GIRK4 Subunits in the Human Heart.

    PubMed

    Li, Ning; Csepe, Thomas A; Hansen, Brian J; Sul, Lidiya V; Kalyanasundaram, Anuradha; Zakharkin, Stanislav O; Zhao, Jichao; Guha, Avirup; Van Wagoner, David R; Kilic, Ahmet; Mohler, Peter J; Janssen, Paul M L; Biesiadecki, Brandon J; Hummel, John D; Weiss, Raul; Fedorov, Vadim V

    2016-08-09

    Adenosine provokes atrial fibrillation (AF) with a higher activation frequency in right atria (RA) versus left atria (LA) in patients, but the underlying molecular and functional substrates are unclear. We tested the hypothesis that adenosine-induced AF is driven by localized reentry in RA areas with highest expression of adenosine A1 receptor and its downstream GIRK (G protein-coupled inwardly rectifying potassium channels) channels (IK,Ado). We applied biatrial optical mapping and immunoblot mapping of various atrial regions to reveal the mechanism of adenosine-induced AF in explanted failing and nonfailing human hearts (n=37). Optical mapping of coronary-perfused atria (n=24) revealed that adenosine perfusion (10-100 µmol/L) produced more significant shortening of action potential durations in RA (from 290±45 to 239±41 ms, 17.3±10.4%; P<0.01) than LA (from 307±24 to 286±23 ms, 6.7±6.6%; P<0.01). In 10 hearts, adenosine induced AF (317±116 s) that, when sustained (≥2 minutes), was primarily maintained by 1 to 2 localized reentrant drivers in lateral RA. Tertiapin (10-100 nmol/L), a selective GIRK channel blocker, counteracted adenosine-induced action potential duration shortening and prevented AF induction. Immunoblotting showed that the superior/middle lateral RA had significantly higher adenosine A1 receptor (2.7±1.7-fold; P<0.01) and GIRK4 (1.7±0.8-fold; P<0.05) protein expression than lateral/posterior LA. This study revealed a 3-fold RA-to-LA adenosine A1 receptor protein expression gradient in the human heart, leading to significantly greater RA versus LA repolarization sensitivity in response to adenosine. Sustained adenosine-induced AF is maintained by reentrant drivers localized in lateral RA regions with the highest adenosine A1 receptor/GIRK4 expression. Selective atrial GIRK channel blockade may effectively treat AF during conditions with increased endogenous adenosine. © 2016 American Heart Association, Inc.

  17. Effects of adenosine and a selective A2A adenosine receptor agonist on hemodynamic and thallium-201 and technetium-99m-sestaMIBI biodistribution and kinetics.

    PubMed

    Mekkaoui, Choukri; Jadbabaie, Farid; Dione, Donald P; Meoli, David F; Purushothaman, Kailasnath; Belardinelli, Luiz; Sinusas, Albert J

    2009-10-01

    The purpose of this study was to compare a selective A(2A) adenosine receptor agonist (regadenoson) with adenosine in clinically relevant canine models with regard to effects on hemodynamics and thallium-201 ((201)Tl) and technetium-99m ((99m)Tc)-sestaMIBI biodistribution and kinetics. The clinical application of vasodilator stress for perfusion imaging requires consideration of the effects of these vasodilating agents on systemic hemodynamics, coronary flow, and radiotracer uptake and clearance kinetics. Sequential imaging and arterial blood sampling was performed on control, anesthetized closed-chest canines (n = 7) to evaluate radiotracer biodistribution and kinetics after either a bolus administration of regadenoson (2.5 microg/kg) or 4.5-min infusion of adenosine (280 microg/kg). The effects of regadenoson on coronary flow and myocardial radiotracer uptake were then evaluated in an open-chest canine model of a critical stenosis (n = 7). Results from ex vivo single-photon emission computed tomography were compared with tissue well-counting. The use of regadenoson compared favorably with adenosine in regard to the duration and magnitude of the hemodynamic effects and the effect on (201)Tl and (99m)Tc-sestaMIBI biodistribution and kinetics. The arterial blood clearance half-time was significantly faster for (99m)Tc-sestaMIBI (regadenoson: 1.4 +/- 0.03 min; adenosine: 1.5 +/- 0.08 min) than for (201)Tl (regadenoson: 2.5 +/- 0.16 min, p < 0.01; adenosine: 2.7 +/- 0.04 min, p < 0.01) for both vasodilator stressors. The relative microsphere flow deficit (0.34 +/- 0.02%) during regadenoson stress was significantly greater than the relative perfusion defect with (99m)Tc-sestaMIBI (0.69 +/- 0.03%, p < 0.001) or (201)Tl (0.53 +/- 0.02%, p < 0.001), although (201)Tl tracked the flow deficit within the ischemic region better than (99m)Tc-sestaMIBI. The perfusion defect score was larger with (201)Tl (22 +/- 2.8% left ventricular) than with (99m)Tc-sestaMIBI (17 +/- 1

  18. Glucocorticoid receptor activation leads to up-regulation of adenosine A1 receptors and down-regulation of adenosine A2 responses in DDT1 MF-2 smooth muscle cells.

    PubMed

    Gerwins, P; Fredholm, B B

    1991-08-01

    The effect of glucocorticoid treatment of DDT1 MF-2 smooth muscle cells on the signaling via two adenosine receptors with opposing actions on cAMP generation was examined. Treatment with dexamethasone caused a dose- and time-dependent increase in the number of adenosine A1 receptors but did not affect the KD or the proportions of receptors in high and low affinity states. The EC50 was 1 nM dexamethasone, and maximal response was achieved after 24 hr. The number of receptors was increased by approximately 50%. Other steroid hormones, including aldosterone, progesterone, testosterone, and estrogen, were much less effective, and addition of the glucocorticoid receptor antagonist RU 486 or the protein synthesis inhibitor cycloheximide prevented the up-regulation, showing that the effect was mediated via a glucocorticoid receptor-specific mechanism that involves protein synthesis. In dexamethasone-treated cells the A1 receptor agonist (-)-N6-phenylisopropyladenosine [(R)-PIA] was 3 times more potent as an inhibitor of cAMP formation induced by isoprenaline than in untreated cells. ADP ribosylation of inhibitory GTP-binding proteins by pertussis toxin completely prevented (R)-PIA from inhibiting cAMP accumulation. A further analysis of the different GTP-binding proteins, including the three Gi subtypes (Gi1, Gi2, and Gi3), revealed no quantitative or qualitative change after dexamethasone treatment. In addition, the adenosine A2 receptors were down-regulated, as indicated by the fact that the ability of the A2 receptor agonist 5'-N-ethylcarboxamidoadenosine to increase cAMP formation was decreased by 20-30% in dexamethasone-treated cells. In summary, we have shown that A1 and A2 receptors on the same cell are differentially regulated by glucocorticoids and that this has functional importance in the regulation of cAMP accumulation.

  19. New perspectives in signaling mediated by receptors coupled to stimulatory G protein: the emerging significance of cAMP efflux and extracellular cAMP-adenosine pathway

    PubMed Central

    Godinho, Rosely O.; Duarte, Thiago; Pacini, Enio S. A.

    2015-01-01

    G protein-coupled receptors (GPCRs) linked to stimulatory G (Gs) proteins (GsPCRs) mediate increases in intracellular cyclic AMP as consequence of activation of nine adenylyl cyclases , which differ considerably in their cellular distribution and activation mechanisms. Once produced, cyclic AMP may act via distinct intracellular signaling effectors such as protein kinase A and the exchange proteins activated by cAMP (Epacs). More recently, attention has been focused on the efflux of cAMP through a specific transport system named multidrug resistance proteins that belongs to the ATP-binding cassette transporter superfamily. Outside the cell, cAMP is metabolized into adenosine, which is able to activate four distinct subtypes of adenosine receptors, members of the GPCR family: A1, A2A, A2B, and A3. Taking into account that this phenomenon occurs in numerous cell types, as consequence of GsPCR activation and increment in intracellular cAMP levels, in this review, we will discuss the impact of cAMP efflux and the extracellular cAMP-adenosine pathway on the regulation of GsPCR-induced cell response. PMID:25859216

  20. New perspectives in signaling mediated by receptors coupled to stimulatory G protein: the emerging significance of cAMP efflux and extracellular cAMP-adenosine pathway.

    PubMed

    Godinho, Rosely O; Duarte, Thiago; Pacini, Enio S A

    2015-01-01

    G protein-coupled receptors (GPCRs) linked to stimulatory G (Gs) proteins (GsPCRs) mediate increases in intracellular cyclic AMP as consequence of activation of nine adenylyl cyclases , which differ considerably in their cellular distribution and activation mechanisms. Once produced, cyclic AMP may act via distinct intracellular signaling effectors such as protein kinase A and the exchange proteins activated by cAMP (Epacs). More recently, attention has been focused on the efflux of cAMP through a specific transport system named multidrug resistance proteins that belongs to the ATP-binding cassette transporter superfamily. Outside the cell, cAMP is metabolized into adenosine, which is able to activate four distinct subtypes of adenosine receptors, members of the GPCR family: A1, A2A, A2B, and A3. Taking into account that this phenomenon occurs in numerous cell types, as consequence of GsPCR activation and increment in intracellular cAMP levels, in this review, we will discuss the impact of cAMP efflux and the extracellular cAMP-adenosine pathway on the regulation of GsPCR-induced cell response.

  1. Administration of Exogenous ATP to Ischemic Skeletal Muscle Induces an Energy Sparing Effect: Role of Adenosine Receptors

    PubMed Central

    Maldonado, Claudio; Pushpakumar, Sathnur B; Perez-Abadia, Gustavo; Arumugam, Sengodagounder; Lane, Andrew N.

    2012-01-01

    Ischemia and reperfusion (IR) injury is a devastating complication that occurs in allotransplantation and replantation of limbs. Over the years, several preservation strategies have been employed to conserve critical levels of intracellular ATP during ischemia to sustain ion gradients across membranes and thus the viability of tissues. Administration of exogenous ATP to ischemic tissues is known to provide beneficial effects during reperfusion, but it is unclear whether it provides protection during ischemia. The purpose of this study was to determine the effect of ATP administration on high-energy phosphate levels in ischemic skeletal muscle and examine the role of purinergic and adenosine receptors in mediating the response to exogenous ATP. Materials and Methods Extensor Digitorum Longus (EDL) muscles of Fischer rats were subjected to ischemia and treated with different concentrations of ATP with or without purinergic and adenosine receptor blockers. 31P-NMR was used to measure the rate of decay of ATP, phosphocreatinine (PCr) and the formation of AMP and acidification. Phosphorylated compounds were analyzed using a simple model of energy metabolism and PCr half-life was used as an index of internal depletion of ATP to distinguish between intracellular and extracellular ATP. Results PCr decay was rapid in all muscle groups and was followed by a gradual ATP decay. The half-life of PCr was significantly longer in ATP-treated muscles compared to vehicle controls, and was maximally prolonged by treating with slow hydrolyzing ATPγS. Purinoceptor (P2X) blockade with ATP treatment significantly increased the half-life of PCr, whereas adenosine receptor blockers blunted the response. Administration of adenosine to ischemic muscles significantly increased the half-life of PCr compared to vehicle controls. Conclusion Exogenous ATP administration to ischemic skeletal muscles appears to spare intracellular energy by acting primarily through adenosine receptors. PMID

  2. Selective Allosteric Enhancement of Agonist Binding and Function at Human A3 Adenosine Receptors by a Series of Imidazoquinoline Derivatives

    PubMed Central

    Gao, Zhan-Guo; Kim, Seong Gon; Soltysiak, Kelly A.; Melman, Neli; Ijzerman, Adriaan P.; Jacobson, Kenneth A.

    2014-01-01

    We have identified a series of 1H-imidazo-[4,5-c]quinolines as selective allosteric enhancers of human A3 adenosine receptors. Several of these compounds potentiated both the potency and maximal efficacy of agonist-induced responses and selectively decreased the dissociation of the agonist N6-(4-amino-3-[125I]iodobenzyl)-5′-N-methylcarboxamidoadenosine from human A3 adenosine receptors. There was no effect on the dissociation of the antagonist [3H]8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2.1-i]purin-5-one (PSB-11) from the A3 receptors, as well as [3H]N6-[(R)-phenylisopropy-l]adenosine from rat brain A1 receptors and [3H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamidoad-enosine from rat striatal A2A receptors, suggesting the selective enhancement of agonist binding at A3 receptors. The analogs were tested as antagonists of competitive binding at human A3 receptors, and Ki values ranging from 120 nM to 101 μM were observed; as for many allosteric modulators of G protein-coupled receptors, an orthosteric effect was also present. The most promising leads from the present set of analogs seem to be the 2-cyclopentyl-1H-imidazo[4,5-c]quinoline derivatives, of which the 4-phenylamino analog DU124183 had the most favorable degree of allosteric modulation versus receptor antagonism. The inhibition of forskolin-stimulated cyclic AMP accumulation in intact cells that express human A3 receptors was employed as a functional index of A3 receptor activation. The enhancer DU124183 caused a marked leftward shift of the concentration-response curve of the A3 receptor agonists in the presence of antagonist and, surprisingly, a potentiation of the maximum agonist efficacy by approximately 30%. Thus, we have identified a novel structural lead for developing allosteric enhancers of A3 adenosine receptors; such enhancers may be useful for treating brain ischemia and other hypoxic conditions. PMID:12065758

  3. Adenosine signaling in normal and sickle erythrocytes and beyond

    PubMed Central

    Zhang, Yujin; Xia, Yang

    2012-01-01

    Sickle cell disease (SCD) is a debilitating hemolytic genetic disorder with high morbidity and mortality affecting millions of individuals worldwide. Although SCD was discovered more than a century ago, no effective mechanism-based prevention and treatment are available due to poorly understood molecular basis of sickling, the fundamental pathogenic process of the disease. SCD patients constantly face hypoxia. One of the best-known signaling molecules to be induced under hypoxic conditions is adenosine. Recent studies demonstrate that hypoxia-mediated elevated adenosine signaling plays an important role in normal erythrocyte physiology. In contrast, elevated adenosine signaling contributes to sickling and multiple life threatening complications including tissue damage, pulmonary dysfunction and priapism. Here, we summarize recent research on the role of adenosine signaling in normal and sickle erythrocytes, progression of the disease and therapeutic implications. In normal erythrocytes, both genetic and pharmacological studies demonstrate that adenosine can enhance 2,3-bisphosphoglycerate (2,3-BPG) production via A2B receptor (ADORA2B) activation, suggesting that elevated adenosine has an unrecognized role in normal erythrocytes to promote O2 release and prevent acute ischemic tissue injury. However, in sickle erythrocytes, the beneficial role of excessive adenosine-mediated 2,3-BPG induction becomes detrimental by promoting deoxygenation, polymerization of sickle hemoglobin and subsequent sickling. Additionally, adenosine signaling via the A2A receptor (ADORA2A) on invariant natural killer T (iNKT) cells inhibits iNKT cell activation and attenuates pulmonary dysfunction in SCD mice. Finally, elevated adenosine coupled with ADORA2BR activation is responsible for priapism, a dangerous complication seen in SCD. Overall, the research reviewed here reveals a differential role of elevated adenosine in normal erythrocytes, sickle erythrocytes, iNK cells and progression

  4. Adenosine signaling in normal and sickle erythrocytes and beyond.

    PubMed

    Zhang, Yujin; Xia, Yang

    2012-08-01

    Sickle cell disease (SCD) is a debilitating hemolytic genetic disorder with high morbidity and mortality affecting millions of individuals worldwide. Although SCD was discovered more than a century ago, no effective mechanism-based prevention and treatment are available due to poorly understood molecular basis of sickling, the fundamental pathogenic process of the disease. SCD patients constantly face hypoxia. One of the best-known signaling molecules to be induced under hypoxic conditions is adenosine. Recent studies demonstrate that hypoxia-mediated elevated adenosine signaling plays an important role in normal erythrocyte physiology. In contrast, elevated adenosine signaling contributes to sickling and multiple life threatening complications including tissue damage, pulmonary dysfunction and priapism. Here, we summarize recent research on the role of adenosine signaling in normal and sickle erythrocytes, progression of the disease and therapeutic implications. In normal erythrocytes, both genetic and pharmacological studies demonstrate that adenosine can enhance 2,3-bisphosphoglycerate (2,3-BPG) production via A(2B) receptor (ADORA2B) activation, suggesting that elevated adenosine has an unrecognized role in normal erythrocytes to promote O(2) release and prevent acute ischemic tissue injury. However, in sickle erythrocytes, the beneficial role of excessive adenosine-mediated 2,3-BPG induction becomes detrimental by promoting deoxygenation, polymerization of sickle hemoglobin and subsequent sickling. Additionally, adenosine signaling via the A(2A) receptor (ADORA2A) on invariant natural killer T (iNKT) cells inhibits iNKT cell activation and attenuates pulmonary dysfunction in SCD mice. Finally, elevated adenosine coupled with ADORA2BR activation is responsible for priapism, a dangerous complication seen in SCD. Overall, the research reviewed here reveals a differential role of elevated adenosine in normal erythrocytes, sickle erythrocytes, iNK cells and

  5. 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

  6. Ligand-dependent cholesterol interactions with the human A2A adenosine receptor

    PubMed Central

    Lee, Ji Young; Patel, Rohan; Lyman, Edward

    2013-01-01

    We present nearly ten microseconds of all-atom simulation data of a G-protein coupled receptor, the human A2A adenosine receptor, bound to four different ligands. Our focus is on binding of cholesterol to the “cholesterol consensus motif,” a cluster of five amino acids on the second and fourth transmembrane helices, which interact with two cholesterols in the intracellular leaflet of the bilayer. We find evidence for a ligand-specific interaction between the CCM and cholesterol, mediated by the rotameric dynamics and configuration of Trp129. Binding of the synthetic agonist UK432097 disrupts hydrogen bonding between Trp129 and Ser47, which activates the rotameric dynamics of Trp129 and disrupts the interaction with one of the two cholesterols. We also investigate the effect of four thermostabilizing mutations, three of which are located on helix two. The conformational stability of helix two has been proposed to be sensitive to interaction with cholesterol in the CCM, suggesting a mechanism for the thermostabilization. However, our data are instead suggestive of a force-field dependent “straightening” of helix two, and therefore offer no basis for rationalizing the effect of the quadruple mutant. PMID:23454349

  7. Structure-Based Screening of Uncharted Chemical Space for Atypical Adenosine Receptor Agonists.

    PubMed

    Rodríguez, David; Chakraborty, Saibal; Warnick, Eugene; Crane, Steven; Gao, Zhan-Guo; O'Connor, Robert; Jacobson, Kenneth A; Carlsson, Jens

    2016-10-21

    Small molecule screening libraries cover only a small fraction of the astronomical number of possible drug-like compounds, limiting the success of ligand discovery efforts. Computational screening of virtual libraries representing unexplored chemical space could potentially bridge this gap. Drug development for adenosine receptors (ARs) as targets for inflammation and cardiovascular diseases has been hampered by the paucity of agonist scaffolds. To identify novel AR agonists, a virtual library of synthetically tractable nucleosides with alternative bases was generated and structure-based virtual screening guided selection of compounds for synthesis. Pharmacological assays were carried out at three AR subtypes for 13 ribosides. Nine compounds displayed significant activity at the ARs, and several of these represented atypical agonist scaffolds. The discovered ligands also provided insights into receptor activation and revealed unknown interactions of endogenous and clinical compounds with the ARs. The results demonstrate that virtual compound databases provide access to bioactive matter from regions of chemical space that are sparsely populated in commercial libraries, an approach transferrable to numerous drug targets.

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  10. Insulin restores gestational diabetes mellitus-reduced adenosine transport involving differential expression of insulin receptor isoforms in human umbilical vein endothelium.

    PubMed

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

    2011-06-01

    To determine whether insulin reverses gestational diabetes mellitus (GDM)-reduced expression and activity of human equilibrative nucleoside transporters 1 (hENT1) in human umbilical vein endothelium cells (HUVECs). Primary cultured HUVECs from full-term normal (n = 44) and diet-treated GDM (n = 44) pregnancies were used. Insulin effect was assayed on hENT1 expression (protein, mRNA, SLC29A1 promoter activity) and activity (initial rates of adenosine transport) as well as endothelial nitric oxide (NO) synthase activity (serine(1177) phosphorylation, l-citrulline formation). Adenosine concentration in culture medium and umbilical vein blood (high-performance liquid chromatography) as well as insulin receptor A and B expression (quantitative PCR) were determined. Reactivity of umbilical vein rings to adenosine and insulin was assayed by wire myography. Experiments were in the absence or presence of l-N(G)-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) or ZM-241385 (an A(2A)-adenosine receptor antagonist). Umbilical vein blood adenosine concentration was higher, and the adenosine- and insulin-induced NO/endothelium-dependent umbilical vein relaxation was lower in GDM. Cells from GDM exhibited increased insulin receptor A isoform expression in addition to the reported NO-dependent inhibition of hENT1-adenosine transport and SLC29A1 reporter repression, and increased extracellular concentration of adenosine and NO synthase activity. Insulin reversed all these parameters to values in normal pregnancies, an effect blocked by ZM-241385 and l-NAME. GDM and normal pregnancy HUVEC phenotypes are differentially responsive to insulin, a phenomenon where insulin acts as protecting factor for endothelial dysfunction characteristic of this syndrome. Abnormal adenosine plasma levels, and potentially A(2A)-adenosine receptors and insulin receptor A, will play crucial roles in this phenomenon in GDM.

  11. The NLRP3 inflammasome is activated by nanoparticles through ATP, ADP and adenosine.

    PubMed

    Baron, L; Gombault, A; Fanny, M; Villeret, B; Savigny, F; Guillou, N; Panek, C; Le Bert, M; Lagente, V; Rassendren, F; Riteau, N; Couillin, I

    2015-02-05

    The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO2) and titanium dioxide (nano-TiO2) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO2 or nano-TiO2 caused a significant increase in P2Y1, P2Y2, A2A and/or A2B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2A or A2B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation.

  12. The NLRP3 inflammasome is activated by nanoparticles through ATP, ADP and adenosine

    PubMed Central

    Baron, L; Gombault, A; Fanny, M; Villeret, B; Savigny, F; Guillou, N; Panek, C; Le Bert, M; Lagente, V; Rassendren, F; Riteau, N; Couillin, I

    2015-01-01

    The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO2) and titanium dioxide (nano-TiO2) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO2 or nano-TiO2 caused a significant increase in P2Y1, P2Y2, A2A and/or A2B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2A or A2B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation. PMID:25654762

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

    PubMed Central

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

    2009-01-01

    Basal ganglia processing results from a balanced activation of direct and indirect striatal efferent pathways, which are controlled by dopamine D1 and D2 receptors, respectively. Adenosine A2A receptors are considered novel 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. PMID:19936569

  14. 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.

  15. Ligand-Dependent Activation and Deactivation of the Human Adenosine A2A Receptor

    PubMed Central

    Li, Jianing; Jonsson, Amanda L.; Beuming, Thijs; Shelley, John C.; Voth, Gregory A.

    2013-01-01

    G protein-coupled receptors (GPCRs) are membrane proteins with critical functions in cellular signal transduction, representing a primary class of drug targets. Acting by direct binding, many drugs modulate GPCR activity and influence the signaling pathways associated with numerous diseases. However, complete details of ligand-dependent GPCR activation/deactivation are difficult to obtain from experiments. Therefore, it remains unclear how ligands modulate a GPCR’s activity. To elucidate the ligand-dependent activation/deactivation mechanism of the human adenosine A2A receptor (AA2AR), a member of the class A GPCRs, we performed large-scale unbiased molecular dynamics and metadynamics simulations of the receptor embedded in a membrane. At the atomic level, we have observed distinct structural states that resemble the active and inactive states. In particular we noted key structural elements changing in a highly concerted fashion during the conformational transitions, including six conformational states of a tryptophan (Trp2466.48). Our findings agree with a previously proposed view, that during activation, this tryptophan residue undergoes a rotameric transition that may be coupled to a series of coherent conformational changes, resulting in the opening of the G protein-binding site. Further, metadynamics simulations provide quantitative evidence for this mechanism, suggesting how ligand binding shifts the equilibrium between the active and inactive states. Our analysis also proposes that a few specific residues are associated with agonism/antagonism, affinity and selectivity, and suggests that the ligand-binding pocket can be thought of as having three distinct regions, providing dynamic features for structure-based design. Additional simulations with AA2AR bound to a novel ligand are consistent with our proposed mechanism. Generally, our study provides insights into the ligand-dependent AA2AR activation/deactivation in addition to what has been found in crystal

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

    PubMed Central

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

    2017-01-01

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

  17. Reinforcing and neurochemical effects of cannabinoid CB1 receptor agonists, but not cocaine, are altered by an adenosine A2A receptor antagonist

    PubMed Central

    Justinová, Zuzana; Ferré, Sergi; Redhi, Godfrey H.; Mascia, Paola; Stroik, Jessica; Quarta, Davide; Yasar, Sevil; Müller, Christa E.; Franco, Rafael; Goldberg, Steven R.

    2010-01-01

    Several recent studies suggest functional and molecular interactions between striatal adenosine A2A and cannabinoid CB1 receptors. Here we demonstrate that A2A receptors selectively modulate reinforcing effects of cannabinoids. We studied effects of A2A receptor blockade on the reinforcing effects of delta-9-tetrahydrocannabinol (THC) and the endogenous CB1 receptor ligand anandamide under a fixed-ratio (FR) schedule of intravenous drug injection in squirrel monkeys. A low dose of the selective adenosine A2A receptor antagonist MSX-3 (1 mg/kg) caused downward shifts of THC and anandamide dose-response curves. In contrast, a higher dose of MSX-3 (3 mg/kg) shifted THC and anandamide dose-response curves to the left. MSX-3 did not modify cocaine or food-pellet self-administration. Also, MSX-3 neither promoted reinstatement of extinguished drug-seeking behavior nor altered reinstatement of drug-seeking behavior by non-contingent priming injections of THC. Finally, using in-vivo microdialysis in freely-moving rats, a behaviorally active dose of MSX-3 significantly counteracted THC-induced, but not cocaine-induced, increases in extracellular dopamine levels in the nucleus accumbens shell. The significant and selective results obtained with the lower dose of MSX-3 suggest that adenosine A2A antagonists acting preferentially at presynaptic A2A receptors might selectively reduce reinforcing effects of cannabinoids that lead to their abuse. However, the appearance of potentiating rather than suppressing effects on cannabinoid reinforcement at the higher dose of MSX-3 would likely preclude the use of such a compound as a medication for cannabis abuse. Adenosine A2A antagonists with more selectivity for presynaptic versus postsynaptic receptors could be potential medications for treatment of cannabis abuse. PMID:21054689

  18. Effects of caffeine on behavioral and inflammatory changes elicited by copper in zebrafish larvae: Role of adenosine receptors.

    PubMed

    Cruz, Fernanda Fernandes; Leite, Carlos Eduardo; Kist, Luiza Wilges; de Oliveira, Giovanna Medeiros; Bogo, Maurício Reis; Bonan, Carla Denise; Campos, Maria Martha; Morrone, Fernanda Bueno

    2017-04-01

    This study investigated the effects of caffeine in the behavioral and inflammatory alterations caused by copper in zebrafish larvae, attempting to correlate these changes with the modulation of adenosine receptors. To perform a survival curve, 7dpf larvae were exposed to 10μM CuSO 4 , combined to different concentrations of caffeine (100μM, 500μM and 1mM) for up to 24h. The treatment with copper showed lower survival rates only when combined with 500μM and 1mM of caffeine. We selected 4 and 24h as treatment time-points. The behavior evaluation was done by analyzing the traveled distance, the number of entries in the center, and the length of permanence in the center and the periphery of the well. The exposure to 10μM CuSO 4 plus 500μM caffeine at 4 and 24h changed the behavioral parameters. To study the inflammatory effects of caffeine, we assessed the PGE 2 levels by using UHPLC-MS/MS, and TNF, COX-2, IL-6 and IL-10 gene expression by RT-qPCR. The expression of adenosine receptors was also evaluated with RT-qPCR. When combined to copper, caffeine altered inflammatory markers depending on the time of exposure. Adenosine receptors expression was significantly increased, especially after 4h exposure to copper and caffeine together or separately. Our results demonstrated that caffeine enhances the inflammation induced by copper by decreasing animal survival, altering inflammatory markers and promoting behavioral changes in zebrafish larvae. We also conclude that alterations in adenosine receptors are related to those effects. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Blockade of adenosine A2A receptors reverses short-term social memory impairments in spontaneously hypertensive rats.

    PubMed

    Prediger, Rui D S; Fernandes, Daniel; Takahashi, Reinaldo N

    2005-04-30

    Spontaneously hypertensive rats (SHR) exhibit impairment across several cognitive domains such as attention, short-term memory and spatial reference memory. These cognitive deficits have been variously attributed to disrupted dopaminergic, cholinergic and adenosinergic neurotransmitter function. However, social memory in SHR has not been investigated. In the present study, we therefore evaluated whether SHR exhibit altered short-term social memory abilities compared to normotensive Wistar rats (WIS) through two experimental paradigms (social recognition and habituation-dishabituation tests). We also compared the performance of SHR and WIS rats in the object recognition test. SHR exhibited significantly impaired performance in both models of social memory, but not in the object recognition test, demonstrating a selective deficit in the ability to recognize a juvenile rat after a short period of time. The administration of acute doses of the non-selective adenosine receptor antagonist caffeine (3.0 or 10.0 mg/kg, i.p.) and the adenosine A2A receptor antagonist 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-yl-amino]ethyl) phenol (ZM241385, 0.5 or 1.0 mg/kg, i.p.) but not the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1.0 or 3.0 mg/kg, i.p.) reversed this social memory impairment in SHR, but these treatments did not alter the hypertension state. These results demonstrate an impairment of short-term social memory in SHR and the involvement of the adenosine A2A receptors in this alteration.

  20. Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes

    PubMed Central

    Ginés, Silvia; Hillion, Joëlle; Torvinen, Maria; Le Crom, Stèphane; Casadó, Vicent; Canela, Enric I.; Rondin, Sofia; Lew, Jow Y.; Watson, Stanley; Zoli, Michele; Agnati, Luigi Francesco; Vernier, Philippe; Lluis, Carmen; Ferré, Sergi; Fuxe, Kjell; Franco, Rafael

    2000-01-01

    The possible molecular basis for the previously described antagonistic interactions between adenosine A1 receptors (A1R) and dopamine D1 receptors (D1R) in the brain have been studied in mouse fibroblast Ltk− cells cotransfected with human A1R and D1R cDNAs or with human A1R and dopamine D2 receptor (long-form) (D2R) cDNAs and in cortical neurons in culture. A1R and D1R, but not A1R and D2R, were found to coimmunoprecipitate in cotransfected fibroblasts. This selective A1R/D1R heteromerization disappeared after pretreatment with the D1R agonist, but not after combined pretreatment with D1R and A1R agonists. A high degree of A1R and D1R colocalization, demonstrated in double immunofluorescence experiments with confocal laser microscopy, was found in both cotransfected fibroblast cells and cortical neurons in culture. On the other hand, a low degree of A1R and D2R colocalization was observed in cotransfected fibroblasts. Pretreatment with the A1R agonist caused coclustering (coaggregation) of A1R and D1R, which was blocked by combined pretreatment with the D1R and A1R agonists in both fibroblast cells and in cortical neurons in culture. Combined pretreatment with D1R and A1R agonists, but not with either one alone, substantially reduced the D1R agonist-induced accumulation of cAMP. The A1R/D1R heteromerization may be one molecular basis for the demonstrated antagonistic modulation of A1R of D1R receptor signaling in the brain. The persistence of A1R/D1R heteromerization seems to be essential for the blockade of A1R agonist-induced A1R/D1R coclustering and for the desensitization of the D1R agonist-induced cAMP accumulation seen on combined pretreatment with D1R and A1R agonists, which indicates a potential role of A1R/D1R heteromers also in desensitization mechanisms and receptor trafficking. PMID:10890919

  1. Hypothermia in mouse is caused by adenosine A1 and A3 receptor agonists and AMP via three distinct mechanisms.

    PubMed

    Carlin, Jesse Lea; Jain, Shalini; Gizewski, Elizabeth; Wan, Tina C; Tosh, Dilip K; Xiao, Cuiying; Auchampach, John A; Jacobson, Kenneth A; Gavrilova, Oksana; Reitman, Marc L

    2017-03-01

    Small mammals have the ability to enter torpor, a hypothermic, hypometabolic state, allowing impressive energy conservation. Administration of adenosine or adenosine 5'-monophosphate (AMP) can trigger a hypothermic, torpor-like state. We investigated the mechanisms for hypothermia using telemetric monitoring of body temperature in wild type and receptor knock out (Adora1 -/- , Adora3 -/- ) mice. Confirming prior data, stimulation of the A 3 adenosine receptor (AR) induced hypothermia via peripheral mast cell degranulation, histamine release, and activation of central histamine H 1 receptors. In contrast, A 1 AR agonists and AMP both acted centrally to cause hypothermia. Commonly used, selective A 1 AR agonists, including N 6 -cyclopentyladenosine (CPA), N 6 -cyclohexyladenosine (CHA), and MRS5474, caused hypothermia via both A 1 AR and A 3 AR when given intraperitoneally. Intracerebroventricular dosing, low peripheral doses of Cl-ENBA [(±)-5'-chloro-5'-deoxy-N 6 -endo-norbornyladenosine], or using Adora3 -/- mice allowed selective stimulation of A 1 AR. AMP-stimulated hypothermia can occur independently of A 1 AR, A 3 AR, and mast cells. A 1 AR and A 3 AR agonists and AMP cause regulated hypothermia that was characterized by a drop in total energy expenditure, physical inactivity, and preference for cooler environmental temperatures, indicating a reduced body temperature set point. Neither A 1 AR nor A 3 AR was required for fasting-induced torpor. A 1 AR and A 3 AR agonists and AMP trigger regulated hypothermia via three distinct mechanisms. Published by Elsevier Ltd.

  2. Adenosine A1 receptor antagonist rolofylline alleviates axonopathy caused by human Tau ΔK280

    PubMed Central

    Dennissen, Frank J. A.; Anglada-Huguet, Marta; Sydow, Astrid; Mandelkow, Eckhard; Mandelkow, Eva-Maria

    2016-01-01

    Accumulation of Tau is a characteristic hallmark of several neurodegenerative diseases but the mode of toxic action of Tau is poorly understood. Here, we show that the Tau protein is toxic due to its aggregation propensity, whereas phosphorylation and/or missorting is not sufficient to cause neuronal dysfunction. Aggregate-prone Tau accumulates, when expressed in vitro at near-endogenous levels, in axons as spindle-shaped grains. These axonal grains contain Tau that is folded in a pathological (MC-1) conformation. Proaggregant Tau induces a reduction of neuronal ATP, concomitant with loss of dendritic spines. Counterintuitively, axonal grains of Tau are not targeted for degradation and do not induce a molecular stress response. Proaggregant Tau causes neuronal and astrocytic hypoactivity and presynaptic dysfunction instead. Here, we show that the adenosine A1 receptor antagonist rolofylline (KW-3902) is alleviating the presynaptic dysfunction and restores neuronal activity as well as dendritic spine levels in vitro. Oral administration of rolofylline for 2-wk to 14-mo-old proaggregant Tau transgenic mice restores the spatial memory deficits and normalizes the basic synaptic transmission. These findings make rolofylline an interesting candidate to combat the hypometabolism and neuronal dysfunction associated with Tau-induced neurodegenerative diseases. PMID:27671637

  3. Sympathetic activity relates to adenosine A(2A) receptor gene variation in blood-injury phobia.

    PubMed

    Hohoff, C; Domschke, K; Schwarte, K; Spellmeyer, G; Vögele, C; Hetzel, G; Deckert, J; Gerlach, A L

    2009-06-01

    Variation in the candidate genes adenosine A(2A) receptor (A(2A)R), catechol-O-methyl-transferase (COMT), and norepinephrine transporter (NET) has been suggested to influence vulnerability to panic disorder. We therefore investigated patients with another anxiety disorder with an even higher heritability, the blood-injury phobia, for association of these variants and used sympathetic measures during venipuncture, which serve as a naturalistic trigger of anxiety and autonomic hyperarousal, as an intermediate phenotype of anxiety. Patients homozygous for the A(2A)R 1976T allele as compared to patients carrying at least one 1976C allele exhibited a significantly increased respiratory rate with a trend towards elevated measures of systolic and diastolic blood pressure and respiratory minute volume. None of the sympathetic measures were influenced by the COMT or NET polymorphisms.This study provides preliminary data suggesting an influence of the A(2A)R 1976C/T polymorphism on sympathetic psychophysiological indicators of anxiety-related arousal in blood-injury phobia and thereby further supports a role of the A(2A)R gene in the pathogenesis of anxiety disorders.

  4. Anticonvulsant effect of AMP by direct activation of adenosine A1 receptor.

    PubMed

    Muzzi, Mirko; Coppi, Elisabetta; Pugliese, Anna Maria; Chiarugi, Alberto

    2013-12-01

    Purinergic neurotransmission mediated by adenosine (Ado) type 1 receptors (A1Rs) plays pivotal roles in negative modulation of epileptic seizures, and Ado is thought to be a key endogenous anticonvulsant. Recent evidence, however, indicates that AMP, the metabolic precursor of Ado, also activate A1Rs. Here, we evaluated the antiepileptic effects of AMP adopting in vitro and in vivo models of epilepsy. We report that AMP reversed the increase in population spike (PS) amplitude and the decrease in PS latency induced by a Mg(2+)-free extracellular solution in CA1 neurons of mouse hippocampal slices. The AMP effects were inhibited by the A1R antagonist DPCPX, but not prevented by inhibiting conversion of AMP into Ado, indicating that AMP inhibited per se sustained hippocampal excitatory neurotransmission by directly activating A1Rs. AMP also reduced seizure severity and mortality in a model of audiogenic convulsion. Of note, the anticonvulsant effects of AMP were potentiated by preventing its conversion into Ado and inhibited by DPCPX. When tested in a model of kainate-induced seizure, AMP prolonged latency of convulsions but had no effects on seizure severity and mortality. Data provide the first evidence that AMP is an endogenous anticonvulsant acting at A1Rs. © 2013.

  5. Inosine Released from Dying or Dead Cells Stimulates Cell Proliferation via Adenosine Receptors.

    PubMed

    Chen, Jin; Chaurio, Ricardo A; Maueröder, Christian; Derer, Anja; Rauh, Manfred; Kost, Andriy; Liu, Yi; Mo, Xianming; Hueber, Axel; Bilyy, Rostyslav; Herrmann, Martin; Zhao, Yi; Muñoz, Luis E

    2017-01-01

    Many antitumor therapies induce apoptotic cell death in order to cause tumor regression. Paradoxically, apoptotic cells are also known to promote wound healing, cell proliferation, and tumor cell repopulation in multicellular organisms. We aimed to characterize the nature of the regenerative signals concentrated in the micromilieu of dead and dying cells. Cultures of viable melanoma B16F10 cells, mouse fibroblasts, and primary human fibroblast-like synoviocytes (FLS) in the presence of dead and dying cells, their supernatants (SNs), or purified agonists and antagonists were used to evaluate the stimulation of proliferation. Viable cell quantification was performed by either flow cytometry of harvested cells or by crystal violet staining of adherent cells. High-performance liquid chromatography and liquid chromatography coupled with mass spectrometry of cell SNs were deployed to identify the nature of growth-promoting factors. Coimplantation of living cells in the presence of SNs collected from dead and dying cells and specific agonists was used to evaluate tumor growth in vivo . The stimulation of proliferation of few surviving cells by bystander dead cells was confirmed for melanoma cells, mouse fibroblasts, and primary FLS. We found that small soluble molecules present in the protein-free fraction of SNs of dead and dying cells were responsible for the promotion of proliferation. The nucleoside inosine released by dead and dying cells acting via adenosine receptors was identified as putative inducer of proliferation of surviving tumor cells after irradiation and heat treatment. Inosine released by dead and dying cells mediates tumor cell proliferation via purinergic receptors. Therapeutic strategies surmounting this pathway may help to reduce the rate of recurrence after radio- and chemotherapy.

  6. Structural Probing of Off-Target G Protein-Coupled Receptor Activities within a Series of Adenosine/Adenine Congeners

    PubMed Central

    Paoletta, Silvia; Tosh, Dilip K.; Salvemini, Daniela; Jacobson, Kenneth A.

    2014-01-01

    We studied patterns of off-target receptor interactions, mostly at G protein-coupled receptors (GPCRs) in the µM range, of nucleoside derivatives that are highly engineered for nM interaction with adenosine receptors (ARs). Because of the considerable interest of using AR ligands for treating diseases of the CNS, we used the Psychoactive Drug Screening Program (PDSP) for probing promiscuity of these adenosine/adenine congeners at 41 diverse receptors, channels and a transporter. The step-wise truncation of rigidified, trisubstituted (at N6, C2, and 5′ positions) nucleosides revealed unanticipated interactions mainly with biogenic amine receptors, such as adrenergic receptors and serotonergic receptors, with affinities as high as 61 nM. The unmasking of consistent sets of structure activity relationship (SAR) at novel sites suggested similarities between receptor families in molecular recognition. Extensive molecular modeling of the GPCRs affected suggested binding modes of the ligands that supported the patterns of SAR at individual receptors. In some cases, the ligand docking mode closely resembled AR binding and in other cases the ligand assumed different orientations. The recognition patterns for different GPCRs were clustered according to which substituent groups were tolerated and explained in light of the complementarity with the receptor binding site. Thus, some likely off-target interactions, a concern for secondary drug effects, can be predicted for analogues of this set of substructures, aiding the design of additional structural analogues that either eliminate or accentuate certain off-target activities. Moreover, similar analyses could be performed for unrelated structural families for other GPCRs. PMID:24859150

  7. 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.

  8. Astrocytic Lrp4 (Low-Density Lipoprotein Receptor-Related Protein 4) Contributes to Ischemia-Induced Brain Injury by Regulating ATP Release and Adenosine-A2AR (Adenosine A2A Receptor) Signaling.

    PubMed

    Ye, Xin-Chun; Hu, Jin-Xia; Li, Lei; Li, Qiang; Tang, Fu-Lei; Lin, Sen; Sun, Dong; Sun, Xiang-Dong; Cui, Gui-Yun; Mei, Lin; Xiong, Wen-Cheng

    2018-01-01

    Lrp4 (low-density lipoprotein receptor-related protein 4) is predominantly expressed in astrocytes, where it regulates glutamatergic neurotransmission by suppressing ATP release. Here, we investigated Lrp4's function in ischemia/stroke-induced brain injury response, which includes glutamate-induced neuronal death and reactive astrogliosis. The brain-specific Lrp4 conditional knockout mice (Lrp4 GFAP-Cre ), astrocytic-specific Lrp4 conditional knockout mice (Lrp4 GFAP-creER ), and their control mice (Lrp4 f/f ) were subjected to photothrombotic ischemia and the transient middle cerebral artery occlusion. After ischemia/stroke, mice or their brain samples were subjected to behavior tests, brain histology, immunofluorescence staining, Western blot, and quantitative real-time polymerase chain reaction. In addition, primary astrocytes and neurons were cocultured with or without oxygen and glucose deprivation and in the presence or absence of the antagonist for adenosine-A 2A R (adenosine A2A receptor) or ATP-P2X7R (P2X purinoceptor 7) signaling. Gliotransmitters, such as glutamate, d-serine, ATP, and adenosine, in the condition medium of cultured astrocytes were also measured. Lrp4, largely expressed in astrocytes, was increased in response to ischemia/stroke. Both Lrp4 GFAP-Cre and Lrp4 GFAP-creER mice showed less brain injury, including reduced neuronal death, and impaired reactive astrogliosis. Mechanistically, Lrp4 conditional knockout in astrocytes increased ATP release and the production of ATP derivative, adenosine, which were further elevated by oxygen and glucose deprivation. Pharmacological inhibition of ATP-P 2 X 7 R or adenosine-A 2A R signaling diminished Lrp4 GFAP-creER 's protective effect. The astrocytic Lrp4 plays an important role in ischemic brain injury response. Lrp4 deficiency in astrocytes seems to be protective in response to ischemic brain injury, likely because of the increased ATP release and adenosine-A 2A R signaling. © 2017 American Heart

  9. Methodical Challenges and a Possible Resolution in the Assessment of Receptor Reserve for Adenosine, an Agonist with Short Half-Life.

    PubMed

    Zsuga, Judit; Erdei, Tamas; Szabó, Katalin; Lampe, Nora; Papp, Csaba; Pinter, Akos; Szentmiklosi, Andras Jozsef; Juhasz, Bela; Szilvássy, Zoltán; Gesztelyi, Rudolf

    2017-05-19

    The term receptor reserve, first introduced and used in the traditional receptor theory, is an integrative measure of response-inducing ability of the interaction between an agonist and a receptor system (consisting of a receptor and its downstream signaling). The underlying phenomenon, i.e., stimulation of a submaximal fraction of receptors can apparently elicit the maximal effect (in certain cases), provides an opportunity to assess the receptor reserve. However, determining receptor reserve is challenging for agonists with short half-lives, such as adenosine. Although adenosine metabolism can be inhibited several ways (in order to prevent the rapid elimination of adenosine administered to construct concentration-effect (E/c) curves for the determination), the consequent accumulation of endogenous adenosine biases the results. To address this problem, we previously proposed a method, by means of which this bias can be mathematically corrected (utilizing a traditional receptor theory-independent approach). In the present investigation, we have offered in silico validation of this method by simulating E/c curves with the use of the operational model of agonism and then by evaluating them using our method. We have found that our method is suitable to reliably assess the receptor reserve for adenosine in our recently published experimental setting, suggesting that it may be capable for a qualitative determination of receptor reserve for rapidly eliminating agonists in general. In addition, we have disclosed a possible interference between FSCPX (8-cyclopentyl- N³ -[3-(4-(fluorosulfonyl)benzoyloxy)propyl]- N ¹-propylxanthine), an irreversible A₁ adenosine receptor antagonist, and NBTI (S-(2-hydroxy-5-nitrobenzyl)-6-thioinosine), a nucleoside transport inhibitor, i.e., FSCPX may blunt the effect of NBTI.

  10. GDNF control of the glutamatergic cortico-striatal pathway requires tonic activation of adenosine A2A Receptors

    PubMed Central

    Gomes, Catarina A.R.V.; Simões, Patrícia F.; Canas, Paula M.; Quiroz, César; Sebastião, Ana M.; Ferré, Sergi; Cunha, Rodrigo A.; Ribeiro, Joaquim A.

    2009-01-01

    Glial cell line-derived neurotrophic factor (GDNF) affords neuroprotection in Parkinson’s disease in accordance with its ability to bolster nigrostriatal innervation. We previously found that GDNF facilitates dopamine release in a manner dependent on adenosine A2A receptor activation. Since motor dysfunction also involves modifications of striatal glutamatergic innervation, we now tested if GDNF and its receptor system, Ret (rearranged during transfection) and GFRα1 (GDNF family receptor alpha 1) controlled the cortico-striatal glutamatergic pathway in an A2A receptor-dependent manner. GDNF (10 ng/ml) enhanced (by ≈13%) glutamate release from rat striatal nerve endings, an effect potentiated (up to ≈ 30%) by the A2A receptor agonist CGS 21680 (10 nM) and prevented by the A2A receptor antagonist, SCH 58261 (50 nM). Triple immunocytochemical studies revealed that Ret and GFRα1 were located in 50% of rat striatal glutamatergic terminals (immunopositive for vesicular glutamate transporters-1/2), where they were found to be co-located with A2A receptors. Activation of the glutamatergic system upon in vivo electrical stimulation of the rat cortico-striatal input induced striatal Ret phosphoprylation that was prevented by pre-treatment with the A2A receptor antagonist, MSX-3 (3 mg/kg). The results provide the first functional and morphological evidence that GDNF controls cortico-striatal glutamatergic pathways in a manner largely dependent on the co-activation of adenosine A2A receptors. PMID:19141075

  11. Singular Location and Signaling Profile of Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Dorsal Striatum.

    PubMed

    Moreno, Estefanía; Chiarlone, Anna; Medrano, Mireia; Puigdellívol, Mar; Bibic, Lucka; Howell, Lesley A; Resel, Eva; Puente, Nagore; Casarejos, María J; Perucho, Juan; Botta, Joaquín; Suelves, Nuria; Ciruela, Francisco; Ginés, Silvia; Galve-Roperh, Ismael; Casadó, Vicent; Grandes, Pedro; Lutz, Beat; Monory, Krisztina; Canela, Enric I; Lluís, Carmen; McCormick, Peter J; Guzmán, Manuel

    2018-04-01

    The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A 2A receptor (A 2A R) and cannabinoid CB 1 receptor (CB 1 R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A 2A R and CB 1 R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A 2A R-CB 1 R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically modified animal models, together with biochemical and pharmacological approaches, we provide a high-resolution expression map and a detailed functional characterization of A 2A R-CB 1 R heteromers in the dorsal striatum. Specifically, our data unveil that the A 2A R-CB 1 R heteromer (i) is essentially absent from corticostriatal projections and striatonigral neurons, and, instead, is largely present in striatopallidal neurons, (ii) displays a striking G protein-coupled signaling profile, where co-stimulation of both receptors leads to strongly reduced downstream signaling, and (iii) undergoes an unprecedented dysfunction in Huntington's disease, an archetypal disease that affects striatal neurons. Altogether, our findings may open a new conceptual framework to understand the role of coordinated adenosine-endocannabinoid signaling in the indirect striatal pathway, which may be relevant in motor function and neurodegenerative diseases.

  12. Sex Differences in the Neurotoxic Effects of Adenosine A1 Receptor Antagonism During Ethanol Withdrawal: Reversal With an A1 Receptor Agonist or an NMDA Receptor Antagonist

    PubMed Central

    Butler, Tracy R.; Smith, Katherine J.; Self, Rachel L.; Braden, Brittany B.; Prendergast, Mark A.

    2009-01-01

    Background Neuronal adaptations that occur during chronic ethanol (EtOH) exposure have been observed to sensitize the brain to excitotoxic insult during withdrawal. The adenosine receptor system warrants further examination in this regard, as recent evidence has implicated adenosine receptor involvement in the behavioral effects of both EtOH exposure and withdrawal. Methods The current studies examined effects of adenosine A1 receptor manipulation on neuronal injury in EtOH-naïve and EtOH-withdrawn male and female rat hippocampal slice cultures. EtOH-naïve and EtOH pretreated (43.1 to 26.9 mM from days 5 to 15 DIV) cultures were exposed to the A1 receptor agonist 2-Chloro-N6-cyclopentyladenosine (CCPA; 10 nM), the A1 receptor antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX;10 nM), or the N-methyl-D-aspartate (NMDA) receptor antagonist D,L,-2-amino-5-phosphovalerate (APV; 20 μM) at 15 days in vitro (DIV). Cytotoxicity was measured in the primary neuronal layers of the dentate gyrus, CA3 and CA1 hippocampal regions by quantification of propidium iodide (PI) fluorescence after 24 hours. Immunohistochemical analysis of A1 receptor abundance was conducted in EtOH-naïve and EtOH pretreated slice cultures at 15 DIV. Results Twenty-four hour exposure to DPCPX in EtOH-naïve slice cultures did not produced neurotoxicity in any region of slice cultures. Though withdrawal from 10 day EtOH exposure produced no toxicity in either male or female slice cultures, exposure to DPCPX during 24 hours of EtOH withdrawal produced a marked increase in PI uptake in all hippocampal culture subregions in female cultures (to ~160% of control values). A significant effect for sex was observed in the CA1 region such that toxicity in females cultures exposed to the A1 antagonist during withdrawal was greater than that observed in male cultures. These effects of DPCPX in EtOH withdrawn female and male slices were prevented by co-exposure to either the A1 agonist CCPA or the NMDA

  13. Adenosine and preeclampsia.

    PubMed

    Salsoso, Rocío; Farías, Marcelo; Gutiérrez, Jaime; Pardo, Fabián; Chiarello, Delia I; Toledo, Fernando; Leiva, Andrea; Mate, Alfonso; Vázquez, Carmen M; Sobrevia, Luis

    2017-06-01

    Adenosine is an endogenous nucleoside with pleiotropic effects in different physiological processes including circulation, renal blood flow, immune function, or glucose homeostasis. Changes in adenosine membrane transporters, adenosine receptors, and corresponding intracellular signalling network associate with development of pathologies of pregnancy, including preeclampsia. Preeclampsia is a cause of maternal and perinatal morbidity and mortality affecting 3-5% of pregnancies. Since the proposed mechanisms of preeclampsia development include adenosine-dependent biological effects, adenosine membrane transporters and receptors, and the associated signalling mechanisms might play a role in the pathophysiology of preeclampsia. Preeclampsia associates with increased adenosine concentration in the maternal blood and placental tissue, likely due to local hypoxia and ischemia (although not directly demonstrated), microthrombosis, increased catecholamine release, and platelet activation. In addition, abnormal expression and function of equilibrative nucleoside transporters is described in foetoplacental tissues from preeclampsia; however, the role of adenosine receptors in the aetiology of this disease is not well understood. Adenosine receptors activation may be related to abnormal trophoblast invasion, angiogenesis, and ischemia/reperfusion mechanisms in the placenta from preeclampsia. These mechanisms may explain only a low fraction of the associated abnormal transformation of spiral arteries in preeclampsia, triggering cellular stress and inflammatory mediators release from the placenta to the maternal circulation. Although increased adenosine concentration in preeclampsia may be a compensatory or adaptive mechanism favouring placental angiogenesis, a poor angiogenic state is found in preeclampsia. Thus, preeclampsia-associated complications might affect the cell response to adenosine due to altered expression and activity of adenosine receptors, membrane transporters

  14. 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

  15. Imidazo[2,1-i]purin-5-ones and related tricyclic water-soluble purine derivatives: potent A(2A)- and A(3)-adenosine receptor antagonists.

    PubMed

    Müller, Christa E; Thorand, Mark; Qurishi, Ramatullah; Diekmann, Martina; Jacobson, Kenneth A; Padgett, William L; Daly, John W

    2002-08-01

    A series of tricyclic imidazo[2,1-i]purinones and ring-enlarged analogues derived from xanthine derivatives have been prepared as adenosine receptor (AR) antagonists. In comparison with xanthines, the tricyclic compounds exhibit increased water solubility due to a basic nitrogen atom, which can be protonated under physiological conditions. Substituents were introduced that confer high affinity for A(2A) or A(3) ARs, respectively. A new capillary electrophoresis method was developed for the determination of the enantiomeric purity of selected chiral products using native and modified beta-cyclodextrins as chiral discriminators. The compounds were investigated in radioligand binding assays at rat brain A(1) and A(2A) ARs. Selected compounds were additionally investigated in radioligand binding assays at human recombinant A(3) ARs and in functional studies (adenylate cyclase assays) at A(1) ARs of rat fat cell membranes, A(2A) ARs of rat PC 12 cell membranes, and mouse A(2B) ARs of NIH 3T3 cell membranes. Structure-activity relationships were similar to those of corresponding xanthine derivatives. The 2-styrylimidazopurinones were less potent at A(2A) ARs as compared to 8-styrylxanthine derivatives. The most potent compound at A(2A) ARs was (S)-1,4-dimethyl-8-ethyl-2-styryl-imidazo[2,1-i]purinone (S-25) exhibiting a K(i) value of 424 nM at rat A(2A) ARs. The compound was highly selective for A(2A) receptors vs A(1) and A(3) ARs. Selectivity vs A(2B) ARs, however, was low. Among the 1-unsubstituted 2-phenyl-imidazo[2,1-i]purin-5-one derivatives, very potent and highly selective antagonists for human A(3) ARs were identified. The most potent A(3) antagonist of the present series was (R)-4-methyl-8-ethyl-2-phenyl-imidazo[2,1-i]purin-5-one (R-24) exhibiting a K(i) value of 2.3 nM and high selectivity for A(3) receptors vs all other AR subtypes.

  16. Adenosine A2A Receptors in the Amygdala Control Synaptic Plasticity and Contextual Fear Memory

    PubMed Central

    Simões, Ana Patrícia; Machado, Nuno J; Gonçalves, Nélio; Kaster, Manuella P; Simões, Ana T; Nunes, Ana; Pereira de Almeida, Luís; Goosens, Ki Ann; Rial, Daniel; Cunha, Rodrigo A

    2016-01-01

    The consumption of caffeine modulates working and reference memory through the antagonism of adenosine A2A receptors (A2ARs) controlling synaptic plasticity processes in hippocampal excitatory synapses. Fear memory essentially involves plastic changes in amygdala circuits. However, it is unknown if A2ARs in the amygdala regulate synaptic plasticity and fear memory. We report that A2ARs in the amygdala are enriched in synapses and located to glutamatergic synapses, where they selectively control synaptic plasticity rather than synaptic transmission at a major afferent pathway to the amygdala. Notably, the downregulation of A2ARs selectively in the basolateral complex of the amygdala, using a lentivirus with a silencing shRNA (small hairpin RNA targeting A2AR (shA2AR)), impaired fear acquisition as well as Pavlovian fear retrieval. This is probably associated with the upregulation and gain of function of A2ARs in the amygdala after fear acquisition. The importance of A2ARs to control fear memory was further confirmed by the ability of SCH58261 (0.1 mg/kg; A2AR antagonist), caffeine (5 mg/kg), but not DPCPX (0.5 mg/kg; A1R antagonist), treatment for 7 days before fear conditioning onwards, to attenuate the retrieval of context fear after 24–48 h and after 7–8 days. These results demonstrate that amygdala A2ARs control fear memory and the underlying process of synaptic plasticity in this brain region. This provides a neurophysiological basis for the association between A2AR polymorphisms and phobia or panic attacks in humans and prompts a therapeutic interest in A2ARs to manage fear-related pathologies. PMID:27312408

  17. Multi-Inhibitory Effects of A2A Adenosine Receptor Signaling on Neutrophil Adhesion Under Flow.

    PubMed

    Yago, Tadayuki; Tsukamoto, Hiroki; Liu, Zhenghui; Wang, Ying; Thompson, Linda F; McEver, Rodger P

    2015-10-15

    A2A adenosine receptor (A2AAR) signaling negatively regulates inflammatory responses in many disease models, but the detailed mechanisms remain unclear. We used the selective A2AAR agonist, ATL313, to examine how A2AAR signaling affects human and murine neutrophil adhesion under flow. Treating neutrophils with ATL313 inhibited selectin-induced, β2 integrin-dependent slow rolling and chemokine-induced, β2 integrin-dependent arrest on ICAM-1. ATL313 inhibited selectin-induced β2 integrin extension, which supports slow rolling, and chemokine-induced hybrid domain "swing-out," which supports arrest. Furthermore, ATL313 inhibited integrin outside-in signaling as revealed by reduced neutrophil superoxide production and spreading on immobilized anti-β2 integrin Ab. ATL313 suppressed selectin-triggered activation of Src family kinases (SFKs) and p38 MAPK, chemokine-triggered activation of Ras-related protein 1, and β2 integrin-triggered activation of SFKs and Vav cytoskeletal regulatory proteins. ATL313 activated protein kinase A and its substrate C-terminal Src kinase, an inhibitor of SFKs. Treating neutrophils with a protein kinase A inhibitor blocked the actions of ATL313. In vivo, ATL313-treated neutrophils rolled faster and arrested much less frequently in postcapillary venules of the murine cremaster muscle after TNF-α challenge. Furthermore, ATL313 markedly suppressed neutrophil migration into the peritoneum challenged with thioglycollate. ATL313 did not affect A2AAR-deficient neutrophils, confirming its specificity. Our findings provide new insights into the anti-inflammatory mechanisms of A2AAR signaling and the potential utility of A2AAR agonists in inflammatory diseases. Copyright © 2015 by The American Association of Immunologists, Inc.

  18. A3 adenosine receptor activation decreases mortality and renal and hepatic injury in murine septic peritonitis.

    PubMed

    Lee, H Thomas; Kim, Mihwa; Joo, Jin Deok; Gallos, George; Chen, Jiang-Fan; Emala, Charles W

    2006-10-01

    The role of A3 adenosine receptors (ARs) in sepsis and inflammation is controversial. In this study, we determined the effects of A3AR modulation on mortality and hepatic and renal dysfunction in a murine model of sepsis. To induce sepsis, congenic A3AR knockout mice (A3AR KO) and wild-type control (A3AR WT) mice were subjected to cecal ligation and double puncture (CLP). A3AR KO mice had significantly worse 7-day survival compared with A3AR WT mice. A3AR KO mice also demonstrated significantly higher elevations in plasma creatinine, alanine aminotransferase, aspartate aminotransferase, keratinocyte-derived chemokine, and TNF-alpha 24 h after induction of sepsis compared with A3AR WT mice. Renal cortices from septic A3AR KO mice exhibited increased mRNA encoding proinflammatory cytokines and enhanced nuclear translocation of NF-kB compared with samples from A3AR WT mice. A3AR WT mice treated with N6-(3-iodobenzyl)ADO-5'N-methyluronamide (IB-MECA; a selective A3AR agonist) or 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191; a selective A3AR antagonist) had improved or worsened 7-day survival after induction of sepsis, respectively. Moreover, A3AR WT mice treated with IB-MECA or MRS-1191 showed acutely improved or worsened, respectively, renal and hepatic function following CLP. IB-MECA significantly reduced mortality in mice lacking the A1AR or A2aAR but not the A3AR, demonstrating specificity of IB-MECA in activating A3ARs and mediating protection against sepsis-induced mortality. We conclude that endogenous or exogenous A3AR activation confers significant protection from murine septic peritonitis primarily by attenuating the hyperacute inflammatory response in sepsis.

  19. The Neuroprotective Benefits of Central Adenosine Receptor Stimulation in a Soman Nerve Agent Rat Model

    DTIC Science & Technology

    2014-04-01

    that bilateral micro - injection of CPA into the cholinergic basal forebrain (BF) and of the adenosine A2AR agonist CGS21680 into the GABAergic...stimulation, we hypothesized that bilateral micro -injection of CPA into the cholinergic basal forebrain (BF) and of the adenosine A2AR agonist...were in home cages. Surgery Using aseptic surgical techniques, animals were prepared first with the insertion of an electronic temperature ID

  20. 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

  1. Activation of Adenosine1 Receptors Induces Antidepressant-Like, Anti-Impulsive Effects on Differential Reinforcement of Low-Rate 72-s Behavior in Rats

    PubMed Central

    2012-01-01

    Stress and psychiatric illness have been associated with a dysregulation of glutamatergic neurotransmission. Recently, positive allosteric modulators (PAMs) of the metabotropic glutamate 2 (mGlu2) receptor have been found to exert antidepressant-like activity in rats performing under a differential reinforcement of low rate (DRL) 72-s schedule. An autoreceptor role at glutamatergic synapses is the most salient physiological role played by the mGlu2 receptor. Adenosine A1 receptors play a heteroreceptor role at many of the same forebrain synapses where mGlu2 autoreceptors are found. Agonists and/or PAMs of mGlu2 receptors act similarly to adenosine A1 receptor agonists with respect to a wide range of electrophysiological, biochemical, and behavioral responses mediated by limbic circuitry thought to play a role in the pathophysiology of neuropsychiatric disease and to mediate therapeutic drug effects. Therefore, the role of adenosine A1 receptor activation on rat DRL 72-s behavior was explored to provide preclinical evidence consistent or inconsistent with potential antidepressant effects. The adenosine A1 receptor agonist N6-cyclohexyladenosine (CHA) increased the reinforcement rate, decreased the response rate, and induced a rightward shift in inter-response time distributions in a dose-dependent fashion similar to most known antidepressant drugs. The adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) blocked these antidepressant-like effects. These novel observations with CHA and DPCPX suggest that activation of adenosine A1 receptors could contribute to antidepressant effects, in addition to previous preclinical reports of anxiolytic and antipsychotic effects. By implication, targeting a dysregulated glutamatergic system may be an important principle in discovering novel antidepressant agents that may also possess anti-impulsive activity. PMID:22323824

  2. Medicinal chemistry of adenosine, P2Y and P2X receptors.

    PubMed

    Jacobson, Kenneth A; Müller, Christa E

    2016-05-01

    Pharmacological tool compounds are now available to define action at the adenosine (ARs), P2Y and P2X receptors. We present a selection of the most commonly used agents to study purines in the nervous system. Some of these compounds, including A1 and A3 AR agonists, P2Y1R and P2Y12R antagonists, and P2X3, P2X4 and P2X7 antagonists, are potentially of clinical use in treatment of disorders of the nervous system, such as chronic pain, neurodegeneration and brain injury. Agonists of the A2AAR and P2Y2R are already used clinically, P2Y12R antagonists are widely used antithrombotics and an antagonist of the A2AAR is approved in Japan for treating Parkinson's disease. The selectivity defined for some of the previously introduced compounds has been revised with updated pharmacological characterization, for example, various AR agonists and antagonists were deemed A1AR or A3AR selective based on human data, but species differences indicated a reduction in selectivity ratios in other species. Also, many of the P2R ligands still lack bioavailability due to charged groups or hydrolytic (either enzymatic or chemical) instability. X-ray crystallographic structures of AR and P2YRs have shifted the mode of ligand discovery to structure-based approaches rather than previous empirical approaches. The X-ray structures can be utilized either for in silico screening of chemically diverse libraries for the discovery of novel ligands or for enhancement of the properties of known ligands by chemical modification. Although X-ray structures of the zebrafish P2X4R have been reported, there is scant structural information about ligand recognition in these trimeric ion channels. In summary, there are definitive, selective agonists and antagonists for all of the ARs and some of the P2YRs; while the pharmacochemistry of P2XRs is still in nascent stages. The therapeutic potential of selectively modulating these receptors is continuing to gain interest in such fields as cancer, inflammation, pain

  3. 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. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Combined neuroprotective action of adenosine A1 and cannabinoid CB1 receptors against NMDA-induced excitotoxicity in the hippocampus.

    PubMed

    Serpa, André; Pinto, Isa; Bernardino, Liliana; Cascalheira, José F

    2015-08-01

    Both adenosine A1 and cannabinoid CB1 receptors trigger similar transduction pathways and protect against neurotoxic insults at the hippocampus, but their combined neuroprotective potential has not been investigated. We set forth to assess the combined action of A1 and CB1 receptors against glutamate NMDA receptor-mediated excitotoxicity at the hippocampus. Cell damage was assessed by measuring propidium iodide (PI) uptake and lactate dehydrogenase (LDH) activity released from cultured hippocampal slices exposed to NMDA. Exposure to NMDA (50 µM) for 1 h increased LDH activity by 92% ± 16%. WIN55212-2 (30 µM), a cannabinoid CB1 receptor agonist, decreased NMDA-induced LDH activity by 53% ± 10% while N6-cyclopentyladenosine (CPA, 100 nM), an adenosine A1 receptor selective agonist, decreased it by 37% ± 11%. The combined inhibitory effect of WIN55212-2 and CPA (88% ± 12%) did not differ from the sum of the individual inhibitory effects of each agonist (90% ± 15%) but was different from the effects of CPA or WIN55212-2 alone. An additive inhibitory effect of co-application of WIN55212-2 (30 µM) and CPA (100 nM) on NMDA (50 µM)-induced PI uptake was also observed in CA3 but not in CA1 area of the hippocampal slice. The results suggest that both CB1 and A1 receptors produce additive cumulative neuroprotection against NMDA-induced excitotoxicity in the hippocampus. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Structure-Activity Relationships of Truncated C2- or C8-Substituted Adenosine Derivatives as Dual Acting A2A and A3 Adenosine Receptor Ligands

    PubMed Central

    Hou, Xiyan; Majik, Mahesh S.; Kim, Kyunglim; Pyee, Yuna; Lee, Yoonji; Alexander, Varughese; Chung, Hwa-Jin; Lee, Hyuk Woo; Chandra, Girish; Lee, Jin Hee; Park, Seul-gi; Choi, Won Jun; Kim, Hea Ok; Phan, Khai; Gao, Zhan-Guo; Jacobson, Kenneth A.; Choi, Sun; Lee, Sang Kook; Jeong, Lak Shin

    2011-01-01

    Truncated N6-substituted-4′-oxo- and 4′-thioadenosine derivatives with C2 or C8 substitution were studied as dual acting A2A and A3 adenosine receptor (AR) ligands. The lithiation-mediated stannyl transfer and palladium-catalyzed cross coupling reactions were utilized for functionalization of the C2 position of 6-chloropurine nucleosides. An unsubstituted 6-amino group and a hydrophobic C2 substituent were required for high affinity at the hA2AAR, but hydrophobic C8 substitution abolished binding at the hA2AAR. However, most of synthesized compounds displayed medium to high binding affinity at the hA3AR, regardless of C2 or C8 substitution, and low efficacy in a functional cAMP assay. Several compounds tended to be full hA2AAR agonists. C2 substitution probed geometrically through hA2AAR-docking, was important for binding in order of hexynyl > hexenyl > hexanyl. Compound 4g was the most potent ligand acting dually as hA2AAR agonist and hA3AR antagonist, which might be useful for treatment of asthma or other inflammatory diseases. PMID:22142423

  6. Positive inotropic and sustained anti-beta-adrenergic effect of diadenosine pentaphosphate in human and guinea pig hearts. Role of dinucleotide receptors and adenosine receptors.

    PubMed

    Arvola, L; Bertelsen, G; Hassaf, D; Ytrehus, K

    2004-11-01

    Diadenosine polyphosphates are present intracellularly and in extracellular fluid due to release from secretory vesicles in platelets, chromaffin cells and other cells. This study investigates effects of diadenosine pentaphosphate (AP5A) on heart muscle function. Contractile force amplitude and action potential duration at 90% repolarization (APD90) were measured after challenge with AP5A 50 microm or isoproterenol 50-70 nM in guinea pig papillary muscles. Isoproterenol was given immediately after AP5A-exposure or after 45 min washout. AP5A was combined with antagonists to the purinergic P2 receptor (suramin 100 microm), the dinucleotide receptor [diinosine pentaphosphate 30 microm (IP5I)] or adenosine receptors [8-(P-sulfophenyl) theophylline 50 microm (8-SPT)]. Results are %-change (mean +/- SEM) from value before exposure. AP5A increased contractile force by 22 +/- 3%* (*P <0.05), and IP5I abolished this. AP5A prolonged APD90 by 7 +/- 2%*. AP5A significantly reduced response to isoproterenol acutely from 31 +/- 4* (controls) to 9 +/- 4% and after 45 min washout from 61 +/- 14* (controls) to 16 +/- 5%. 8-SPT abolished the sustained effect. Increase in contractile force by AP5A was confirmed in human atria trabecula preparations. AP5A increased contractile force and prolonged APD90. Contractile force increased by stimulation of the dinucleotide receptor in guinea pig myocardium. The sustained anti-beta-adrenergic effect of AP5A was due to adenosine receptor stimulation.

  7. Structure-Based Scaffold Repurposing for G Protein-Coupled Receptors: Transformation of Adenosine Derivatives into 5HT2B/5HT2CSerotonin Receptor Antagonists.

    PubMed

    Tosh, Dilip K; Ciancetta, Antonella; Warnick, Eugene; Crane, Steven; Gao, Zhan-Guo; Jacobson, Kenneth A

    2016-12-22

    Adenosine derivatives developed to activate adenosine receptors (ARs) revealed micromolar activity at serotonin 5HT 2B and 5HT 2C receptors (5HTRs). We explored the structure-activity relationship at 5HT 2 Rs and modeled receptor interactions in order to optimize affinity and simultaneously reduce AR affinity. Depending on N 6 substitution, small 5'-alkylamide modification maintained 5HT 2B R affinity, which was enhanced upon ribose substitution with rigid bicyclo[3.1.0]hexane (North (N)-methanocarba), e.g., N 6 -dicyclopropylmethyl 4'-CH 2 OH derivative 14 (K i 11 nM). 5'-Methylamide 23 was 170-fold selective as antagonist for 5HT 2B R vs 5HT 2C R. 5'-Methyl 25 and ethyl 26 esters potently antagonized 5HT 2 Rs with moderate selectivity in comparison to ARs; related 6-N,N-dimethylamino analogue 30 was 5HT 2 R-selective. 5' position flexibility of substitution was indicated in 5HT 2B R docking. Both 5'-ester and 5'-amide derivatives displayed in vivo t 1/2 of 3-4 h. Thus, we used G protein-coupled receptor modeling to repurpose nucleoside scaffolds in favor of binding at nonpurine receptors as novel 5HT 2 R antagonists, with potential for cardioprotection, liver protection, or central nervous system activity.

  8. Insulin-Increased L-Arginine Transport Requires A2A Adenosine Receptors Activation in Human Umbilical Vein Endothelium

    PubMed Central

    Guzmán-Gutiérrez, Enrique; Westermeier, Francisco; Salomón, Carlos; González, Marcelo; Pardo, Fabián; Leiva, Andrea; Sobrevia, Luis

    2012-01-01

    Adenosine causes vasodilation of human placenta vasculature by increasing the transport of arginine via cationic amino acid transporters 1 (hCAT-1). This process involves the activation of A2A adenosine receptors (A2AAR) in human umbilical vein endothelial cells (HUVECs). Insulin increases hCAT-1 activity and expression in HUVECs, and A2AAR stimulation increases insulin sensitivity in subjects with insulin resistance. However, whether A2AAR plays a role in insulin-mediated increase in L-arginine transport in HUVECs is unknown. To determine this, we first assayed the kinetics of saturable L-arginine transport (1 minute, 37°C) in the absence or presence of nitrobenzylthioinosine (NBTI, 10 µmol/L, adenosine transport inhibitor) and/or adenosine receptors agonist/antagonists. We also determined hCAT-1 protein and mRNA expression levels (Western blots and quantitative PCR), and SLC7A1 (for hCAT-1) reporter promoter activity. Insulin and NBTI increased the extracellular adenosine concentration, the maximal velocity for L-arginine transport without altering the apparent K m for L-arginine transport, hCAT-1 protein and mRNA expression levels, and SLC7A1 transcriptional activity. An A2AAR antagonist ZM-241385 blocked these effects. ZM241385 inhibited SLC7A1 reporter transcriptional activity to the same extent in cells transfected with pGL3-hCAT-1−1606 or pGL3-hCAT-1−650 constructs in the presence of NBTI + insulin. However, SLC7A1 reporter activity was increased by NBTI only in cells transfected with pGL3-hCAT-1−1606, and the ZM-241385 sensitive fraction of the NBTI response was similar in the absence or in the presence of insulin. Thus, insulin modulation of hCAT-1 expression and activity requires functional A2AAR in HUVECs, a mechanism that may be applicable to diseases associated with fetal insulin resistance, such as gestational diabetes. PMID:22844517

  9. Adenosine A1 receptor antagonist mitigates deleterious effects of sleep deprivation on adult neurogenesis and spatial reference memory in rats.

    PubMed

    Chauhan, G; Ray, K; Sahu, S; Roy, K; Jain, V; Wadhwa, M; Panjwani, U; Kishore, K; Singh, S B

    2016-11-19

    Sleep deprivation (SD) upsurges intracellular levels of adenosine, impairs adult neuronal cell proliferation (NCP) and cognition while caffeine, a non-selective adenosine A1 receptor (A1R) antagonist improves cognition and adult NCP during SD. We examined the selective antagonistic effects of adenosine A1R using 8-cyclopentyl-1,3-dimethylxanthine (8-CPT) on impairment of spatial reference memory and adult NCP during 48h SD. Adult male Sprague Dawley rats were sleep deprived for 48h, using an automatic cage vibrating stimulus based on animal activity. Spatial reference memory was tested as a measure of cognitive performance employing Morris Water Maze. Rats were given 8-CPT dissolved in 50% dimethyl sulfoxide (DMSO), twice daily (10mg/kg, i.p.) along with 5-bromo-2-deoxyuridine (BrdU) (50mg/kg/day, i.p.). The rats treated with 8-CPT showed significantly short mean latency and path-length to reach the platform compared to the SD rats. Consistent with these findings, 8-CPT-treated group was found to have significantly increased the number of BrdU, Ki-67 and doublecortin (DCX) positive cells. However, no significant difference was seen in NeuN expression in the Dentate Gyrus (DG). Brain-derived neurotropic factor (BDNF) expression in the DG and CA1 region was observed to decrease significantly after SD and be rescued by 8-CPT treatment. Furthermore, latency to reach platform showed a negative correlation with number of BrdU, DCX type-1 cells and BDNF expression in DG. Thus, it may be concluded that treatment with 8-CPT, an adenosine A1R antagonist during SD mitigates SD induced decline in spatial reference memory and adult NCP possibly via up regulation of BDNF levels in DG and CA1 regions. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  10. Adenylated dinucleotide binding to the adenosine 5',5'''-P1,P4-tetraphosphate mouse heart receptor.

    PubMed

    Hilderman, R H; Lilien, J E; Zimmerman, J K; Tate, D H; Dimmick, M A; Jones, G B

    1994-04-29

    We have demonstrated specific adenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) receptors at heart cell surfaces. Optimal Ap4A binding requires receptor activation. Other Investigators have demonstrated that Ap5A and Ap6A act as vasopressors. We now compare the binding of Ap4A, Ap5A and Ap6A on heart membranes to determine if all three ligands bind to the same receptor and their relative avidities. Anti-Ap4A receptor antibodies inhibit the binding of all three ligands. SDS-PAGE analysis of Ap4A, Ap5A and Ap6A cross-linked to membranes reveals that all three are attached to a 30 kDa peptide. The specific activity for binding to unactivated membranes is similar for all three ligands. However, after receptor activation there is a 3.4x increase in Ap4A binding and a 32.5x decrease in the KD; values remain unchanged for Ap5A and Ap6A. These data indicate that Ap4A, Ap5A and Ap6A bind to the same receptor on cardiac membranes but receptor activation enhances only Ap4A binding.

  11. Adenosine-Induced Atrial Fibrillation: Localized Reentrant Drivers in Lateral Right Atria due to Heterogeneous Expression of Adenosine A1 Receptors and GIRK4 Subunits in the Human Heart

    PubMed Central

    Li, Ning; Csepe, Thomas A.; Hansen, Brian J.; Sul, Lidiya V.; Kalyanasundaram, Anuradha; Zakharkin, Stanislav O.; Zhao, Jichao; Guha, Avirup; Van Wagoner, David R.; Kilic, Ahmet; Mohler, Peter J; Janssen, Paul ML; Biesiadecki, Brandon; Hummel, John D; Weiss, Raul; Fedorov, Vadim V.

    2016-01-01

    Background Adenosine provokes atrial fibrillation (AF) with a higher activation frequency in right atria (RA) versus left atria (LA) in patients, but the underlying molecular and functional substrates are unclear. We tested the hypothesis that adenosine-induced AF is driven by localized reentry in RA areas with highest expression of adenosine A1 receptor (A1R) and its downstream GIRK channels (IK,Ado). Methods We applied bi-atrial optical mapping and immunoblot mapping of various atrial regions to reveal the mechanism of adenosine-induced AF in explanted failing and non-failing human hearts (n=37). Results Optical mapping of coronary-perfused atria (n=24) revealed that adenosine perfusion (10–100μM) produced more significant shortening of action potential durations (APD80) in RA (from 290±45ms to 239±41ms, 17.3±10.4%; p<0.01) than LA (from 307±24ms to 286±23ms, 6.7±6.6%; p<0.01). In ten hearts, adenosine induced AF (317±116 sec) that, when sustained (≥2 min), was primarily maintained by one/two localized reentrant drivers in lateral RA. Tertiapin (10–100nM), a selective GIRK channel blocker, counteracted adenosine-induced APD shortening and prevented AF induction. Immunoblotting showed that the superior/middle lateral RA had significantly higher A1R (2.7±1.7 fold; p<0.01) and GIRK4 (1.7±0.8 fold; p<0.05) protein expression than lateral/posterior LA. Conclusions This study revealed a three-fold RA-to-LA A1R protein expression gradient in the human heart, leading to significantly greater RA vs. LA repolarization sensitivity in response to adenosine. Sustained adenosine-induced AF is maintained by reentrant drivers localized in lateral RA regions with the highest A1R/GIRK4 expression. Selective atrial GIRK channel blockade may effectively treat AF during conditions with increased endogenous adenosine. PMID:27462069

  12. Adenosine: Direct and Indirect Actions on Gastric Acid Secretion

    PubMed Central

    Arin, Rosa M.; Gorostidi, Adriana; Navarro-Imaz, Hiart; Rueda, Yuri; Fresnedo, Olatz; Ochoa, Begoña

    2017-01-01

    Composed by a molecule of adenine and a molecule of ribose, adenosine is a paradigm of recyclable nucleoside with a multiplicity of functions that occupies a privileged position in the metabolic and regulatory contexts. Adenosine is formed continuously in intracellular and extracellular locations of all tissues. Extracellular adenosine is a signaling molecule, able to modulate a vast range of physiologic responses in many cells and organs, including digestive organs. The adenosine A1, A2A, A2B, and A3 receptors are P1 purinergic receptors, G protein-coupled proteins implicated in tissue protection. This review is focused on gastric acid secretion, a process centered on the parietal cell of the stomach, which contains large amounts of H+/K+-ATPase, the proton pump responsible for proton extrusion during acid secretion. Gastric acid secretion is regulated by an extensive collection of neural stimuli and endocrine and paracrine agents, which act either directly at membrane receptors of the parietal cell or indirectly through other regulatory cells of the gastric mucosa, as well as mechanic and chemic stimuli. In this review, after briefly introducing these points, we condense the current body of knowledge about the modulating action of adenosine on the pathophysiology of gastric acid secretion and update its significance based on recent findings in gastric mucosa and parietal cells in humans and animal models. PMID:29018360

  13. Renal interstitial adenosine is increased in angiotensin II-induced hypertensive rats.

    PubMed

    Franco, Martha; Bautista, Rocio; Pérez-Méndez, Oscar; González, Lidia; Pacheco, Ursino; Sánchez-Lozada, L G; Santamaría, José; Tapia, Edilia; Monreal, Rogelio; Martínez, Flavio

    2008-01-01

    Since marked renal vasoconstriction is observed in angiotensin II (ANG II)-mediated hypertensive rats, we studied the possible interaction between ANG II and adenosine in this model. ANG II was infused into male Wistar rats through osmotic minipumps (435 ng x kg(-1) x min(-1)) for 14 days. In sham and ANG II groups, renal tissue and interstitial adenosine were measured; both increased to a similar twofold extent in the ANG II-treated rats (31.40 +/- 4 vs. 62.0 +/- 8.4 nM, sham vs. ANG II, interstitial adenosine; P< 0.001). The latter decreased by 47% with the specific blockade of 5'-nucleotidase. Glomerular hemodynamics demonstrated marked renal vasoconstriction in the angiotensin-treated group, which was reverted by an adenosine A(1)-receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine, 10 mug.kg(-1) x min(-1)). 5'-Nucleotidase and adenosine deaminase (ADA) activities were measured in the cytosolic and membrane fractions. Only the membrane ADA activity decreased from 1,202 +/- 80 to 900 +/- 50 mU/mg protein in the ANG II-treated rats (P< 0.05), as well as in their protein and mRNA expression. Despite the adenosine elevation, A(1) and A(2b) receptor protein did not change; in contrast, downregulation was observed in A(2a) receptor and upregulation in A(3) receptor. A similar pattern was found in the cortex and in the medulla; mRNA significantly decreased only in the A(3) receptor in both segments. These results suggest that the elevation of renal tissue and interstitial adenosine contributes to the renal vasoconstriction observed in the ANG II-induced hypertension and that it is mediated by a decrease in the activity and expression of ADA, increased production of adenosine, and an induced imbalance in adenosine receptors.

  14. Crystal structure of the adenosine A2A receptor bound to an antagonist reveals a potential allosteric pocket

    PubMed Central

    Sun, Bingfa; Bachhawat, Priti; Chu, Matthew Ling-Hon; Wood, Martyn; Ceska, Tom; Sands, Zara A.; Mercier, Joel; Lebon, Florence; Kobilka, Tong Sun; Kobilka, Brian K.

    2017-01-01

    The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson’s disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl d-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR–BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A2AR–BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1–bound A2AR–BRIL prevented formation of the lattice observed with the ZM241385–bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson’s disease. PMID:28167788

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

    PubMed

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

    2013-08-01

    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. End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the mouse phrenic-nerve diaphragm preparations using conventional intracellular electrophysiological techniques. 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 Cd(2+) , the removal of extracellular Ca(2+) 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. 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. © 2013 The British Pharmacological Society.

  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. Crystal structure of the adenosine A 2A receptor bound to an antagonist reveals a potential allosteric pocket

    SciTech Connect

    Sun, Bingfa; Bachhawat, Priti; Chu, Matthew Ling-Hon

    2017-02-06

    The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson’s disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl D-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR–BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phasemore » diffusion. Whereas A2AR–BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1–bound A2AR–BRIL prevented formation of the lattice observed with the ZM241385–bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson’s disease.« less

  18. 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.

  19. Extracellular ATP and adenosine: The Yin and Yang in immune responses?

    PubMed

    Faas, M M; Sáez, T; de Vos, P

    2017-06-01

    Extracellular adenosine 5'-triphosphate (ATP) and adenosine molecules are intimately involved in immune responses. ATP is mostly a pro-inflammatory molecule and is released during hypoxic condition and by necrotic cells, as well as by activated immune cells and endothelial cells. However, under certain conditions, for instance at low concentrations or at prolonged exposure, ATP may also have anti-inflammatory properties. Extracellular ATP can activate both P2X and P2Y purinergic receptors. Extracellular ATP can be hydrolyzed into adenosine in a two-step enzymatic process involving the ectonucleotidases CD39 (ecto-apyrase) and CD73. These enzymes are expressed by many cell types, including endothelial cells and immune cells. The counterpart of ATP is adenosine, which is produced by breakdown of intra- or extracellular ATP. Adenosine has mainly anti-inflammatory effects by binding to the adenosine, or P1, receptors (A1, A2A, A2B, and A3). These receptors are also expressed in many cells, including immune cells. The final effect of ATP and adenosine in immune responses depends on the fine regulatory balance between the 2 molecules. In the present review, we will discuss the current knowledge on the role of these 2 molecules in the immune responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Role of Adenosine Receptor A2A in Traumatic Optic Neuropathies

    DTIC Science & Technology

    2015-02-01

    interruption of trophic support, oxidative stress, and increased extracellular glutamate levels that result in excitotoxicity. These stimuli associated...processes, including interrup tion of trophic support, oxidative stress, and increased extracellular glu tamate levels that result in excitotoxicity. These...the pathogenesis of DR (Ibrahim et al., 2011a). Adenosine is centrally involved in the signaling cascade of related events, including anti

  1. Adenosine A(2A) receptor gene (ADORA2A) variants may increase autistic symptoms and anxiety in autism spectrum disorder.

    PubMed

    Freitag, Christine M; Agelopoulos, Konstantin; Huy, Ellen; Rothermundt, Matthias; Krakowitzky, Petra; Meyer, Jobst; Deckert, Jürgen; von Gontard, Alexander; Hohoff, Christa

    2010-01-01

    Autism spectrum disorders (ASDs) are heterogeneous disorders presenting with increased rates of anxiety. The adenosine A(2A) receptor gene (ADORA2A) is associated with panic disorder and is located on chromosome 22q11.23. Its gene product, the adenosine A(2A) receptor, is strongly expressed in the caudate nucleus, which also is involved in ASD. As autistic symptoms are increased in individuals with 22q11.2 deletion syndrome, and large 22q11.2 deletions and duplications have been observed in ASD individuals, in this study, 98 individuals with ASD and 234 control individuals were genotyped for eight single-nucleotide polymorphisms in ADORA2A. Nominal association with the disorder was observed for rs2236624-CC, and phenotypic variability in ASD symptoms was influenced by rs3761422, rs5751876 and rs35320474. In addition, association of ADORA2A variants with anxiety was replicated for individuals with ASD. Findings point toward a possible mediating role of ADORA2A variants on phenotypic expression in ASD that need to be replicated in a larger sample.

  2. Adenosine A1 receptor-mediated suppression of carbamazepine-resistant seizure-like events in human neocortical slices.

    PubMed

    Klaft, Zin-Juan; Hollnagel, Jan-Oliver; Salar, Seda; Calişkan, Gürsel; Schulz, Steffen B; Schneider, Ulf C; Horn, Peter; Koch, Arend; Holtkamp, Martin; Gabriel, Siegrun; Gerevich, Zoltan; Heinemann, Uwe

    2016-05-01

    The need for alternative pharmacologic strategies in treatment of epilepsies is pressing for about 30% of patients with epilepsy who do not experience satisfactory seizure control with present treatments. In temporal lobe epilepsy (TLE) even up to 80% of patients are pharmacoresistant, and surgical resection of the ictogenic tissue is only possible for a minority of TLE patients. In this study we investigate purinergic modulation of drug-resistant seizure-like events (SLEs) in human temporal cortex slices. Layer V/VI field potentials from a total of 77 neocortical slices from 17 pharmacoresistant patients were recorded to monitor SLEs induced by application of 8 mM [K(+) ] and 50 μm bicuculline. Activating A1 receptors with a specific agonist completely suppressed SLEs in 73% of human temporal cortex slices. In the remaining slices, incidence of SLEs was markedly reduced. Because a subportion of slices can be pharmacosensitive, we tested effects of an A1 agonist, in slices insensitive to a high dose of carbamazepine (50 μm). Also in these cases the A1 agonist was equally efficient. Moreover, ATP and adenosine blocked or modulated SLEs, an effect mediated not by P2 receptors but rather by adenosine A1 receptors. Selective activation of A1 receptors mediates a strong anticonvulsant action in human neocortical slices from pharmacoresistant patients. We propose that our human slice model of seizure-like activity is a feasible option for future studies investigating new antiepileptic drug (AED) candidates. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

  3. 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. Copyright © 2016 IBRO. All rights reserved.

  4. 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

  5. Caffeine, Adenosine Receptors and Estrogen in Toxin Models of Parkinson’s Disease

    DTIC Science & Technology

    2009-10-01

    disease. W81XWH-04-1-0881 Annual Report (Year 5) - 6 - B. Environmental Neurotoxin Exposure in Military Service – By characterizing the...modeling how gender and estrogen status interact with environmental exposures of relevance to the military (i.e., neurotoxins , caffeine). A better...conditional knockout reagents developed under this award to other environmentally relevant purine targets in PD, including adenosine metabolites like

  6. Role of Adenosine Receptor A2A in Traumatic Optic Neuropathies (Addendum)

    DTIC Science & Technology

    2016-03-01

    provides seizure suppression in various models of epilepsy (Ugarkar et al., 2000); 3) inhibition of AK in hippocampal slicesincreases endogenous...Diabetes 2003;52:506–11. Boison D. Adenosine kinase, epilepsy and stroke: mechanisms and therapies. Trends Pharmacol Sci 2006;27:652–8. Bong GW...Remessy AB, Al-ShabraweyM, Khalifa Y, Tsai NT, Caldwell RB, Liou GI. Neuroprotective and blood–retinal barrier-preserving effects of cannabidiol in

  7. Adenosine 2A Receptor Antagonist Prevented and Reversed Liver Fibrosis in a Mouse Model of Ethanol-Exacerbated Liver Fibrosis

    PubMed Central

    Chiang, Dian J.; Roychowdhury, Sanjoy; Bush, Katelyn; McMullen, Megan R.; Pisano, Sorana; Niese, Kathryn; Olman, Mitchell A.; Pritchard, Michele T.; Nagy, Laura E.

    2013-01-01

    The effect of moderate alcohol consumption on liver fibrosis is not well understood, but evidence suggests that adenosine may play a role in mediating the effects of moderate ethanol on tissue injury. Ethanol increases the concentration of adenosine in the liver. Adenosine 2A receptor (A2AR) activation is known to enhance hepatic stellate cell (HSC) activation and A2AR deficient mice are protected from fibrosis in mice. Making use of a novel mouse model of moderate ethanol consumption in which female C57BL/6J mice were allowed continued access to 2% (vol/vol) ethanol (11% calories) or pair-fed control diets for 2 days, 2 weeks or 5 weeks and superimposed with exposure to CCl4, we tested the hypothesis that moderate ethanol consumption increases fibrosis in response to carbon tetrachloride (CCl4) and that treatment of mice with an A2AR antagonist prevents and/or reverses this ethanol-induced increase in liver fibrosis. Neither the expression or activity of CYP2E1, required for bio-activation of CCl4, nor AST and ALT activity in the plasma were affected by ethanol, indicating that moderate ethanol did not increase the direct hepatotoxicity of CCl4. However, ethanol feeding enhanced HSC activation and exacerbated liver fibrosis upon exposure to CCl4. This was associated with an increased sinusoidal angiogenic response in the liver. Treatment with A2AR antagonist both prevented and reversed the ability of ethanol to exacerbate liver fibrosis. Conclusion Moderate ethanol consumption exacerbates hepatic fibrosis upon exposure to CCl4. A2AR antagonism may be a potential pharmaceutical intervention to decrease hepatic fibrosis in response to ethanol. PMID:23874883

  8. Adenosine A2A receptor deletion affects social behaviors and anxiety in mice: Involvement of anterior cingulate cortex and amygdala.

    PubMed

    López-Cruz, Laura; Carbó-Gas, Maria; Pardo, Marta; Bayarri, Pilar; Valverde, Olga; Ledent, Catherine; Salamone, John D; Correa, Mercè

    2017-03-15

    Blockade of adenosine A 2A receptors can potentiate motivation to work for natural reinforcers such as food. Conspecific interaction is a potent natural reinforcer in social animals that can be manifested as preference for social exploration versus other sources of novel stimulation. Deficiencies in this type of motivated behavior (social withdrawal) have been seen in several pathologies such as autism and depression. However, the role of A 2A receptors in motivation for social interaction has not been widely explored. Social interaction paradigms evaluate the natural preference of animals for exploring other conspecifics, and the ability to differentiate between familiar versus novel ones. Anxiety is one of the factors that can induce avoidance of social interaction. In the present study, adenosine A 2A knockout (A 2A KO) and wild-type (WT) mice were assessed for social and anxiety-related behaviors. c-Fos immunoreactivity was evaluated as a measure of neuronal activation in brain areas involved in different aspects of motivation and emotional processes. Although A 2A KO mice showed an anxious profile, they displayed higher levels of sociability and were less sensitive to social novelty. WT mice displayed a typical pattern of social recognition 24h later, but not A 2A KO mice, which explored equally both conspecifics. There were no differences between strains in aggressiveness, perseverance or social odor preferences. c-Fos immunoreactivity in A 2A KO mice was higher in anterior cingulate and amygdala compared to WT mice. Thus, A 2A receptors appear to be potential targets for the improvement of pathologies related to social function. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Characteristics of P2X7-like receptor activated by adenosine triphosphate in HIT-T15 cells.

    PubMed

    Lee, Dong Hyeon; Kim, Eun Gi; Park, Kyu-Sang; Jeong, Seong-Woo; Kong, In Deok; Lee, Joong-Woo

    2007-07-01

    The study examined the presence of a P2X7 receptor subtype and its functional roles in pancreatic beta cells. In a hamster beta-cell line, HIT-T15 cells, purinergic stimulation was investigated using fluorometry, electrophysiology, flow cytometry, and electrophoresis. Adenosine triphosphate (ATP) and 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) increased in the intracellular free Ca2+ concentration, with an EC50 of 398.0 and 136.6 microM, respectively. Preincubation with oxidized ATP, a P2X7 receptor antagonist, inhibited the ATP- and BzATP-induced increase in the intracellular Ca2+ level. The BzATP-induced increase in the intracellular Ca2+ level was dependent on the extracellular Ca2+ concentration. The extracellular Mg2+ had a significant effect on the ATP-induced increase in the intracellular Ca2+ level. The ATP also induced depolarization like high potassium chloride. In the voltage-clamp experiments, ATP evoked inward currents, which were reversed at almost 0 mV. The ATP stimulated the slow influx of ethidium bromide, indicating permeability to larger molecules. Flow cytometry showed that the number of hypodiploid cells (A0), which are indicative of apoptosis, increased when the cells were exposed to ATP for 24 hours. The ATP also induced DNA fragmentation. These results suggest that the HIT-T15 cells have endogenous P2X7-like receptors and that purinergic stimulation increased the level of intracellular Ca2+, depolarization, inward current, permeability, and apoptosis.

  10. Inosine reduces pain-related behavior in mice: involvement of adenosine A1 and A2A receptor subtypes and protein kinase C pathways.

    PubMed

    Nascimento, Francisney P; Figueredo, Sonia M; Marcon, Rodrigo; Martins, Daniel F; Macedo, Sérgio J; Lima, Denise A N; Almeida, Rúbia C; Ostroski, Rosana M; Rodrigues, Ana Lúcia S; Santos, Adair Roberto Soares

    2010-08-01

    Inosine, an endogenous purine, is the first metabolite of adenosine in a reaction catalyzed by adenosine deaminase. This study aimed to investigate the antinociceptive effects of inosine against several models of pain in mice and rats. In mice, inosine given by systemic or central routes inhibited acetic acid-induced nociception. Furthermore, inosine also decreased the late phase of formalin-induced licking and the nociception induced by glutamate. Inosine produced inhibition (for up to 4 h) of mechanical allodynia induced by complete Freund's adjuvant (CFA) injected into the mouse's paw. Given chronically for 21 days, inosine reversed the mechanical allodynia caused by CFA. Moreover, inosine also reduced the thermal (cold stimuli) and mechanical allodynia caused by partial sciatic nerve ligation (PSNL) for 4 h; when inosine was chronically administered, it decreased the mechanical allodynia induced by PSNL for 22 days. Antinociception caused by inosine in the acetic acid test was attenuated by treatment of mice with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; a selective adenosine A(1) receptor antagonist), 8-phenyltheophylline (8-PT; a nonselective adenosine A(1) receptor antagonist), and 4-{2- [7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-yl- amino]ethyl}phenol (ZM241385; a selective adenosine A(2A) receptor antagonist). In rats, inosine inhibited the mechanical and heat hyperalgesia induced by bradykinin and phorbol 12-myristate 13-acetate, without affecting similar responses caused by prostaglandin E(2) or forskolin. These results indicate that inosine induces antinociceptive, antiallodynic, and antihyperalgesic effects in rodents. The precise mechanisms through which inosine produces antinociception are currently under investigation, but involvement of adenosine A(1) and A(2A) receptors and blockade of the protein kinase C pathway seem to largely account for inosine's antinociceptive effect.

  11. Striatal adenosine A2A receptor neurons control active-period sleep via parvalbumin neurons in external globus pallidus

    PubMed Central

    Qu, Wei-Min; Yang, Su-Rong; Cherasse, Yoan; Lazarus, Michael; Schiffmann, Serge N; d'Exaerde, Alban de Kerchove

    2017-01-01

    Dysfunction of the striatum is frequently associated with sleep disturbances. However, its role in sleep-wake regulation has been paid little attention even though the striatum densely expresses adenosine A2A receptors (A2ARs), which are essential for adenosine-induced sleep. Here we showed that chemogenetic activation of A2AR neurons in specific subregions of the striatum induced a remarkable increase in non-rapid eye movement (NREM) sleep. Anatomical mapping and immunoelectron microscopy revealed that striatal A2AR neurons innervated the external globus pallidus (GPe) in a topographically organized manner and preferentially formed inhibitory synapses with GPe parvalbumin (PV) neurons. Moreover, lesions of GPe PV neurons abolished the sleep-promoting effect of striatal A2AR neurons. In addition, chemogenetic inhibition of striatal A2AR neurons led to a significant decrease of NREM sleep at active period, but not inactive period of mice. These findings reveal a prominent contribution of striatal A2AR neuron/GPe PV neuron circuit in sleep control. PMID:29022877

  12. Cardiac‐Restricted Overexpression of the A2A‐Adenosine Receptor in FVB Mice Transiently Increases Contractile Performance and Rescues the Heart Failure Phenotype in Mice Overexpressing the A1‐Adenosine Receptor

    PubMed Central

    Chan, Tung O.; Funakoshi, Hajime; Song, Jianliang; Zhang, Xue‐Qian; Wang, JuFang; Chung, Paul H.; DeGeorge, Jr, Brent R.; Li, Xue; Zhang, Jin; Herrmann, David E.; Diamond, Maura; Hamad, Eman; Houser, Steven R.; Koch, Walter J.; Cheung, Joseph Y.; Feldman, Arthur M.

    2008-01-01

    Abstract In the heart, adenosine binds to pharmacologically distinct G‐protein‐coupled receptors (A1‐R, A2A‐R, and A3‐R). While the role of A1‐and A3‐Rs in the heart has been clarifed, the effect of genetically manipulating the A2A‐R has not been defned. Thus, we created mice overexpressing a cardiac‐restricted A2A‐R transgene. Mice with both low (Lo) and high (Hi) levels of A2A‐R overexpression demonstrated an increase in cardiac contractility at 12 weeks. These changes were associated with a signifcantly higher systolic but not diastolic [Ca2+]i, higher maximal contraction amplitudes, and a signifcantly enhanced sarcoplasmic reticulum Ca2+ uptake activity. At 20 weeks, the effects of A2A‐R overexpression on cardiac contractility diminished. The positive effects elicited by A2A‐R overexpression differ from the heart failure phenotype we observed with A1‐R overexpresson. Interestingly, coexpression of A2A‐R TGHi, but not A2A‐R TGLo, enhanced survival, prevented the development of left ventricular dysfunction and heart failure, and improved Ca2+ handling in mice overexpressing the A1‐R. These results suggest that adenosine‐mediated signaling in the heart requires a balance between A1‐ and A2A‐Rs—a fnding that may have important implications for the ongoing clinical evaluation of adenosine receptor subtype‐specifc agonists and antagonists for the treatment of cardiovascular diseases. PMID:20354569

  13. The novel N-methyl-D-aspartate (NMDA) antagonist CGS 19755 prevents ischemia-induced reductions of adenosine A1, NMDA, and PCP receptors in gerbil brain.

    PubMed

    Jarvis, M F; Murphy, D E; Williams, M; Gerhardt, S C; Boast, C A

    1988-01-01

    Transient brain ischemia results in a selective destruction of cell bodies within the hippocampus and cortex. This cellular destruction appears to be mediated through a release of endogenous exictatory amino acids following the ischemic episode, since the neurotoxic effects of ischemia can be attenuated by compounds that have antagonist activity at N-methyl-D-aspartate (NMDA) receptors. In the present study, the protective effects of a novel NMDA receptor antagonist, CGS 19755, were further evaluated by using quantitative autoradiography to characterize adenosine A1, NMDA, PCP, and benzodiazepine receptors in ischemic gerbil brain. Bilateral carotid artery occlusion (20 minutes) resulted in marked decreases (30-60%) in adenosine A1, NMDA, and PCP, but not benzodiazepine, receptors in gerbil forebrain. Postischemic treatment with CGS 19755 was found to completely block the ischemia-induced decreases in brain adenosine and NMDA receptors. [3H]TCP binding in ischemic gerbil brain was also elevated by CGS 19755 treatment; significant differences remained, however, between the CGS 19755-treated and control gerbils. These results indicate that transient brain ischemia produces significant and selective alterations in gerbil forebrain receptor systems. The observed decreases in radioligand binding are probably reflective of an ischemia-induced destruction of forebrain structures. However, there is some evidence that transient ischemia can also cause long-term changes in the affinity of some receptor systems. The postischemic efficacy of CGS 19755 appears to be due to its ability to block the neurotoxic effects of transient ischemia.

  14. Lack of effect of adenosine on the function of rodent osteoblasts and osteoclasts in vitro.

    PubMed

    Hajjawi, Mark O R; Patel, Jessal J; Corcelli, Michelangelo; Arnett, Timothy R; Orriss, Isabel R

    2016-06-01

    Extracellular ATP, signalling through P2 receptors, exerts well-documented effects on bone cells, inhibiting mineral deposition by osteoblasts and stimulating the formation and resorptive activity of osteoclasts. The aims of this study were to determine the potential osteotropic effects of adenosine, the hydrolysis product of ATP, on primary bone cells in vitro. We determined the effect of exogenous adenosine on (1) the growth, alkaline phosphatase (TNAP) activity and bone-forming ability of osteoblasts derived from the calvariae of neonatal rats and mice and the marrow of juvenile rats and (2) the formation and resorptive activity of osteoclasts from juvenile mouse marrow. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed marked differences in the expression of P1 receptors in osteoblasts from different sources. Whilst mRNA for the A1 and A2B receptors was expressed by all primary osteoblasts, A2A receptor expression was limited to rat bone marrow and mouse calvarial osteoblasts and the A3 receptor to rat bone marrow osteoblasts. We found that adenosine had no detectable effects on cell growth, TNAP activity or bone formation by rodent osteoblasts in vitro. The analogue 2-chloroadenosine, which is hydrolysed more slowly than adenosine, had no effects on rat or mouse calvarial osteoblasts but increased TNAP activity and bone formation by rat bone marrow osteoblasts by 30-50 % at a concentration of 1 μM. Osteoclasts were found to express the A2A, A2B and A3 receptors; however, neither adenosine (≤100 μM) nor 2-chloroadenosine (≤10 μM) had any effect on the formation or resorptive activity of mouse osteoclasts in vitro. These results suggest that adenosine, unlike ATP, is not a major signalling molecule in the bone.

  15. Adenosine A1 receptor mRNA expression and the effects of systemic theophylline administration on respiratory function 4 months after C2 hemisection.

    PubMed

    Nantwi, Kwaku D; Basura, Gregory J; Goshgarian, Harry G

    2003-01-01

    Previous studies from our laboratory have demonstrated that in an animal model of acute cervical spinal cord injury (SCI), respiratory function can be restored by theophylline. We also have shown that respiratory recovery occurs spontaneously after prolonged postinjury survival periods when a hemidiaphragm is paralyzed by an ipsilateral upper cervical (C2) spinal cord hemisection. Theophylline mediates functional recovery by central nervous system adenosine A1 receptor antagonism; however, it is unclear whether adenosine receptors are altered after prolonged postinjury periods and whether theophylline can further enhance restored respiratory function that occurs spontaneously. To assess putative effects of systemic theophylline administration on further enhancing spontaneous respiratory muscle recovery 4 months after C2 hemisection in rats and to determine whether adenosine A1 receptor mRNA expression is altered in these animals. Electrophysiologic assessment of respiratory activity in the phrenic nerves was conducted in C2 hemisected rats 4 months after hemisection under standardized conditions. Immediately thereafter, rats were killed and the cervical spinal cords were prepared for adenosine A1 receptor mRNA expression by in situ hybridization. Spontaneous recovery of respiratory activity in the ipsilateral phrenic nerve was detected in a majority (15/20) of C2 hemisected animals and amounted to 44.06% +/- 2.38% when expressed as a percentage of activity in the homolateral phrenic nerve in noninjured animals. At the optimal dosage used in the acute studies, theophylline (15 mg/kg) did not enhance, but rather unexpectedly blocked, recovered respiratory activity in 4 out of 5 animals tested. At dosages of 5 mg/kg and 2.5 mg/kg, the drug blocked recovered respiratory activity in 3 out of 4 and 3 out of 5 animals tested, respectively. Quantitative analysis of adenosine A1 receptor mRNA expression did not reveal a significant difference between experimental animals

  16. Frog secretions and hunting magic in the upper Amazon: identification of a peptide that interacts with an adenosine receptor.

    PubMed Central

    Daly, J W; Caceres, J; Moni, R W; Gusovsky, F; Moos, M; Seamon, K B; Milton, K; Myers, C W

    1992-01-01

    A frog used for "hunting magic" by several groups of Panoan-speaking Indians in the borderline between Brazil and Peru is identified as Phyllomedusa bicolor. This frog's skin secretion, which the Indians introduce into the body through fresh burns, is rich in peptides. These include vasoactive peptides, opioid peptides, and a peptide that we have named adenoregulin, with the sequence GLWSKIKEVGKEAAKAAAKAAGKAALGAVSEAV as determined from mass spectrometry and Edman degradation. The natural peptide may contain a D amino acid residue, since it is not identical in chromatographic properties to the synthetic peptide. Adenoregulin enhances binding of agonists to A1 adenosine receptors; it is accompanied in the skin secretion by peptides that inhibit binding. The vasoactive peptide sauvagine, the opioid peptides, and adenoregulin and related peptides affect behavior in mice and presumably contribute to the behavioral sequelae observed in humans. Images PMID:1438301

  17. QSAR of adenosine receptor antagonists. Part 3: Exploring physicochemical requirements for selective binding of 1,2,4-triazolo[5,1-i]purine derivatives with human adenosine A3 receptor subtype.

    PubMed

    Roy, Kunal; Leonard, J Thomas; Sengupta, Chandana

    2004-07-16

    Considering potential of selective adenosine A3 receptor antagonists in the development of prospective therapeutic agents, an attempt has been made to explore selectivity requirements of 1,2,4-triazolo[5,1-i]purine derivatives for binding with cloned human adenosine A3 receptor subtype. In this study, partition coefficient (logP) values of the molecules (calculated by Crippen's fragmentation method) and Wang-Ford charges of the common atoms of the triazolopurine nucleus (calculated from molecular electrostatic potential surface of energy minimized geometry using AM1 technique) were used as independent variables along with suitable dummy parameters. The best equation describing A3 binding affinity [n=29, Q2=0.796, Ra2=0.853, R2=0.874, R=0.935, s=0.342, F=41.5 (df 4,24), SDEP=0.396] showed parabolic relation with logP (optimum value being 4.134). Further, it was found that an aromatic substituent conjugated with the triazole nucleus should be present at R2 position for A3 binding affinity. Again, high negative charges on N2 and N4 are conducive to the binding affinity. While exploring selectivity requirements of the compounds for binding with A3 receptor over that with A2A receptor, the selectivity relation [n=23, Q2=0.909, Ra2=0.918, R2=0.933, R=0.966, s=0.401, F=62.4 (df 4,18), SDEP=0.412] showed that an aromatic R2 substituent conjugated with the triazole nucleus contributes significantly to the selectivity. Again, presence of a 4-substituted-phenyl ring (except 4-OH-phenyl and 4-CH3-phenyl) at R2 position also increases selectivity. Further, charge difference between N2 and N11 (negative charge on the former should be higher and that on the latter should be less) contributes significantly to the selectivity. In addition, negative charge on N7 is conducive while presence of substituents like propyl, butyl, pentyl or phenyl at R1 position is detrimental for the A3 selectivity.

  18. Caffeine acts through neuronal adenosine A2A receptors to prevent mood and memory dysfunction triggered by chronic stress

    PubMed Central

    Kaster, Manuella P.; Machado, Nuno J.; Silva, Henrique B.; Nunes, Ana; Ardais, Ana Paula; Santana, Magda; Baqi, Younis; Müller, Christa E.; Rodrigues, Ana Lúcia S.; Porciúncula, Lisiane O.; Chen, Jiang Fan; Tomé, Ângelo R.; Agostinho, Paula; Canas, Paula M.; Cunha, Rodrigo A.

    2015-01-01

    The consumption of caffeine (an adenosine receptor antagonist) correlates inversely with depression and memory deterioration, and adenosine A2A receptor (A2AR) antagonists emerge as candidate therapeutic targets because they control aberrant synaptic plasticity and afford neuroprotection. Therefore we tested the ability of A2AR to control the behavioral, electrophysiological, and neurochemical modifications caused by chronic unpredictable stress (CUS), which alters hippocampal circuits, dampens mood and memory performance, and enhances susceptibility to depression. CUS for 3 wk in adult mice induced anxiogenic and helpless-like behavior and decreased memory performance. These behavioral changes were accompanied by synaptic alterations, typified by a decrease in synaptic plasticity and a reduced density of synaptic proteins (synaptosomal-associated protein 25, syntaxin, and vesicular glutamate transporter type 1), together with an increased density of A2AR in glutamatergic terminals in the hippocampus. Except for anxiety, for which results were mixed, CUS-induced behavioral and synaptic alterations were prevented by (i) caffeine (1 g/L in the drinking water, starting 3 wk before and continued throughout CUS); (ii) the selective A2AR antagonist KW6002 (3 mg/kg, p.o.); (iii) global A2AR deletion; and (iv) selective A2AR deletion in forebrain neurons. Notably, A2AR blockade was not only prophylactic but also therapeutically efficacious, because a 3-wk treatment with the A2AR antagonist SCH58261 (0.1 mg/kg, i.p.) reversed the mood and synaptic dysfunction caused by CUS. These results herald a key role for synaptic A2AR in the control of chronic stress-induced modifications and suggest A2AR as candidate targets to alleviate the consequences of chronic stress on brain function. PMID:26056314

  19. 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.

  20. Adenosine receptors regulate susceptibility to noise-induced neural injury in the mouse cochlea and hearing loss.

    PubMed

    Vlajkovic, Srdjan M; Ambepitiya, Kaushi; Barclay, Meagan; Boison, Detlev; Housley, Gary D; Thorne, Peter R

    2017-03-01

    Our previous studies have shown that the stimulation of A 1 adenosine receptors in the inner ear can mitigate the loss of sensory hair cells and hearing loss caused by exposure to traumatic noise. Here, we focus on the role of adenosine receptors (AR) in the development of noise-induced neural injury in the cochlea using A 1 AR and A 2A AR null mice (A 1 AR -/- and A 2A AR -/- ). Wildtype (WT) and AR deficient mice were exposed to octave band noise (8-16 kHz, 100 dB SPL) for 2 h to induce cochlear injury and hearing loss. Auditory thresholds and input/output functions were assessed using auditory brainstem responses (ABR) before and two weeks post-exposure. The loss of outer hair cells (OHC), afferent synapses and spiral ganglion neurons (SGN) were assessed by quantitative histology. A 1 AR -/- mice (6-8 weeks old) displayed a high frequency hearing loss (ABR threshold shift and reduced ABR wave I and II amplitudes). This hearing loss was further aggravated by acute noise exposure and exceeded the hearing loss in the WT and A 2A AR -/- mice. All mice experienced the loss of OHC, synaptic ribbons and SGN after noise exposure, but the loss of SGN was significantly higher in A 1 AR -/- mice than in the A 2A AR -/- and WT genotypes. The A 2A AR -/- demonstrated better preservation of OHC and afferent synapses and the minimal loss of SGN after noise exposure. The findings suggest that the loss of A 1 AR expression results in an increased susceptibility to cochlear neural injury and hearing loss, whilst absence of A 2A AR increases cochlear resistance to acoustic trauma. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Integrating Pharmacophore into Membrane Molecular Dynamics Simulations to Improve Homology Modeling of G Protein-coupled Receptors with Ligand Selectivity: A2A Adenosine Receptor as an Example.

    PubMed

    Zeng, Lingxiao; Guan, Mengxin; Jin, Hongwei; Liu, Zhenming; Zhang, Liangren

    2015-12-01

    Homology modeling has been applied to fill in the gap in experimental G protein-coupled receptors structure determination. However, achievement of G protein-coupled receptors homology models with ligand selectivity remains challenging due to structural diversity of G protein-coupled receptors. In this work, we propose a novel strategy by integrating pharmacophore and membrane molecular dynamics (MD) simulations to improve homology modeling of G protein-coupled receptors with ligand selectivity. To validate this integrated strategy, the A2A adenosine receptor (A2A AR), whose structures in both active and inactive states have been established, has been chosen as an example. We performed blind predictions of the active-state A2A AR structure based on the inactive-state structure and compared the performance of different refinement strategies. The blind prediction model combined with the integrated strategy identified ligand-receptor interactions and conformational changes of key structural elements related to the activation of A2 A AR, including (i) the movements of intracellular ends of TM3 and TM5/TM6; (ii) the opening of ionic lock; (iii) the movements of binding site residues. The integrated strategy of pharmacophore with molecular dynamics simulations can aid in the optimization in the identification of side chain conformations in receptor models. This strategy can be further investigated in homology modeling and expand its applicability to other G protein-coupled receptor modeling, which should aid in the discovery of more effective and selective G protein-coupled receptor ligands. © 2015 John Wiley & Sons A/S.

  2. 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

  3. Adenosine A(2A) receptor up-regulates retinal wave frequency via starburst amacrine cells in the developing rat retina.

    PubMed

    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

    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 A(2A) receptor (A(2A)R) regulates retinal waves and whether A(2A)R regulation of retinal waves acts via presynaptic SACs. We showed that A(2A)R was expressed in the inner plexiform layer and ganglion cell layer of the developing rat retina. Knockdown of A(2A)R decreased the frequency of spontaneous Ca²⁺ transients, suggesting that endogenous A(2A)R may up-regulate wave frequency. To investigate whether A(2A)R 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 A(2A)R (A2AR-WT) in SACs, suggesting that A(2A)R may up-regulate retinal waves via presynaptic SACs. Subsequent patch-clamp recordings on RGCs revealed that presynaptic A(2A)R-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 A(2A)R may not affect the membrane properties of postsynaptic RGCs. In contrast, by expressing the C-terminal truncated A(2A)R mutant (A(2A)R-ΔC) in SACs, the wave frequency was reduced compared to the A(2A)R-WT, but was similar to the control, suggesting that the full-length A(2A)R in SACs is required for A(2A)R up-regulation of retinal waves. A(2A)R up-regulates the frequency of retinal waves via presynaptic SACs, requiring its full-length protein structure. Thus, by

  4. Exploring an interaction of adenosine A2A receptor variability with coffee and tea intake in Parkinson's disease.

    PubMed

    Tan, E K; Lu, Z Y; Fook-Chong, S M C; Tan, E; Shen, H; Chua, E; Yih, Y; Teo, Y Y; Zhao, Y

    2006-09-05

    Caffeine is an adenosine receptor A1 and A2A receptor antagonist and a putative functional genetic variant of the A2A receptor (2592C > Tins) mediates caffeine-induced anxiety. Here we investigated the potential interaction of this A2A genetic variant with the quantity of coffee and tea intake and their relationship with the risk of PD. A total of 441 subjects consisting of 222 PD and 219 race, gender and age matched controls were included. A multivariate analysis of the variables including the 2592C > Tins A2A genotypes, age of onset, gender, and the quantity of tea and coffee intake, interaction of the A2A genotypes with coffee intake, interaction of A2A genotypes with tea intake demonstrated the quantity of coffee intake to be significantly associated with PD (P < 0.0005, OR = 0.922, 95% CI: 0.881, 0.964). However, there was no significant interaction of the A2A genotypes with the quantity of coffee and tea intake in modulating the risk of PD. The dose dependent protective effect of coffee intake in PD was independent of the 2592C > Tins A2A genotype suggesting that the pharmacogenetic action of caffeine in PD may be mediated differently from other caffeine-induced neurologic syndromes.

  5. Beneficial effects of a novel agonist of the adenosine A2A receptor on monocrotaline-induced pulmonary hypertension in rats

    PubMed Central

    Alencar, Allan K N; Pereira, Sharlene L; Montagnoli, Tadeu L; Maia, Rodolfo C; Kümmerle, Arthur E; Landgraf, Sharon S; Caruso-Neves, Celso; Ferraz, Emanuelle B; Tesch, Roberta; Nascimento, José H M; de Sant'Anna, Carlos M R; Fraga, Carlos A M; Barreiro, Eliezer J; Sudo, Roberto T; Zapata-Sudo, Gisele

    2013-01-01

    Background and Purpose Pulmonary arterial hypertension (PAH) is characterized by enhanced pulmonary vascular resistance, right ventricular hypertrophy and increased right ventricular systolic pressure. Here, we investigated the effects of a N-acylhydrazone derivative, 3,4-dimethoxyphenyl-N-methyl-benzoylhydrazide (LASSBio-1359), on monocrotaline (MCT)-induced pulmonary hypertension in rats. Experimental Approach PAH was induced in male Wistar rats by a single i.p. injection of MCT (60 mg·kg−1) and 2 weeks later, oral LASSBio-1359 (50 mg·kg−1) or vehicle was given once daily for 14 days. Echocardiography was used to measure cardiac function and pulmonary artery dimensions, with histological assay of vascular collagen. Studies of binding to human recombinant adenosine receptors (A1, A2A, A3) and of docking with A2A receptors were also performed. Key Results MCT administration induced changes in vascular and ventricular structure and function, characteristic of PAH. These changes were reversed by treatment with LASSBio-1359. MCT also induced endothelial dysfunction in pulmonary artery, as measured by diminished relaxation of pre-contracted arterial rings, and this dysfunction was reversed by LASSBio-1359. In pulmonary artery rings from normal Wistar rats, LASSBio-1359 induced relaxation, which was decreased by the adenosine A2A receptor antagonist, ZM 241385. In adenosine receptor binding studies, LASSBio-1359 showed most affinity for the A2A receptor and in the docking analyses, binding modes of LASSBio-1359 and the A2A receptor agonist, CGS21680, were very similar. Conclusion and Implications In rats with MCT-induced PAH, structural and functional changes in heart and pulmonary artery were reversed by treatment with oral LASSBio-1359, most probably through the activation of adenosine A2A receptors. PMID:23530610

  6. Functional histamine H3 and adenosine A2A receptor heteromers in recombinant cells and rat striatum.

    PubMed

    Márquez-Gómez, Ricardo; Robins, Meridith T; Gutiérrez-Rodelo, Citlaly; Arias, Juan-Manuel; Olivares-Reyes, Jesús-Alberto; van Rijn, Richard M; Arias-Montaño, José-Antonio

    2018-03-01

    In the striatum, histamine H 3 receptors (H 3 Rs) are co-expressed with adenosine A 2A receptors (A 2A Rs) in the cortico-striatal glutamatergic afferents and the GABAergic medium-sized spiny neurons that originate the indirect pathway of the basal ganglia. This location allows H 3 Rs and A 2A Rs to regulate the striatal GABAergic and glutamatergic transmission. However, whether these receptors can physically interact has not yet been assessed. To test this hypothesis, a heteromer-selective in vitro assay was used to detect functional complementation between a chimeric A 2A R 302 -Gα qi4 and wild-type H 3 Rs in transfected HEK-293T cells. H 3 R activation with the agonist RAMH resulted in Ca 2+ mobilization (pEC 50 7.31 ± 0.23; maximal stimulation, Emax 449 ± 25% of basal) indicative of receptor heterodimerization. Functional H 3 R-A 2A R heteromers were confirmed by co-immunoprecipitation and observations of differential cAMP signaling when both receptors were co-expressed in the same cells. In membranes from rat striatal synaptosomes, H 3 R activation decreased A 2A R affinity for the agonist CGS-21680 (pKi values 8.10 ± 0.04 and 7.70 ± 0.04). Moreover, H 3 Rs and A 2A Rs co-immunoprecipitated in protein extracts from striatal synaptosomes. These results support the existence of a H 3 R-A 2A R heteromer with possible physiological implications for the modulation of the intra-striatal transmission. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Impact of purification conditions and history on A 2A adenosine receptor activity: The role of CHAPS and lipids

    SciTech Connect

    Naranjo, Andrea N.; McNeely, Patrick M.; Katsaras, John

    2016-05-27

    The adenosine A 2A receptor (A 2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A 2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A 2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilizationmore » in DDM, DDM/CHAPS, or DHPC micelles, although A 2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A 2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. Furthermore, the studies presented in this paper also underline the importance of the protein’s purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.« less

  8. ADP-induced bladder contractility is mediated by P2Y12 receptor and temporally regulated by ectonucleotidases and adenosine signaling

    PubMed Central

    Yu, Weiqun; Sun, Xiaofeng; Robson, Simon C.; Hill, Warren G.

    2014-01-01

    Purinergic signaling comprises one key pathway in modulating bladder smooth muscle (BSM) contractility, disorders of which become highly prevalent with aging. ADP was first observed to modulate BSM contractility >40 yr ago, yet the underlying molecular mechanism still remains unclear. Here, we demonstrate, using myography, that ADP and ADPβS dose-dependently induce mouse BSM contraction, and ADP-induced BSM contraction is blocked by a selective P2Y12 receptor (P2Y12R) antagonist, PSB 0739 (25 μM), but is unaffected by P2Y1 and P2Y13 receptor antagonists. P2Y12R in BSM exhibits distinct pharmacological properties that are different from P2Y12R in platelets. After an immediate contraction, prolonged exposure to ADP causes BSM to become refractory to further ADP-mediated contraction. However, in mice lacking ectonucleotidases Entpd1 (ATP→ADP→AMP) or Nt5e (AMP→adenosine), or by inhibiting adenosine signaling, the refractory response was altered, resulting in repeated BSM contractions in response to repeated ADP (0.1-1 mM) stimulation. Our data indicate that P2Y12R undergoes slow desensitization; ADP-P2Y12 signaling is tightly regulated by Entpd1/Nt5e activity and adenosine receptors; and ADP-adenosine signaling play an important role in modulating P2X-mediated BSM contraction. The identification of P2Y12R in BSM, and the current clinical availability of P2Y12R inhibitors, such as clopidogrel, offers potentially novel treatment strategies for bladder contractility disorders.—Yu, W., Sun, X., Robson, S. C., Hill, W. G. ADP-induced bladder contractility is mediated by P2Y12 receptor and temporally regulated by ectonucleotidases and adenosine signaling. PMID:25208846

  9. Extracellular ATP differentially affects epileptiform activity via purinergic P2X7 and adenosine A1 receptors in naive and chronic epileptic rats.

    PubMed

    Klaft, Zin-Juan; Schulz, Steffen B; Maslarova, Anna; Gabriel, Siegrun; Heinemann, Uwe; Gerevich, Zoltan

    2012-11-01

    Adenosine is considered an endogenous anticonvulsant. However, much less is known about the putative effects of its precursor, ATP, on epilepsy. Therefore, we tested whether ATP and its receptors are able to modulate epileptiform activity in the medial entorhinal cortex of the rat. Recurrent epileptiform discharges (REDs) were induced by elevating extracellular potassium concentration combined with application of bicuculline in brain slices from naive and pilocarpine-treated chronic epileptic rats. Field potentials were recorded from layer V/VI of the medial entorhinal cortex. REDs in slices from naive animals had a higher incidence and a shorter duration than in slices from chronic epileptic animals. Exogenous application of ATP reversibly reduced the incidence of REDs in naive and chronic epileptic slices via activation of adenosine A(1) receptors without discernible P2 receptor effects. This effect was stronger in slices from chronic epileptic rats. In slices from naive rats, the P2X7 receptor antagonist A 740003 slightly but significantly reduced the amplitude of slow field potentials of REDs. In slices from chronic epileptic rats, none of the P2 receptor antagonists affected the parameters of REDs. Our results suggest that endogenously released ATP differentially modulates REDs by activation of A(1) and P2X7 receptors. Although it has a minor proepileptic effect by direct activation of P2X7 receptors, its metabolite adenosine reduces the epileptiform activity via activation of A(1) receptors. The exact effect of ATP on neural activity depends on the actual activity of ectonucleotidases and the expression level of the purinergic receptors, which both alter during epileptogenesis. In addition, our data suggest that P2X7 receptor antagonists have a minor antiepileptic effect. Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.

  10. The protective effect of adenosine A2A receptor antagonism in cerebral ischemia.

    PubMed

    Pedata, F; Gianfriddo, M; Turchi, D; Melani, A

    2005-03-01

    We reviewed our most recent work on the protective effect of adenosine A(2A)antagonism in cerebral ischemia. Focal ischemia was produced in rats by introducing a nylon monofilament pre-coated with silicone through the external carotid artery to occlude the right MCA at its origin. A(2A) antagonism was found protective in the model of permanent focal ischemia induced by the monofilament technique. This methodology provides the possibility of evaluating the protection against the outflow of excitatory amino acids and against an acute motor disturbance, i.e.contralateral turning to the ischemic side in the first hours after ischemia in awake rats. Hours later, a definite neurological deficit and necrotic neuronal damage can be evaluated. Our results suggest that A(2A) antagonism may be protective from the earliest up to several hours after the ischemic event.

  11. Involvement of purinergic receptors and NOD-like receptor-family protein 3-inflammasome pathway in the adenosine triphosphate-induced cytokine release from macrophages.

    PubMed

    Gicquel, Thomas; Victoni, Tatiana; Fautrel, Alain; Robert, Sacha; Gleonnec, Florence; Guezingar, Marie; Couillin, Isabelle; Catros, Véronique; Boichot, Elisabeth; Lagente, Vincent

    2014-04-01

    Adenosine triphosphate (ATP) has been described as a danger signal activating the NOD-like receptor-family protein 3 (NLRP3)-inflammasome leading to the pro-inflammatory cytokine, interleukin (IL)-1β, release in the lung. The NLRP3-inflammasome pathway has been previously described to be involved in experimental collagen deposition and the development of pulmonary fibrosis. The aim of the present study was to investigate the role of the NLRP3 inflammasome pathway and P2X7 purinergic receptor in the activation of human macrophages in vitro by ATP. We showed that adenosine 5'-[γ-thio]triphosphate tetralithium salt (ATPγS) and 2',3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP), two stable analogs of ATP, are able to potentiate the release of IL-1β from human monocyte-derived macrophages induced by low concentration of lipopolysaccharide (LPS). However, in the same conditions no increase in IL-1α and IL-6 was observed. Immunochemistry has shown that human macrophages natively express NLRP3 and purinergic P2X7 receptors (P2X7 R). NLRP3 and IL-1β mRNA expression were induced from LPS-primed macrophages, but also after 5-h treatment of BzATP as analysed by reverse transcription quantitative polymerase chain reaction. However, other inflammasome pathways (NLRP1, NLRP2, NLRC4, NLRP6 and AIM2) and P2X7 R were not induced by BzATP. We observed that P2X7 R antagonists, A-438079 and A-740003, were able to reduce the release of IL-1β, but not of IL-1α and IL-6 from macrophages stimulated by ATPγS or BzATP. The present results showed the involvement of the P2X7 R-NLRP3 inflammasome pathway in the secretion of IL-1β from ATP-stimulated human macrophages, and suggest that P2X7 R were not involved in IL-1α and IL-6 release. This study also points out that repression of the P2X7 R represents a novel potential therapeutic approach to control fibrosis in lung injury. © 2014 Wiley Publishing Asia Pty Ltd.

  12. The neuronal Ca(2+) -binding protein 2 (NECAB2) interacts with the adenosine A(2A) receptor and modulates the cell surface expression and function of the receptor.

    PubMed

    Canela, Laia; Luján, Rafael; Lluís, Carme; Burgueño, Javier; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Ciruela, Francisco

    2007-09-01

    Heptaspanning membrane also known as G protein-coupled receptors (GPCR) do interact with a variety of intracellular proteins whose function is regulate receptor traffic and/or signaling. Using a yeast two-hybrid screen, NECAB2, a neuronal calcium binding protein, was identified as a binding partner for the adenosine A(2A) receptor (A(2A)R) interacting with its C-terminal domain. Co-localization, co-immunoprecipitation and pull-down experiments showed a close and specific interaction between A(2A)R and NECAB2 in both transfected HEK-293 cells and also in rat striatum. Immunoelectron microscopy detection of NECAB2 and A(2A)R in the rat striatopallidal structures indicated that both proteins are co-distributed in the same glutamatergic nerve terminals. The interaction of NECAB2 with A(2A)R modulated the cell surface expression, the ligand-dependent internalization and the receptor-mediated activation of the MAPK pathway. Overall, these results show that A(2A)R interacts with NECAB2 in striatal neurones co-expressing the two proteins and that the interaction is relevant for A(2A)R function.

  13. Antidepressant activity of the adenosine A2A receptor antagonist, istradefylline (KW-6002) on learned helplessness in rats.

    PubMed

    Yamada, Koji; Kobayashi, Minoru; Shiozaki, Shizuo; Ohta, Teruko; Mori, Akihisa; Jenner, Peter; Kanda, Tomoyuki

    2014-07-01

    Istradefylline, an adenosine A2A receptor antagonist, improves motor function in animal models of Parkinson's disease (PD) and in patients with PD. In addition, some A2A antagonists exert antidepressant-like activity in rodent models of depression, such as the forced swim and the tail suspension tests. We have investigated the effect of istradefylline on depression-like behaviors using the rat learned helplessness (LH) model. Acute, as well as chronic, oral administration of istradefylline significantly improved the inescapable shock (IES)-induced escape deficit with a degree of efficacy comparable to chronic treatment with the tricyclic antidepressant desipramine and the selective serotonin (5-HT) reuptake inhibitor, fluoxetine. Both the A1/A2A receptor nonspecific antagonist theophylline and the moderately selective antagonist CGS15943, but not the A1 selective antagonist DPCPX, ameliorated the IES-induced escape deficit. The enhancement of escape response by istradefylline was reversed by a local injection of the A2A specific agonist CGS21680 either into the nucleus accumbens, the caudate-putamen, or the paraventricular nucleus of the hypothalamus, but not by the A1 specific agonist R-PIA into the nucleus accumbens. Moreover, neither the 5-HT2A/2C receptor antagonist methysergide or the adrenergic α 2 antagonist yohimbine, nor the β-adrenergic antagonist propranolol, affected the improvement of escape response induced by istradefylline. Istradefylline exerts antidepressant-like effects via modulation of A2A receptor activity which is independent of monoaminergic transmission in the brain. Istradefylline may represent a novel treatment option for depression in PD as well as for the motor symptoms.

  14. Absence of the Adenosine A2A Receptor Confers Pulmonary Arterial Hypertension and Increased Pulmonary Vascular Remodeling in Mice

    PubMed Central

    Xu, M.H.; Gong, Y.S.; Su, M.S.; Dai, Z.Y.; Dai, S.S.; Bao, S.Z.; Li, N.; Zheng, R.Y.; He, J.C.; Chen, J.F.; Wang, X.T.

    2011-01-01

    Background Pulmonary arterial hypertension (PAH) is characterized by sustained elevation of pulmonary vascular resistance resulting from endothelial and smooth muscle cell dysfunction and collagen deposition in pulmonary vascular walls. In this study, we investigated the role of the adenosine A2A receptor (A2AR) in the development of PAH by determining the effect of genetic inactivation of A2ARs on pulmonary vascular remodeling in mice. Methods and Results We characterized hemodynamic, histological and ultrastructural changes in pulmonary vascular remodeling in A2AR knockout (KO) mice compared with their wild-type (WT) littermates after exposure to normoxia and hypoxic conditions. After exposure to normoxia, compared to WT mice, A2AR KO mice displayed: (1) increased right ventricular systolic pressures and an elevated ratio of the right ventricle over left ventricle plus septum (Fulton index), (2) increased wall area and thickness as well as enhanced smooth muscle actin immunoreactivity in pulmonary resistance vessels, (3) increased proliferating cell nuclear antigen-positive cells in pulmonary resistance vessels and (4) increased smooth muscle cells hypertrophy and collagen deposition in the adventitia of pulmonary arteriole walls as revealed by electron microscope. By contrast, histological analysis revealed no features of hypertensive nephropathy in A2AR KO mice and there was no significant difference in systemic blood pressure, and left ventricular masses among the 3 genotypes. Furthermore, following chronic exposure to hypoxia, A2AR KO mice exhibited exacerbated elevation in right ventricular systolic pressure, hypertrophy of pulmonary resistance vessels and increased cell proliferation in pulmonary resistance vessels, compared to WT littermates. Thus, genetic inactivation of A2ARs selectively produced PAH and associated increased smooth muscle proliferation and collagen deposition. Conclusions Extracellular adenosine acting at A2ARs represents an important

  15. A partial agonist of the A(1)-adenosine receptor selectively slows AV conduction in guinea pig hearts.

    PubMed

    Wu, L; Belardinelli, L; Zablocki, J A; Palle, V; Shryock, J C

    2001-01-01

    The use of full agonists of the A(1)-adenosine receptor (A(1)-ADOR) as antiarrhythmic agents is limited by their actions to cause high-grade atrioventricular (AV) block, profound bradycardia, atrial fibrillation, and vasodilation. It may be possible to avoid these undesired actions by use of partial agonists. We determined the effects of CVT-2759, a potential partial agonist of A(1)-ADORs, on guinea pig hearts. CVT-2759 (0.1-100 microM) increased the S-H interval of the isolated heart from 45 +/- 1 to 60 +/- 3 ms (P < 0. 01) with a half-maximal effect at 3.1 microM. CVT-2759 did not cause second-degree AV block. CVT-2759 significantly attenuated the actions of the full agonists N(6)-cyclopentyladenosine and adenosine. CVT-2759 caused a moderate slowing of atrial rate by 10 microM. In contrast, CVT-2759 was a full agonist to decrease cAMP content of rat adipocytes and Fischer rat thyroid line 5 cells. Results of radioligand binding assays indicated that CVT-2759 stabilized a high-affinity, G protein-coupled state of the A(1)-ADOR in membranes prepared from rat adipocytes but not in membranes prepared from the guinea pig brain. The results suggest that a weak A(1)-ADOR agonist, such as CVT-2759, may be useful to slow AV nodal conduction and thereby ventricular rate without causing AV block, bradycardia, atrial arrhythmias, or vasodilation.

  16. Selecting an optimal number of binding site waters to improve virtual screening enrichments against the adenosine A2A receptor.

    PubMed

    Lenselink, Eelke B; Beuming, Thijs; Sherman, Woody; van Vlijmen, Herman W T; IJzerman, Adriaan P

    2014-06-23

    A major challenge in structure-based virtual screening (VS) involves the treatment of explicit water molecules during docking in order to improve the enrichment of active compounds over decoys. Here we have investigated this in the context of the adenosine A2A receptor, where water molecules have previously been shown to be important for achieving high enrichment rates with docking, and where the positions of some binding site waters are known from a high-resolution crystal structure. The effect of these waters (both their presence and orientations) on VS enrichment was assessed using a carefully curated set of 299 high affinity A2A antagonists and 17,337 decoys. We show that including certain crystal waters greatly improves VS enrichment and that optimization of water hydrogen positions is needed in order to achieve the best results. We also show that waters derived from a molecular dynamics simulation - without any knowledge of crystallographic waters - can improve enrichments to a similar degree as the crystallographic waters, which makes this strategy applicable to structures without experimental knowledge of water positions. Finally, we used decision trees to select an ensemble of structures with different water molecule positions and orientations that outperforms any single structure with water molecules. The approach presented here is validated against independent test sets of A2A receptor antagonists and decoys from the literature. In general, this water optimization strategy could be applied to any target with waters-mediated protein-ligand interactions.

  17. Resetting microbiota by Lactobacillus reuteri inhibits T reg deficiency–induced autoimmunity via adenosine A2A receptors

    PubMed Central

    Hoang, Thomas K.; Tian, Xiangjun; Luo, Meng; Zhou, Jain; Tatevian, Nina; Molina, Jose G.; Blackburn, Michael R.; Gomez, Thomas H.

    2017-01-01

    Regulatory T (T reg) cell deficiency causes lethal, CD4+ T cell–driven autoimmune diseases. Stem cell transplantation is used to treat these diseases, but this procedure is limited by the availability of a suitable donor. The intestinal microbiota drives host immune homeostasis by regulating the differentiation and expansion of T reg, Th1, and Th2 cells. It is currently unclear if T reg cell deficiency–mediated autoimmune disorders can be treated by targeting the enteric microbiota. Here, we demonstrate that Foxp3+ T reg cell deficiency results in gut microbial dysbiosis and autoimmunity over the lifespan of scurfy (SF) mouse. Remodeling microbiota with Lactobacillus reuteri prolonged survival and reduced multiorgan inflammation in SF mice. L. reuteri changed the metabolomic profile disrupted by T reg cell deficiency, and a major effect was to restore levels of the purine metabolite inosine. Feeding inosine itself prolonged life and inhibited multiorgan inflammation by reducing Th1/Th2 cells and their associated cytokines. Mechanistically, the inhibition of inosine on the differentiation of Th1 and Th2 cells in vitro depended on adenosine A2A receptors, which were also required for the efficacy of inosine and of L. reuteri in vivo. These results reveal that the microbiota–inosine–A2A receptor axis might represent a potential avenue for combatting autoimmune diseases mediated by T reg cell dysfunction. PMID:27994068

  18. Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics

    PubMed Central

    Hothersall, J. Daniel; Guo, Dong; Sarda, Sunil; Sheppard, Robert J.; Chen, Hongming; Keur, Wesley; Waring, Michael J.; IJzerman, Adriaan P.; Hill, Stephen J.; Dale, Ian L.

    2017-01-01

    The duration of action of adenosine A2A receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t1/2 > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy. PMID:27803241

  19. The antidepressant-like effect of inosine in the FST is associated with both adenosine A1 and A 2A receptors.

    PubMed

    Kaster, Manuella P; Budni, Josiane; Gazal, Marta; Cunha, Mauricio P; Santos, Adair R S; Rodrigues, Ana Lúcia S

    2013-09-01

    Inosine is an endogenous purine nucleoside, which is formed during the breakdown of adenosine. The adenosinergic system was already described as capable of modulating mood in preclinical models; we now explored the effects of inosine in two predictive models of depression: the forced swim test (FST) and tail suspension test (TST). Mice treated with inosine displayed higher anti-immobility in the FST (5 and 50 mg/kg, intraperitoneal route (i.p.)) and in the TST (1 and 10 mg/kg, i.p.) when compared to vehicle-treated groups. These antidepressant-like effects started 30 min and lasted for 2 h after intraperitoneal administration of inosine and were not accompanied by any changes in the ambulatory activity in the open-field test. Both adenosine A1 and A2A receptor antagonists prevented the antidepressant-like effect of inosine in the FST. In addition, the administration of an adenosine deaminase inhibitor (1 and 10 mg/kg, i.p.) also caused an antidepressant-like effect in the FST. These results indicate that inosine possesses an antidepressant-like effect in the FST and TST probably through the activation of adenosine A1 and A2A receptors, further reinforcing the potential of targeting the purinergic system to the management of mood disorders.

  20. Effects of a Proprietary Standardized Orthosiphon stamineus Ethanolic Leaf Extract on Enhancing Memory in Sprague Dawley Rats Possibly via Blockade of Adenosine A2A Receptors

    PubMed Central

    Choudhary, Yogendra; Choudhary, Vandana Kotak; Bommu, Praveen; Wong, Hoi Jin

    2015-01-01

    The aim of the study was to explore a propriety standardized ethanolic extract from leaves of Orthosiphon stamineus Benth in improving impairments in short-term social memory in vivo, possibly via blockade of adenosine A2A receptors (A2AR). The ethanolic extract of O. stamineus leaves showed significant in vitro binding activity of A2AR with 74% inhibition at 150 μg/ml and significant A2AR antagonist activity with 98% inhibition at 300 μg/mL. A significant adenosine A1 receptor (A1R) antagonist activity with 100% inhibition was observed at 300 μg/mL. Its effect on learning and memory was assessed via social recognition task using Sprague Dawley rats whereby the ethanolic extract of O. stamineus showed significant (p < 0.001) change in recognition index (RI) at 300 mg/kg and 600 mg/kg p.o and 120 mg/kg i.p., respectively, compared to the vehicle control. In comparison, the ethanolic extract of Polygonum minus aerial parts showed small change in inflexion; however, it remained insignificant in RI at 200 mg/kg p.o. Our findings suggest that the ethanolic extract of O. stamineus leaves improves memory by reversing age-related deficits in short-term social memory and the possible involvement of adenosine A1 and adenosine A2A as a target bioactivity site in the restoration of memory. PMID:26649059

  1. Insulin requires A1 adenosine receptors expression to reverse gestational diabetes-increased L-arginine transport in human umbilical vein endothelium.

    PubMed

    Guzmán-Gutiérrez, Enrique; Armella, Axel; Toledo, Fernando; Pardo, Fabián; Leiva, Andrea; Sobrevia, Luis

    2016-03-01

    Gestational diabetes mellitus (GDM) associates with increased L-arginine transport and extracellular concentration of adenosine in human umbilical vein endothelial cells (HUVECs). In this study we aim to determine whether insulin reverses GDM-increased L-arginine transport requiring adenosine receptors expression in HUVECs. Primary cultured HUVECs from full-term normal (n = 38) and diet-treated GDM (n = 38) pregnancies were used. Insulin effect was assayed on human cationic amino acid transporter 1 (hCAT1) expression (protein, mRNA, SLC7A1 promoter activity) and activity (initial rates of L-arginine transport) in the absence or presence of adenosine receptors agonists or antagonists. A1 adenosine receptors (A1AR) and A2AAR expression (Western blot, quantitative PCR) was determined. Experiments were done in cells expressing or siRNA-suppressed expression of A1AR or A2AAR. HUVECs from GDM exhibit higher maximal transport capacity (maximal velocity (V max)/apparent Michaelis Menten constant (K m), V max/K m), which is blocked by insulin by reducing the V max to values in cells from normal pregnancies. Insulin also reversed the GDM-associated increase in hCAT-1 protein abundance and mRNA expression, and SLC7A1 promoter activity for the fragment -606 bp from the transcription start point. Insulin effects required A1AR, but not A2AAR expression and activity in this cell type. In the absence of insulin, GDM-increased hCAT-1 expression and activity required A2AAR expression and activity. HUVECs from GDM pregnancies exhibit a differential requirement of A1AR or A2AAR depending on the level of insulin, a phenomenon that represent a condition where adenosine or analogues of this nucleoside could be acting as helpers of insulin biological effects in GDM.

  2. Nucleus accumbens and effort-related functions: behavioral and neural markers of the interactions between adenosine A2A and dopamine D2 receptors.

    PubMed

    Farrar, A M; Segovia, K N; Randall, P A; Nunes, E J; Collins, L E; Stopper, C M; Port, R G; Hockemeyer, J; Müller, C E; Correa, M; Salamone, J D

    2010-04-14

    Nucleus accumbens dopamine (DA) is a critical component of the brain circuitry regulating work output in reinforcement-seeking behavior and effort-related choice behavior. Moreover, there is evidence of an interaction between DA D(2) and adenosine A(2A) receptor function. Systemic administration of adenosine A(2A) antagonists reverses the effects of D(2) antagonists on tasks that assess effort related choice. The present experiments were conducted to determine if nucleus accumbens is a brain locus at which adenosine A(2A) and DA D(2) antagonists interact to regulate effort-related choice behavior. A concurrent fixed ratio 5 (FR5)/chow feeding procedure was used; with this procedure, rats can choose between completing an FR5 lever-pressing requirement for a preferred food (i.e., high carbohydrate operant pellets) or approaching and consuming a freely available food (i.e., standard rodent chow). Rats trained with this procedure spend most of their time pressing the lever for the preferred food, and eat very little of the concurrently available chow. Intracranial injections of the selective DA D(2) receptor antagonist eticlopride (1.0, 2.0, 4.0 microg) into nucleus accumbens core, but not a dorsal control site, suppressed FR5 lever-pressing and increased consumption of freely available chow. Either systemic or intra-accumbens injections of the adenosine A(2A) receptor antagonist MSX-3 reversed these effects of eticlopride on effort-related choice. Intra-accumbens injections of eticlopride also increased local expression of c-Fos immunoreactivity, and this effect was attenuated by co-administration of MSX-3. Adenosine and DA systems interact to regulate instrumental behavior and effort-related processes, and nucleus accumbens is an important locus for this interaction. These findings may have implications for the treatment of psychiatric symptoms such as psychomotor slowing, anergia and fatigue. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

  3. Ligand-dependent oligomerization of dopamine D(2) and adenosine A(2A) receptors in living neuronal cells.

    PubMed

    Vidi, Pierre-Alexandre; Chemel, Benjamin R; Hu, Chang-Deng; Watts, Val J

    2008-09-01

    Adenosine A(2A) and dopamine D(2) receptors (A(2A) and D(2)) associate in homo- and heteromeric complexes in the striatum, providing a structural basis for their mutual antagonism. At the cellular level, the portion of receptors engaging in homo- and heteromers, as well as the effect of persistent receptor activation or antagonism on the cell oligomer repertoire, are largely unknown. We have used bimolecular fluorescence complementation (BiFC) to visualize A(2A) and D(2) oligomerization in the Cath.a differentiated neuronal cell model. Receptor fusions to BiFC fluorescent protein fragments retained their function when expressed alone or in A(2A)/A(2A), D(2)/D(2), and A(2A)/D(2) BiFC pairs. Robust fluorescence complementation reflecting A(2A)/D(2) heteromers was detected at the cell membrane as well as in endosomes. In contrast, weaker BiFC signals, largely confined to intracellular domains, were detected with A(2A)/dopamine D(1) BiFC pairs. Multicolor BiFC was used to simultaneously visualize A(2A) and D(2) homo- and heteromers in living cells and to examine drug-induced changes in receptor oligomers. Prolonged D(2) stimulation with quinpirole lead to the internalization of D(2)/D(2) and A(2A)/D(2) oligomers and resulted in decreased A(2A)/D(2) relative to A(2A)/A(2A) oligomer formation. Opposing effects were observed in cells treated with D(2) antagonists or with the A(2A) agonist 5'-N-methylcarboxamidoadenosine (MECA). Subsequent radioreceptor binding analysis indicated that the drug-induced changes in oligomer formation were not readily explained by alterations in receptor density. These observations support the hypothesis that long-term drug exposure differentially alters A(2A)/D(2) receptor oligomerization and provide the first demonstration for the use of BiFC to monitor drug-modulated GPCR oligomerization.

  4. Modulation of gene expression of adenosine and metabotropic glutamate receptors in rat's neuronal cells exposed to L-glutamate and [60]fullerene.

    PubMed

    Giust, Davide; Da Ros, Tatiana; Martín, Mairena; Albasanz, José Luis

    2014-08-01

    L-Glutamate (L-Glu) has been often associated not only to fundamental physiological roles, as learning and memory, but also to neuronal cell death and the genesis and development of important neurodegenerative diseases. Herein we studied the variation in the adenosine and metabotropic glutamate receptors expression induced by L-Glu treatment in rat's cortical neurons. The possibility to have structural alteration of the cells induced by L-Glu (100 nM, 1 and 10 microM) has been addressed, studying the modulation of microtubule associated protein-2 (MAP-2) and neurofilament heavy polypeptide (NEFH), natively associated proteins to the dendritic shape maintenance. Results showed that the proposed treatments were not destabilizing the cells, so the L-Glu concentrations were acceptable to investigate fluctuation in receptors expression, which were studied by RT-PCR. Interestingly, C60 fullerene derivative t3ss elicited a protective effect against glutamate toxicity, as demonstrated by MTT assay. In addition, t3ss compound exerted a different effect on the adenosine and metabotropic glutamate receptors analyzed. Interestingly, A(2A) and mGlu1 mRNAs were significantly decreased in conditions were t3ss neuroprotected cortical neurons from L-Glu toxicity. In summary, t3ss protects neurons from glutamate toxicity in a process that appears to be associated with the modulation of the gene expression of adenosine and metabotropic glutamate receptors.

  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.

    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 extendedmore » 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.« less

  6. Activation of P2Y6 Receptors Facilitates Nonneuronal Adenosine Triphosphate and Acetylcholine Release from Urothelium with the Lamina Propria of Men with Bladder Outlet Obstruction.

    PubMed

    Silva, Isabel; Ferreirinha, Fátima; Magalhães-Cardoso, Maria Teresa; Silva-Ramos, Miguel; Correia-de-Sá, Paulo

    2015-10-01

    Deregulation of purinergic bladder signaling may contribute to persistent detrusor overactivity in patients with bladder outlet obstruction. Activation of uridine diphosphate sensitive P2Y6 receptors increases voiding frequency in rats indirectly by releasing adenosine triphosphate from the urothelium. To our knowledge this mechanism has never been tested in the human bladder. We examined the role of the uridine diphosphate sensitive P2Y6 receptor on tetrodotoxin insensitive nonneuronal adenosine triphosphate and [(3)H]acetylcholine release from the human urothelium with the lamina propria of control organ donors and patients with benign prostatic hyperplasia. The adenosine triphosphate-to-[(3)H]acetylcholine ratio was fivefold higher in mucosal urothelium/lamina propria strips from benign prostatic hyperplasia patients than control men. The selective P2Y6 receptor agonist PSB0474 (100 nM) augmented by a similar amount adenosine triphosphate and [(3)H]acetylcholine release from mucosal urothelium/lamina propria strips from both groups of individuals. The facilitatory effect of PSB0474 was prevented by MRS2578 (50 nM) and by carbenoxolone (10 μM), which block P2Y6 receptor and pannexin-1 hemichannels, respectively. Blockade of P2X3 (and/or P2X2/3) receptors with A317491 (100 nM) also attenuated release facilitation by PSB0474 in control men but not in patients with benign prostatic hyperplasia. Immunolocalization studies showed that P2Y6, P2X2 and P2X3 receptors were present in choline acetyltransferase positive urothelial cells. In contrast to P2Y6 staining, choline acetyltransferase, P2X2 and P2X3 immunoreactivity decreased in the urothelium of benign prostatic hyperplasia patients. Activation of P2Y6 receptor amplifies mucosal adenosine triphosphate release underlying bladder overactivity in patients with benign prostatic hyperplasia. Therefore, we propose selective P2Y6 receptor blockade as a novel therapeutic strategy to control persistent storage symptoms in

  7. Extracellular adenosine sensing-a metabolic cell death priming mechanism downstream of p53.

    PubMed

    Long, Jaclyn S; Crighton, Diane; O'Prey, James; Mackay, Gillian; Zheng, Liang; Palmer, Timothy M; Gottlieb, Eyal; Ryan, Kevin M

    2013-05-09

    Tumor cells undergo changes in metabolism to meet their energetic and anabolic needs. It is conceivable that mechanisms exist to sense these changes and link them to pathways that eradicate cells primed for cancer development. We report that the tumor suppressor p53 activates a cell death priming mechanism that senses extracellular adenosine. Adenosine, the backbone of ATP, accumulates under conditions of cellular stress or altered metabolism. We show that its receptor, A2B, is upregulated by p53. A2B expression has little effect on cell viability, but ligand engagement activates a caspase- and Puma-dependent apoptotic response involving downregulation of antiapoptotic Bcl-2 proteins. Stimulation of A2B also significantly enhances cell death mediated by p53 and upon accumulation of endogenous adenosine following chemotherapeutic drug treatment and exposure to hypoxia. Since extracellular adenosine also accumulates within many solid tumors, this distinct p53 function links programmed cell death to both a cancer- and therapy-associated metabolic change. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Deletion of striatal adenosine A2A receptor spares latent inhibition and prepulse inhibition but impairs active avoidance learning

    PubMed Central

    Singer, Philipp; Wei, Catherine J.; Chen, Jiang-Fan; Boison, Detlev; Yee, Benjamin K.

    2013-01-01

    Following early clinical leads, the adenosine A2AR receptor (A2AR) has continued to attract attention as a potential novel target for treating schizophrenia; especially against the negative and cognitive symptoms of the disease because of A2AR’s unique modulatory action over glutamatergic in addition to dopaminergic signaling. Through the antagonistic interaction with the dopamine D2 receptor, and by regulating glutamate release and N-methyl-d-aspartate receptor function, striatal A2AR is ideally positioned to fine-tune the dopamine-glutamate balance whose disturbance is implicated in the pathophysiology of schizophrenia. However, the precise function of striatal A2ARsin the regulation of schizophrenia-relevant behavior is poorly understood. Here, we tested the impact of conditional striatum-specific A2AR knockout (st-A2AR-KO) on latent inhibition (LI) and prepulse inhibition (PPI) – behavior that is tightly regulated by striatal dopamine and glutamate. These are two common cross-species translational tests for the assessment of selective attention and sensorimotor gating deficits reported in schizophrenia patients; and enhanced performance in these tests is associated with antipsychotic drug action. We found that neither LI nor PPI was significantly affected in st-A2AR-KO mice; although a deficit in active avoidance learning was identified in these animals. The latter phenotype, however, was not replicated in another form of aversive conditioning – conditioned taste aversion. Hence, the present study shows that neither learned inattention (as measured by LI) nor sensory gating (as indexed by PPI) requires the integrity of striatal A2ARs– a finding that may undermine the hypothesized importance of A2AR in the genesis and/or treatment of schizophrenia. PMID:23276608

  9. GPCR Ligand Dendrimer (GLiDe) Conjugates: Adenosine Receptor Interactions of a Series of Multivalent Xanthine Antagonists

    PubMed Central

    Kecskés, Angela; Tosh, Dilip K.; Wei, Qiang; Gao, Zhan-Guo; Jacobson, Kenneth A.

    2011-01-01

    Previously, G protein–coupled receptor (GPCR) agonists were tethered from polyamidoamine (PAMAM) dendrimers to provide high receptor affinity and selectivity. Here we prepared GPCR Ligand Dendrimer (GLiDe) conjugates from a potent adenosine receptor (AR) antagonist; such agents are of interest for treating Parkinson’s disease, asthma, and other conditions. Xanthine amine congener (XAC) was appended with an alkyne group on an extended C8 substituent for coupling by Cu(I)-catalyzed click chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. These conjugates also contained triazole-linked PEG groups (8 or 22 moieties per 64 terminal positions) for increasing water-solubility and optionally prosthetic groups for spectroscopic characterization and affinity labeling. Human AR binding affinity increased progressively with the degree of xanthine substitution to reach Ki values in the nM range. The order of affinity of each conjugate was hA2AAR > hA3AR > hA1AR, while the corresponding monomer was ranked hA2AAR > hA1AR ≥ hA3AR. The antagonist activity of the most potent conjugate 14 (34 xanthines per dendrimer) was examined at the Gi-coupled A1AR. Conjugate 14 at 100 nM right-shifted the AR agonist concentration-response curve in a cyclic AMP functional assay in a parallel manner, but at 10 nM (lower than its Ki value) it significantly suppressed the maximal agonist effect in calcium mobilization. This is the first systematic probing of a potent AR antagonist tethered on a dendrimer and its activity as a function of variable loading. PMID:21539392

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

    PubMed

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

    2016-01-01

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

  11. Modulation of G protein-coupled adenosine receptors by strategically functionalized agonists and antagonists immobilized on gold nanoparticles.

    PubMed

    Jayasekara, P Suresh; Phan, Khai; Tosh, Dilip K; Kumar, T Santhosh; Moss, Steven M; Zhang, Guofeng; Barchi, Joseph J; Gao, Zhan-Guo; Jacobson, Kenneth A

    2013-06-01

    Gold nanoparticles (AuNPs) allow the tuning of pharmacokinetic and pharmacodynamic properties by active or passive targeting of drugs for cancer and other diseases. We have functionalized gold nanoparticles by tethering specific ligands, agonists and antagonists, of adenosine receptors (ARs) to the gold surface as models for cell surface interactions with G protein-coupled receptors (GPCRs). The AuNP conjugates with chain-extended AR ligands alone (PEGylated nucleosides and nonnucleosides, anchored to the Au via thioctic acid) were found to be insoluble in water due to hydrophobic entities in the ligand. Therefore, we added a second, biologically inactive pendant moiety to increase the water solubility, consisting of a PEGylated chain terminating in a carboxylic or phosphate group. The purity and stability of the immobilized biologically active ligand were examined by ultrafiltration and HPLC. Pharmacological receptor binding studies on these GPCR ligand-derivatized AuNPs (2-5 nm in diameter), performed using membranes of mammalian cells stably expressing human A1, A2A, and A3ARs, showed that the desired selectivity was retained with K(i) values (nanomolar) of A3AR agonist 21b and A2AAR antagonists 24 and 26a of 14 (A3), 34 (A2A), and 69 (A2A), respectively. The corresponding monomers displayed K i values of 37, 61, and 1,420 nM, respectively. In conclusion, we have synthesized stable, water-soluble AuNP derivatives of tethered A3 and A2AAR ligands that retain the biological properties of their monomeric ligands and are intended for therapeutic and imaging applications. This is the first prototypical application to gold carriers of small molecule (nonpeptide) GPCR ligands, which are under investigation for treatment of cancer and inflammatory diseases.

  12. Adenosine A1 Receptor Protects Against Cisplatin Ototoxicity by Suppressing the NOX3/STAT1 Inflammatory Pathway in the Cochlea

    PubMed Central

    Kaur, Tejbeer; Borse, Vikrant; Sheth, Sandeep; Sheehan, Kelly; Ghosh, Sumana; Tupal, Srinivasan; Jajoo, Sarvesh; Mukherjea, Debashree; Rybak, Leonard P.

    2016-01-01

    Cisplatin is a commonly used antineoplastic agent that produces ototoxicity that is mediated in part by increasing levels of reactive oxygen species (ROS) via the NOX3 NADPH oxidase pathway in the cochlea. Recent studies implicate ROS generation in mediating inflammatory and apoptotic processes and hearing loss by activating signal transducer and activator of transcription (STAT1). In this study, we show that the adenosine A1 receptor (A1AR) protects against cisplatin ototoxicity by suppressing an inflammatory response initiated by ROS generation via NOX3 NADPH oxidase, leading to inhibition of STAT1. Trans-tympanic administration of the A1AR agonist R-phenylisopropyladenosine (R-PIA) inhibited cisplatin-induced ototoxicity, as measured by auditory brainstem responses and scanning electron microscopy in male Wistar rats. This was associated with reduced NOX3 expression, STAT1 activation, tumor necrosis factor-α (TNF-α) levels, and apoptosis in the cochlea. In vitro studies in UB/OC-1 cells, an organ of Corti immortalized cell line, showed that R-PIA reduced cisplatin-induced phosphorylation of STAT1 Ser727 (but not Tyr701) and STAT1 luciferase activity by suppressing the ERK1/2, p38, and JNK mitogen-activated protein kinase (MAPK) pathways. R-PIA also decreased the expression of STAT1 target genes, such as TNF-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced cisplatin-mediated apoptosis. These data suggest that the A1AR provides otoprotection by suppressing NOX3 and inflammation in the cochlea and could serve as an ideal target for otoprotective drug therapy. SIGNIFICANCE STATEMENT Cisplatin is a widely used chemotherapeutic agent for the treatment of solid tumors. Its use results in significant and permanent hearing loss, for which no US Food and Drug Administration-approved treatment is currently available. In this study, we targeted the cochlear adenosine A1 receptor (A1AR) by trans-tympanic injections of the agonist R

  13. Polyamidoamine (PAMAM) Dendrimer Conjugates of Clickable Agonists of the A3 Adenosine Receptor and Coactivation of the P2Y14 Receptor by a Tethered Nucleotide

    SciTech Connect

    Tosh, Dilip, K.; Yoo, Lena S.; Chinn, Moshe

    2010-01-01

    We previously synthesized a series of potent and selective A{sub 3} adenosine receptor (AR) agonists (North-methanocarba nucleoside 5{prime}-uronamides) containing dialkyne groups on extended adenine C2 substituents. We coupled the distal alkyne of a 2-octadiynyl nucleoside by Cu(I)-catalyzed 'click' chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. A{sub 3}AR activation was preserved in these multivalent conjugates, which bound with apparent Ki of 0.1-0.3 nM. They were substituted with nucleoside moieties, solely or in combination with water-solubilizing carboxylic acid groups derived from hexynoic acid. A comparison with various amide-linked dendrimers showed that triazole-linked conjugates displayed selectivity and enhanced A{sub 3}ARmore » affinity. We prepared a PAMAM dendrimer containing equiproportioned peripheral azido and amino groups for conjugation of multiple ligands. A bifunctional conjugate activated both A{sub 3} and P2Y{sub 14} receptors (via amide-linked uridine-5{prime}-diphosphoglucuronic acid), with selectivity in comparison to other ARs and P2Y receptors. This is the first example of targeting two different GPCRs with the same dendrimer conjugate, which is intended for activation of heteromeric GPCR aggregates. Synergistic effects of activating multiple GPCRs with a single dendrimer conjugate might be useful in disease treatment.« less

  14. Polyamidoamine (PAMAM) Dendrimer Conjugates of “Clickable” Agonists of the A3 Adenosine Receptor and Coactivation of the P2Y14 Receptor by a Tethered Nucleotide

    PubMed Central

    Tosh, Dilip K.; Yoo, Lena S.; Chinn, Moshe; Hong, Kunlun; Kilbey, S. Michael; Barrett, Matthew O.; Fricks, Ingrid P.; Harden, T. Kendall; Gao, Zhan-Guo; Jacobson, Kenneth A.

    2010-01-01

    We previously synthesized a series of potent and selective A3 adenosine receptor (AR) agonists (North-methanocarba nucleoside 5′-uronamides) containing dialkyne groups on extended adenine C2 substituents. We coupled the distal alkyne of a 2-octadiynyl nucleoside by Cu(I)-catalyzed “click” chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. A3AR activation was preserved in these multivalent conjugates, which bound with apparent Ki 0.1–0.3 nM. They were substituted with nucleoside moieties, solely or in combination with water-solubilizing carboxylic acid groups derived from hexynoic acid. A comparison with various amide-linked dendrimers showed that triazole-linked conjugates displayed selectivity and enhanced A3AR affinity. We prepared a PAMAM dendrimer containing equiproportioned peripheral azido and amino groups for conjugation of multiple ligands. A bifunctional conjugate activated both A3 and P2Y14 receptors (via amide-linked uridine-5′-diphosphoglucuronic acid), with selectivity in comparison to other ARs and P2Y receptors. This is the first example of targeting two different GPCRs with the same dendrimer conjugate, which is intended for activation of heteromeric GPCR aggregates. Synergistic effects of activating multiple GPCRs with a single dendrimer conjugate might be useful in disease treatment. PMID:20121074

  15. H(N), N, C(α), C(β) and C' assignments of the intrinsically disordered C-terminus of human adenosine A2A receptor.

    PubMed

    Tossavainen, Helena; Hellman, Maarit; Piirainen, Henni; Jaakola, Veli-Pekka; Permi, Perttu

    2015-10-01

    The C-terminus of the human adenosine A2A receptor differs from the other human adenosine receptors by its exceptional length and lack of a canonical cysteine residue. We have previously structurally characterized this C-terminal domain and its interaction with calmodulin. It was shown to be structurally disordered and flexible, and to bind calmodulin with high affinity in a calcium-dependent manner. Interaction with calmodulin takes place at the N-terminal end of the A2A C-terminal domain without major conformational changes in the latter. NMR was one of the biophysical methods used in the study. Here we present the H(N), N, C(α), C(β) and C' chemical shift assignments of the free form of the C-terminus residues 293-412, used in the NMR spectroscopic characterization of the domain.

  16. Role of the NR2A/2B subunits of the N-methyl-D-aspartate receptor in glutamate-induced glutamic acid decarboxylase alteration in cortical GABAergic neurons in vitro.

    PubMed

    Monnerie, H; Hsu, F-C; Coulter, D A; Le Roux, P D

    2010-12-29

    The vulnerability of brain neuronal cell subpopulations to neurologic insults varies greatly. Among cells that survive a pathological insult, for example ischemia or brain trauma, some may undergo morphological and/or biochemical changes that may compromise brain function. The present study is a follow-up of our previous studies that investigated the effect of glutamate-induced excitotoxicity on the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67)'s expression in surviving DIV 11 cortical GABAergic neurons in vitro [Monnerie and Le Roux, (2007) Exp Neurol 205:367-382, (2008) Exp Neurol 213:145-153]. An N-methyl-D-aspartate receptor (NMDAR)-mediated decrease in GAD expression was found following glutamate exposure. Here we examined which NMDAR subtype(s) mediated the glutamate-induced change in GAD protein levels. Western blotting techniques on cortical neuron cultures showed that glutamate's effect on GAD proteins was not altered by NR2B-containing diheteromeric (NR1/NR2B) receptor blockade. By contrast, blockade of triheteromeric (NR1/NR2A/NR2B) receptors fully protected against a decrease in GAD protein levels following glutamate exposure. When receptor location on the postsynaptic membrane was examined, extrasynaptic NMDAR stimulation was observed to be sufficient to decrease GAD protein levels similar to that observed after glutamate bath application. Blocking diheteromeric receptors prevented glutamate's effect on GAD proteins after extrasynaptic NMDAR stimulation. Finally, NR2B subunit examination with site-specific antibodies demonstrated a glutamate-induced, calpain-mediated alteration in NR2B expression. These results suggest that glutamate-induced excitotoxic NMDAR stimulation in cultured GABAergic cortical neurons depends upon subunit composition and receptor location (synaptic vs. extrasynaptic) on the neuronal membrane. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered

  17. A1 adenosine receptors in the striatum play a role in the memory impairment caused by sleep deprivation through downregulation of the PKA pathway.

    PubMed

    Oliveira, S L B; Oliveira, M G M; Hipolide, D C

    2018-03-31

    Sleep deprivation is known to affect memory formation, but how it interacts with different memory systems is not completely understood. Adenosine, a homeostatic regulator of sleep that has an increased extracellular concentration during sleep deprivation, is one of the neuromodulators that may be involved in this interaction. The A 1 adenosine receptor is involved in both sleep regulation and memory formation. Among other pathways, the A 1 receptor decreases cAMP levels in the cytosol and thus also regulates protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) activity. To verify the role of the A 1 receptor in the memory impairment caused by sleep deprivation, we tested the effect of 96 h of sleep deprivation (SD) and the administration of DPCPX, an A 1 receptor antagonist on male Wistar rats prior to the training sessions for two memory tasks that relies on the hippocampal function: the multiple trial inhibitory avoidance (MTIA) task, which also requires the striatum, and the contextual fear conditioning (CFC) task, which does not. We also evaluated the effect of SD, DPCPX and the MTIA training session on the protein expression levels of the A1 receptor, PKA phosphorylation and EPAC activity in both the hippocampus and the striatum. Sleep deprivation impaired the performance in the test sessions of both tasks; DPCPX was able to prevent the impairment in the MTIA test but not in the CFC test. SD increased A 1 receptor protein expression levels in the striatum but not in the hippocampus and also decreased PKA phosphorylation in both structures; DPCPX prevented this decrease in the striatum, but not in the hippocampus. Finally, SD had no effect on EPAC activity in either of the structures. These results indicate that the A 1 adenosine receptors play a role in the memory impairment caused by sleep deprivation in tasks that involve the striatum through modulation of the cAMP/PKA pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. A genetic variation in the adenosine A2A receptor gene (ADORA2A) contributes to individual sensitivity to caffeine effects on sleep.

    PubMed

    Rétey, J V; Adam, M; Khatami, R; Luhmann, U F O; Jung, H H; Berger, W; Landolt, H-P

    2007-05-01

    Caffeine is the most widely used stimulant in Western countries. Some people voluntarily reduce caffeine consumption because it impairs the quality of their sleep. Studies in mice revealed that the disruption of sleep after caffeine is mediated by blockade of adenosine A2A receptors. Here we show in humans that (1) habitual caffeine consumption is associated with reduced sleep quality in self-rated caffeine-sensitive individuals, but not in caffeine-insensitive individuals; (2) the distribution of distinct c.1083T>C genotypes of the adenosine A2A receptor gene (ADORA2A) differs between caffeine-sensitive and -insensitive adults; and (3) the ADORA2A c.1083T>C genotype determines how closely the caffeine-induced changes in brain electrical activity during sleep resemble the alterations observed in patients with insomnia. These data demonstrate a role of adenosine A2A receptors for sleep in humans, and suggest that a common variation in ADORA2A contributes to subjective and objective responses to caffeine on sleep.

  20. Adenosine A2A receptor inhibition restores the normal transport of endothelial glutamate transporters in the brain.

    PubMed

    Bai, Wei; Li, Ping; Ning, Ya-Lei; Peng, Yan; Xiong, Ren-Ping; Yang, Nan; Chen, Xing; Zhou, Yuan-Guo

    2018-04-15

    Excitatory amino acid transporters (EAATs) on cerebral vascular endothelial cells play an important role in maintaining glutamate homeostasis in the brain. The dysfunction of endothelial EAATs is an important reason for the dramatically elevated brain glutamate levels after brain injury, such as traumatic brain injury (TBI). The adenosine A 2A receptor (A 2A R) plays an important role in regulating the brain glutamate level after brain injury; however, researchers have not clearly determined whether this role was related to its ability to regulate endothelial EAATs. Activation of A 2A R in vitro not only decreased the PKA- and glutamate level-dependent strengthening of the interaction between NKA-α1 and the FXYD1 subunit and the subsequent decrease in the activity of Na + /K + -ATPases (NKAs) but also enhanced its interaction with EAATs and ultimately aggravated the reverse transport function of endothelial EAATs under oxygen-glucose deprivation (OGD) conditions. Conversely, inhibition of A 2A R restored the normal transport of EAAT. Moreover, A 2A R inhibition increased NKA activity and decreased its interaction with EAATs in isolated brain capillaries after TBI, further confirming its role in endothelial EAATs in vivo. Based on our results, A 2A R played an important role in regulating endothelial EAAT function, and strategies that restore the normal transport of endothelial EAATs through the inhibition of A 2A R might serve as an effective treatment for brain injury. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Activation of the adenosine A2A receptor attenuates experimental autoimmune encephalomyelitis and is associated with increased intracellular calcium levels.

    PubMed

    Liu, Yumei; Zou, Haifeng; Zhao, Ping; Sun, Bo; Wang, Jinghua; Kong, Qingfei; Mu, Lili; Zhao, Sihan; Wang, Guangyou; Wang, Dandan; Zhang, Yao; Zhao, Jiaying; Yin, Pengqi; Liu, Lei; Zhao, Xiuli; Li, Hulun

    2016-08-25

    Multiple sclerosis (MS) is a common autoimmune disease that inevitably causes inflammatory nerve demyelination. However, an effective approach to prevent its course is still lacking and urgently needed. Recently, the adenosine A2A receptor (A2AR) has emerged as a novel inflammation regulator. Manipulation of A2AR activity may suppress the MS process and protect against nerve damage. To test this hypothesis, we treated murine experimental autoimmune encephalomyelitis (EAE), a model for MS, with the selective A2AR agonist, CGS21680 (CGS). We evaluated the effects of CGS on the pathological features of EAE progression, including CNS cellular infiltration, inflammatory cytokine expression, lymphocyte proliferation, and cell surface markers. Treatment with CGS significantly suppressed specific lymphocyte proliferation, reduced infiltration of CD4(+) T lymphocytes, and attenuated the expression of inflammatory cytokines, which in turn inhibited the EAE progression. For the first time, we demonstrate that CGS can increase the intracellular calcium concentration ([Ca(2+)]i) in murine lymphocytes, which may be the mechanism underlying the suppressive effects of CGS-induced A2AR activation on EAE progression. Our findings strongly suggest that A2AR is a potential therapeutic target for MS and provide insight into the mechanism of action of A2AR agonists, which may offer a therapeutic option for this disease. Copyright © 2016. Published by Elsevier Ltd.

  2. Purine (N)-Methanocarba Nucleoside Derivatives Lacking an Exocyclic Amine as Selective A3 Adenosine Receptor Agonists

    PubMed Central

    2016-01-01

    Purine (N)-methanocarba-5′-N-alkyluronamidoriboside A3 adenosine receptor (A3AR) agonists lacking an exocyclic amine resulted from an unexpected reaction during a Sonogashira coupling and subsequent aminolysis. Because the initial C6-Me and C6-styryl derivatives had unexpectedly high A3AR affinity, other rigid nucleoside analogues lacking an exocyclic amine were prepared. Of these, the C6-Me-(2-phenylethynyl) and C2-(5-chlorothienylethynyl) analogues were particularly potent, with human A3AR Ki values of 6 and 42 nM, respectively. Additionally, the C2-(5-chlorothienyl)-6-H analogue was potent and selective at A3AR (MRS7220, Ki 60 nM) and also completely reversed mouse sciatic nerve mechanoallodynia (in vivo, 3 μmol/kg, po). The lack of a C6 H-bond donor while maintaining A3AR affinity and efficacy could be rationalized by homology modeling and docking of these hypermodified nucleosides. The modeling suggests that a suitable combination of stabilizing features can partially compensate for the lack of an exocyclic amine, an otherwise important contributor to recognition in the A3AR binding site. PMID:26890707

  3. Theobromine suppresses adipogenesis through enhancement of CCAAT-enhancer-binding protein β degradation by adenosine receptor A1.

    PubMed

    Mitani, Takakazu; Watanabe, Shun; Yoshioka, Yasukiyo; Katayama, Shigeru; Nakamura, Soichiro; Ashida, Hitoshi

    2017-12-01

    Theobromine, a methylxanthine derived from cacao beans, reportedly has various health-promoting properties but molecular mechanism by which effects of theobromine on adipocyte differentiation and adipogenesis remains unclear. In this study, we aimed to clarify the molecular mechanisms of the anti-adipogenic effect of theobromine in vitro and in vivo. ICR mice (4week-old) were administered with theobromine (0.1g/kg) for 7days. Theobromine administration attenuated gains in body and epididymal adipose tissue weights in mice and suppressed expression of adipogenic-associated genes in mouse adipose tissue. In 3T3-L1 preadipocytes, theobromine caused degradation of C/EBPβ protein by the ubiquitin-proteasome pathway. Pull down assay showed that theobromine selectively interacts with adenosine receptor A1 (AR1), and AR1 knockdown inhibited theobromine-induced C/EBPβ degradation. Theobromine increased sumoylation of C/EBPβ at Lys133. Expression of the small ubiquitin-like modifier (SUMO)-specific protease 2 (SENP2) gene, coding for a desumoylation enzyme, was suppressed by theobromine. In vivo knockdown studies showed that AR1 knockdown in mice attenuated the anti-adipogenic effects of theobromine in younger mice. Theobromine suppresses adipocyte differentiation and induced C/EBPβ degradation by increasing its sumoylation. Furthermore, the inhibition of AR1 signaling is important for theobromine-induced C/EBPβ degradation. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Mechanistic insights into allosteric regulation of the A 2A adenosine G protein-coupled receptor by physiological cations

    DOE PAGES

    Ye, Libin; Neale, Chris Andrew; Sljoka, Adnan; ...

    2018-04-10

    Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19F NMR is used to delineate the effects of cations on functional states of the adenosine A 2A GPCR. While Na + reinforces an inactive ensemble and a partial-agonist stabilized state, Ca 2+ and Mg 2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridgemore » specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. Lastly, an understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.« less

  5. Calcium and adenosine triphosphate control of cellular pathology: asparaginase-induced pancreatitis elicited via protease-activated receptor 2.

    PubMed

    Peng, Shuang; Gerasimenko, Julia V; Tsugorka, Tatiana; Gryshchenko, Oleksiy; Samarasinghe, Sujith; Petersen, Ole H; Gerasimenko, Oleg V

    2016-08-05

    Exocytotic secretion of digestive enzymes from pancreatic acinar cells is elicited by physiological cytosolic Ca(2+) signals, occurring as repetitive short-lasting spikes largely confined to the secretory granule region, that stimulate mitochondrial adenosine triphosphate (ATP) production. By contrast, sustained global cytosolic Ca(2+) elevations decrease ATP levels and cause necrosis, leading to the disease acute pancreatitis (AP). Toxic Ca(2+) signals can be evoked by products of alcohol and fatty acids as well as bile acids. Here, we have investigated the mechanism by which l-asparaginase evokes AP. Asparaginase is an essential element in the successful treatment of acute lymphoblastic leukaemia, the most common type of cancer affecting children, but AP is a side-effect occurring in about 5-10% of cases. Like other pancreatitis-inducing agents, asparaginase evoked intracellular Ca(2+) release followed by Ca(2+) entry and also substantially reduced Ca(2+) extrusion because of decreased intracellular ATP levels. The toxic Ca(2+) signals caused extensive necrosis. The asparaginase-induced pathology depended on protease-activated receptor 2 and its inhibition prevented the toxic Ca(2+) signals and necrosis. We tested the effects of inhibiting the Ca(2+) release-activated Ca(2+) entry by the Ca(2+) channel inhibitor GSK-7975A. This markedly reduced asparaginase-induced Ca(2+) entry and also protected effectively against the development of necrosis.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'. © 2016 The Authors.

  6. Adenosine triphosphate release and purinergic (P2) receptor-mediated secretion in small and large mouse cholangiocytes.

    PubMed

    Woo, Kangmee; Sathe, Meghana; Kresge, Charles; Esser, Victoria; Ueno, Yoshiyuki; Venter, Julie; Glaser, Shannon S; Alpini, Gianfranco; Feranchak, Andrew P

    2010-11-01

    Adenosine triphosphate (ATP) is released from cholangiocytes into bile and is a potent secretogogue by increasing intracellular Ca²(+) and stimulating fluid and electrolyte secretion via binding purinergic (P2) receptors on the apical membrane. Although morphological differences exist between small and large cholangiocytes (lining small and large bile ducts, respectively), the role of P2 signaling has not been previously evaluated along the intrahepatic biliary epithelium. The aim of these studies therefore was to characterize ATP release and P2-signaling pathways in small (MSC) and large (MLC) mouse cholangiocytes. The findings reveal that both MSCs and MLCs express P2 receptors, including P2X4 and P2Y2. Exposure to extracellular nucleotides (ATP, uridine triphosphate, or 2',3'-O-[4-benzoyl-benzoyl]-ATP) caused a rapid increase in intracellular Ca²(+) concentration and in transepithelial secretion (I(sc)) in both cell types, which was inhibited by the Cl(-) channel blockers 5-nitro-2-(-3-phenylpropylamino)-benzoic acid (NPPB) or niflumic acid. In response to mechanical stimulation (flow/shear or cell swelling secondary to hypotonic exposure), both MSCs and MLCs exhibited a significant increase in the rate of exocytosis, which was paralleled by an increase in ATP release. Mechanosensitive ATP release was two-fold greater in MSCs compared to MLCs. ATP release was significantly inhibited by disruption of vesicular trafficking by monensin in both cell types. These findings suggest the existence of a P2 signaling axis along intrahepatic biliary ducts with the "upstream" MSCs releasing ATP, which can serve as a paracrine signaling molecule to "downstream" MLCs stimulating Ca²(+)-dependent secretion. Additionally, in MSCs, which do not express the cystic fibrosis transmembrane conductance regulator, Ca²(+)-activated Cl(-) efflux in response to extracellular nucleotides represents the first secretory pathway clearly identified in these cholangiocytes derived from the

  7. Adenosine A1 receptor activation modulates N-methyl-d-aspartate (NMDA) preconditioning phenotype in the brain.

    PubMed

    Constantino, Leandra C; Pamplona, Fabrício A; Matheus, Filipe C; Ludka, Fabiana K; Gomez-Soler, Maricel; Ciruela, Francisco; Boeck, Carina R; Prediger, Rui D; Tasca, Carla I

    2015-04-01

    N-methyl-d-aspartate (NMDA) preconditioning is induced by subtoxic doses of NMDA and it promotes a transient state of resistance against subsequent lethal insults. Interestingly, this mechanism of neuroprotection depends on adenosine A1 receptors (A1R), since blockade of A1R precludes this phenomenon. In this study we evaluated the consequences of NMDA preconditioning on the hippocampal A1R biology (i.e. expression, binding properties and functionality). Accordingly, we measured A1R expression in NMDA preconditioned mice (75mg/kg, i.p.; 24h) and showed that neither the total amount of receptor, nor the A1R levels in the synaptic fraction was altered. In addition, the A1R binding affinity to the antagonist [(3)H] DPCPX was slightly increased in total membrane extracts of hippocampus from preconditioned mice. Next, we evaluated the impact of NMDA preconditioning on A1R functioning by measuring the A1R-mediated regulation of glutamate uptake into hippocampal slices and on behavioral responses in the open field and hot plate tests. NMDA preconditioning increased glutamate uptake into hippocampal slices without altering the expression of glutamate transporter GLT-1. Interestingly, NMDA preconditioning also induced antinociception in the hot plate test and both effects were reversed by post-activation of A1R with the agonist CCPA (0.2mg/kg, i.p.). NMDA preconditioning or A1R modulation did not alter locomotor activity in the open field. Overall, the results described herein provide new evidence that post-activation of A1R modulates NMDA preconditioning-mediated responses, pointing to the importance of the cross-talk between glutamatergic and adenosinergic systems to neuroprotection. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2014-10-01

    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 μm(2)/s, respectively. FCS analysis of a green fluorescent protein (GFP)-tagged A3AR exhibited a single diffusing species (0.105 μm(2)/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 © FASEB.

  9. Up-regulation of striatal adenosine A2A receptors with iron deficiency in rats. Effects on locomotion and cortico-striatal neurotransmission

    PubMed Central

    Quiroz, César; Pearson, Virginia; Gulyani, Seema; Allen, Richard; Earley, Christopher; Ferré, Sergi

    2010-01-01

    Brain iron deficiency leads to altered dopaminergic function in experimental animals, which can provide a mechanistic explanation for iron deficiency-related human sensory-motor disorders, such as Restless Legs Syndrome (RLS). However, mechanisms linking both conditions have not been determined. Considering the strong modulation exerted by adenosine on dopamine signaling, one connection could involve changes in adenosine receptor expression or function. In the striatum, presynaptic A2A receptors are localized in glutamatergic terminals contacting GABAergic dynorphinergic neurons and their function can be analyzed by the ability of A2A receptor antagonists to block the motor output induced by cortical electrical stimulation. Postsynaptic A2A receptors are localized in the dendritic field of GABAergic enkephalinergic neurons and their function can be analyzed by studying the ability of A2A receptor antagonists to produce locomotor activity and to counteract striatal ERK1/2 phosphorylation induced by cortical electrical stimulation. Increased density of striatal A2A receptors was found in rats fed during three weeks with an iron-deficient diet during the post-weaning period. In iron-deficient rats, the selective A2A receptor antagonist MSX-3, at doses of 1 and 3 mg/kg, was more effective at blocking motor output induced by cortical electrical stimulation (presynaptic A2A receptor-mediated effect) and at enhancing locomotor activation and blocking striatal ERK phosphorylation induced by cortical electrical stimulation (postsynaptic A2A receptor-mediated effects). These results indicate that brain iron deficiency induces a functional up-regulation of both striatal pre- and postsynaptic A2A receptor, which could be involved in sensory-motor disorders associated with iron deficiency such as RLS. PMID:20385128

  10. Up-regulation of striatal adenosine A(2A) receptors with iron deficiency in rats: effects on locomotion and cortico-striatal neurotransmission.

    PubMed

    Quiroz, César; Pearson, Virginia; Gulyani, Seema; Allen, Richard; Earley, Christopher; Ferré, Sergi

    2010-07-01

    Brain iron deficiency leads to altered dopaminergic function in experimental animals, which can provide a mechanistic explanation for iron deficiency-related human sensory-motor disorders, such as Restless Legs Syndrome (RLS). However, mechanisms linking both conditions have not been determined. Considering the strong modulation exerted by adenosine on dopamine signaling, one connection could involve changes in adenosine receptor expression or function. In the striatum, presynaptic A(2A) receptors are localized in glutamatergic terminals contacting GABAergic dynorphinergic neurons and their function can be analyzed by the ability of A(2A) receptor antagonists to block the motor output induced by cortical electrical stimulation. Postsynaptic A(2A) receptors are localized in the dendritic field of GABAergic enkephalinergic neurons and their function can be analyzed by studying the ability of A(2A) receptor antagonists to produce locomotor activity and to counteract striatal ERK1/2 phosphorylation induced by cortical electrical stimulation. Increased density of striatal A(2A) receptors was found in rats fed during 3 weeks with an iron-deficient diet during the post-weaning period. In iron-deficient rats, the selective A(2A) receptor antagonist MSX-3, at doses of 1 and 3 mg/kg, was more effective at blocking motor output induced by cortical electrical stimulation (presynaptic A(2A) receptor-mediated effect) and at enhancing locomotor activation and blocking striatal ERK phosphorylation induced by cortical electrical stimulation (postsynaptic A(2A) receptor-mediated effects). These results indicate that brain iron deficiency induces a functional up-regulation of both striatal pre- and postsynaptic A(2A) receptor, which could be involved in sensory-motor disorders associated with iron deficiency such as RLS. Copyright 2010. Published by Elsevier Inc.

  11. Caffeine, Adenosine Receptors and Estrogen in Toxin Models of Parkinson’s Disease

    DTIC Science & Technology

    2005-10-01

    right). Adjacent coronal brain sections were processed for A2A receptor immunohistochemsitry (B) with a HRP-coupled secondary antibody yielding a...declines steadily with increasing intake of caffeine or of coffee (but not decaffeinated coffee). The mechanisms that underlie this epidemiological...ad Sju - 5261.[7 of decaffeinated coffee was not associated with an Furthermore. A2A knockout mice (lacking function- altered risk of PD). clearly

  12. Caffeine, Adenosine Receptors and Estrogen in Toxin Models of Parkinson’s Disease

    DTIC Science & Technology

    2007-10-01

    through blockade of brain A2A (not A1) receptors to protect dopaminergic neurons in both acute (MPTP) and chronic ( paraquat /maneb) toxin models of PD...neuroprotective effect. To explore this possibility in a chronic dual pesticide ( paraquat + maneb) model of PD we first had to determine whether caffeine is...protective in this model. C57Bl/6 mice were treated twice weekly with paraquat (Pq) and maneb (Mb) i.p. for 8 weeks. Ten min prior to each Pq+Mb

  13. Enhanced recovery of breathing capacity from combined adenosine 2A receptor inhibition and daily acute intermittent hypoxia after chronic cervical spinal injury

    PubMed Central

    Navarrete-Opazo, A.; Dougherty, B.J.; Mitchell, G.S.

    2016-01-01

    Daily acute intermittent hypoxia (dAIH) improves breathing capacity after C2 spinal hemisection (C2HS) in rats. Since C2HS disrupts spinal serotonergic innervation below the injury, adenosine-dependent mechanisms underlie dAIH-induced functional recovery 2 weeks post-injury. We hypothesized that dAIH-induced functional recovery converts from an adenosine-dependent to a serotonin-dependent, adenosine-constrained mechanism with chronic injury. Eight weeks post-C2HS, rats began dAIH (10, 5-min episodes, 10.5% O2; 5-min intervals; 7 days) followed by AIH 3× per week (3×wAIH) for 8 additional weeks with/without systemic A2A receptor inhibition (KW6002) on each AIH exposure day. Tidal volume (VT) and bilateral diaphragm (Dia) and T2 external intercostal motor activity were assessed in unanesthetized rats breathing air and during maximum chemoreflex stimulation (MCS: 7% CO2, 10.5% O2). Nine weeks post-C2HS, dAIH increased VT versus time controls (p < 0.05), an effect enhanced by KW6002 (p < 0.05). dAIH increased bilateral Dia activity (p < 0.05), and KW6002 enhanced this effect in contralateral (p < 0.05) and ipsilateral Dia activity (p < 0.001), but not T2 inspiratory activity. Functional benefits of combined AIH plus systemic A2A receptor inhibition were maintained for 4 weeks. Thus, in rats with chronic injuries: 1) dAIH improves VT and bilateral diaphragm activity; 2) VT recovery is enhanced by A2A receptor inhibition; and 3) functional recovery with A2A receptor inhibition and AIH “reminders” last 4 weeks. Combined dAIH and A2A receptor inhibition may be a simple, safe, and effective strategy to accelerate/enhance functional recovery of breathing capacity in patients with respiratory impairment from chronic spinal injury. PMID:27079999

  14. Genetic basis of apnoea of prematurity and caffeine treatment response: role of adenosine receptor polymorphisms: genetic basis of apnoea of prematurity.

    PubMed

    Kumral, Abdullah; Tuzun, Funda; Yesilirmak, Didem Cemile; Duman, Nuray; Ozkan, Hasan

    2012-07-01

    Caffeine treatment reduces the frequency of apnoea of prematurity (AOP) and eliminates the need for mechanical ventilation by acting as a nonspecific inhibitor of adenosine A1 and adenosine 2A receptors. Patients with AOP have demonstrated variant responses to caffeine therapy. We proposed to investigate the role of A1 and 2A polymorphisms in the development of AOP and individual differences in caffeine response. Secondly, we aimed to determine whether these polymorphisms have any effect on bronchopulmonary dysplasia (BPD) development. Cord blood samples were collected from infants born with gestational ages between 24 and 34 weeks. Two groups were defined: patients without apnoea (n = 60) and patients with apnoea (n = 55). Patients with apnoea were divided into two subgroups: a caffeine-responsive group (n = 30) and an unresponsive group (n = 25). Six single-nucleotide polymorphisms were chosen for genotyping. Patients with apnoea over 28 weeks of gestational age who responded to the caffeine treatment were found to carry the rs16851030 C/C genotype rather than the C/T or T/T genotype. Logistic regression analysis showed a significant correlation between rs35320474-C/T and T/T genotypes and apnoea and BPD development. Our results indicate a role for adenosine receptor gene polymorphisms in susceptibility to AOP and BPD and in interindividual variability to caffeine response. © 2012 The Author(s)/Acta Paediatrica © 2012 Foundation Acta Paediatrica.

  15. Retinoic acid receptor-related orphan receptor α-induced activation of adenosine monophosphate-activated protein kinase results in attenuation of hepatic steatosis.

    PubMed

    Kim, Eun-Jin; Yoon, Young-Sil; Hong, Suckchang; Son, Ho-Young; Na, Tae-Young; Lee, Min-Ho; Kang, Hyun-Jin; Park, Jinyoung; Cho, Won-Jea; Kim, Sang-Gun; Koo, Seung-Hoi; Park, Hyeung-geun; Lee, Mi-Ock

    2012-05-01

    There is increasing evidence that the retinoic acid receptor-related orphan receptor α (RORα) plays an important role in the regulation of metabolic pathways, particularly of fatty acid and cholesterol metabolism; however, the role of RORα in the regulation of hepatic lipogenesis has not been studied. Here, we report that RORα attenuates hepatic steatosis, probably via activation of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) and repression of the liver X receptor α (LXRα). First, RORα and its activator, cholesterol sulfate (CS), induced phosphorylation of AMPK, which was accompanied by the activation of serine-threonine kinase liver kinase B1 (LKB1). Second, the activation of RORα, either by transient transfection or CS treatment, decreased the TO901317-induced transcriptional expression of LXRα and its downstream target genes, such as the sterol regulatory element binding protein-1 (SREBP-1) and fatty acid synthase. RORα interacted physically with LXRα and inhibited the LXRα response element in the promoter of LXRα, indicating that RORα interrupts the autoregulatory activation loop of LXRα. Third, infection with adenovirus encoding RORα suppressed the lipid accumulation that had been induced by a free-fatty-acid mixture in cultured cells. Furthermore, we observed that the level of expression of the RORα protein was decreased in the liver of mice that were fed a high-fat diet. Restoration of RORα via tail-vein injection of adenovirus (Ad)-RORα decreased the high-fat-diet-induced hepatic steatosis. Finally, we synthesized thiourea derivatives that activated RORα, thereby inducing activation of AMPK and repression of LXRα. These compounds decreased hepatic triglyceride levels and lipid droplets in the high-fat-diet-fed mice. We found that RORα induced activation of AMPK and inhibition of the lipogenic function of LXRα, which may be key phenomena that provide the beneficial effects of RORα against hepatic steatosis

  16. Caffeine Neuroprotective Mechanism Against β-Amyloid Neurotoxicity in SHSY5Y Cell Line: Involvement of Adenosine, Ryanodine, and N-Methyl-D-Aspartate Receptors.

    PubMed

    Keshavarz, Mojtaba; Farrokhi, Majid Reza; Amiri, Atena

    2017-12-01

    Purpose: Some reports have shown neuroprotective effects of caffeine in several neurodegenerative disorders. However, its mechanism of action is not completely clear. Therefore, the aim of this study was to explore the interference of ryanodine, N-methyl-D-aspartate (NMDA) and adenosine modulators with the neuroprotective effects of caffeine against β-amyloid (Aβ) neurotoxicity in the SHSY5Y cells. Methods: The SHSY5Y cells were treated with Aβ23-35 (20µM) and/or caffeine (0.6 and 1mM), or both for 24 hours. Adenosine (20, 40, 60, 80, 100µM), NMDA (20, 50, 70, 90µM), dantrolene (2, 4, 6, 8, 10µM) were also added to the medium and incubated for 24 hours. The cell viability was measured via the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) method. The data were analyzed using one-way ANOVA followed by Bonferroni test. Results: Caffeine at all the used concentrations (0.6, 0.8, 0.9, 1, and 3mM) significantly protected neuronal cells against Aβ neurotoxicity. Adenosine at the concentrations of 20, 40, 80 and 100μM diminished the neuroprotective effects of caffeine (0.6 and 1mM) against Aβ neurotoxicity. NMDA at the concentrations of 20, 50, 70 and 90μM blocked caffeine (0.6 and 1mM) neuroprotective effects. Dantrolene at the concentration of 2, 4, 6, 8 and 10μM diminished the neuroprotective effects of caffeine (0.6mM) and at the concentrations of 2 and 10μM impede caffeine (1mM) neuroprotection against Aβ neurotoxicity. Conclusion: Caffeine produced neuroprotective effect against Aβ neurotoxicity. Blockade of adenosine and NMDA receptors, as well as the activation of ryanodine receptors, may contribute to the neuroprotective effects of caffeine.

  17. Ribavirin suppresses hepatic lipogenesis through inosine monophosphate dehydrogenase inhibition: Involvement of adenosine monophosphate-activated protein kinase-related kinases and retinoid X receptor α.

    PubMed

    Satoh, Shinya; Mori, Kyoko; Onomura, Daichi; Ueda, Youki; Dansako, Hiromichi; Honda, Masao; Kaneko, Shuichi; Ikeda, Masanori; Kato, Nobuyuki

    2017-08-01

    Ribavirin (RBV) has been widely used as an antiviral reagent, specifically for patients with chronic hepatitis C. We previously demonstrated that adenosine kinase, which monophosphorylates RBV into the metabolically active form, is a key determinant for RBV sensitivity against hepatitis C virus RNA replication. However, the precise mechanism of RBV action and whether RBV affects cellular metabolism remain unclear. Analysis of liver gene expression profiles obtained from patients with advanced chronic hepatitis C treated with the combination of pegylated interferon and RBV showed that the adenosine kinase expression level tends to be lower in patients who are overweight and significantly decreases with progression to advanced fibrosis stages. In our effort to investigate whether RBV affects cellular metabolism, we found that RBV treatment under clinically achievable concentrations suppressed lipogenesis in hepatic cells. In this process, guanosine triphosphate depletion through inosine monophosphate dehydrogenase inhibition by RBV and adenosine monophosphate-activated protein kinase-related kinases, especially microtubule affinity regulating kinase 4, were required. In addition, RBV treatment led to the down-regulation of retinoid X receptor α (RXRα), a key nuclear receptor in various metabolic processes, including lipogenesis. Moreover, we found that guanosine triphosphate depletion in cells induced the down-regulation of RXRα, which was mediated by microtubule affinity regulating kinase 4. Overexpression of RXRα attenuated the RBV action for suppression of lipogenic genes and intracellular neutral lipids, suggesting that down-regulation of RXRα was required for the suppression of lipogenesis in RBV action. Conclusion : We provide novel insights about RBV action in lipogenesis and its mechanisms involving inosine monophosphate dehydrogenase inhibition, adenosine monophosphate-activated protein kinase-related kinases, and down-regulation of RXRα. RBV may be a

  18. MicroRNA-206 is involved in the pathogenesis of ulcerative colitis via regulation of adenosine A3 receptor.

    PubMed

    Wu, Weiyun; He, Yanting; Feng, Xiao; Ye, Shicai; Wang, Hao; Tan, Wenkai; Yu, Caiyuan; Hu, Juxiang; Zheng, Rong; Zhou, Yu

    2017-01-03

    Increasing evidence suggests that miRNAs are widely dysregulated in ulcerative colitis (UC), potentially affecting UC pathogenesis, diagnosis, and therapy. microRNA (miR) -206 has been reported to be upregulated in UC; however, its function and role in UC remain unknown. Here, we elucidate the function of miR-206 in the pathogenesis of UC. In patients with active-UC, miR-206 and adenosine A3 receptor (A3AR) levels were significantly upregulated and downregulated, respectively, and were inversely correlated. A3AR was expressed in the colon mucosa (particularly in colon epithelial-cell membranes). In HT-29 cells, miR-206 downregulated A3AR mRNA/protein expression by directly targeting the A3AR 3'-UTR; miR-206 overexpression and knockdown respectively increased and decreased TNF-α-induced nuclear NF-κB/p65, p-IκB-α, IKKα, p-IKKα and IL-8/IL-1β secretion. However, A3AR-siRNA reversed the miR-206 inhibitory effect. Furthermore, miR-206 increased dextran sodium sulphate-induced colitis severity (i.e., increased bodyweight loss, DAI score, colon shrinkage, and MPO activity), which was partially ameliorated by miR-206-antagomir treatment. miR-206-agomir treatment potently suppressed A3AR expression and increased NF-κB signalling and downstream cytokine (TNF-α/IL-8/IL-1β) expression in the mouse colon, in contrast to miR-206-antagomir administration. Taken together, our results demonstrated that miR-206 has a proinflammatory role in UC by downregulating A3AR expression and activating NF-κB signalling.

  19. A novel mechanism of renal blood flow autoregulation and the autoregulatory role of A1 adenosine receptors in mice.

    PubMed

    Just, Armin; Arendshorst, William J

    2007-11-01

    Autoregulation of renal blood flow (RBF) is mediated by a fast myogenic response (MR; approximately 5 s), a slower tubuloglomerular feedback (TGF; approximately 25 s), and potentially additional mechanisms. A1 adenosine receptors (A1AR) mediate TGF in superficial nephrons and contribute to overall autoregulation, but the impact on the other autoregulatory mechanisms is unknown. We studied dynamic autoregulatory responses of RBF to rapid step increases of renal artery pressure in mice. MR was estimated from autoregulation within the first 5 s, TGF from that at 5-25 s, and a third mechanism from 25-100 s. Genetic deficiency of A1AR (A1AR-/-) reduced autoregulation at 5-25 s by 50%, indicating a residual fourth mechanism resembling TGF kinetics but independent of A1AR. MR and third mechanism were unaltered in A1AR-/-. Autoregulation in A1AR-/- was faster at 5-25 than at 25-100 s suggesting two separate mechanisms. Furosemide in wild-type mice (WT) eliminated the third mechanism and enhanced MR, indicating TGF-MR interaction. In A1AR-/-, furosemide did not further impair autoregulation at 5-25 s, but eliminated the third mechanism and enhanced MR. The resulting time course was the same as during furosemide in WT, indicating that A1AR do not affect autoregulation during furosemide inhibition of TGF. We conclude that at least one novel mechanism complements MR and TGF in RBF autoregulation, that is slower than MR and TGF and sensitive to furosemide, but not mediated by A1AR. A fourth mechanism with kinetics similar to TGF but independent of A1AR and furosemide might also contribute. A1AR mediate classical TGF but not TGF-MR interaction.

  20. Adenosine receptor A2A deficiency in leukocytes increases arterial neointima formation in apolipoprotein E-deficient mice.

    PubMed

    Wang, Huan; Zhang, Weiyu; Tang, Rong; Zhu, Chuhong; Bucher, Christoph; Blazar, Bruce R; Geng, Jian-Guo; Zhang, Chunxiang; Linden, Joel; Wu, Chaodong; Huo, Yuqing

    2010-05-01

    To use the mice deficient in both adenosine receptor A(2A)(A(2A)R(-/-)) and apolipoprotein E (apoE(-/-)) to investigate the role of A(2A)R in mediating the interactions of leukocytes with injured arterial walls and the formation of arterial neointima induced by a guide wire. In apoE(-/-) mice, A(2A)R deficiency increased the size of the arterial neointima in injured carotid arteries by 83%. Arterial neointima formation was also enhanced in chimeric mice that underwent bone marrow transplantation (these mice lacked A(2A)R in their bone marrow-derived cells). Epifluorescence intravital microscopy showed that neutrophil rolling and adherence to the injured arterial area were enhanced by 80% and 110% in A(2A)R(-/-)/apoE(-/-) mice, respectively. This phenomenon occurred even though the protein levels of homing molecules on A(2A)R-deficient neutrophils were unchanged from those of wild-type neutrophils. A(2A)R-deficient neutrophils exhibited an increase in the phosphorylation of p38 mitogen-activated protein kinase, P-selectin glycoprotein ligand-1 (PSGL-1) clustering, and the affinity of b(2) integrins. The inhibition of p38 phosphorylation abrogated the increased PSGL-1 clustering and beta(2) integrin affinity, thus reversing the increased homing ability of A(2A)R-deficient leukocytes. A(2A)R plays a complex role in inflammation and tissue injury. The deficiency of A(2A)R enhances the homing ability of leukocytes and increases the formation of the arterial neointima after injury. A(2A)R antagonists are being tested for the treatment of neurodegenerative and other chronic diseases. An evaluation of the effect of A(2A)R antagonists on arterial restenosis after arterial angioplasty should be conducted.

  1. Proximal tubule sphingosine kinase-1 has a critical role in A1 adenosine receptor-mediated renal protection from ischemia

    PubMed Central

    Park, Sang Won; Kim, Mihwa; Kim, Joo Yun; Brown, Kevin M.; Haase, Volker H.; D’Agati, Vivette D.; Lee, H. Thomas

    2012-01-01

    Renal ischemia reperfusion injury is a major cause of acute kidney injury. We previously found that renal A1 adenosine receptor (A1AR) activation attenuated multiple cell death pathways including necrosis, apoptosis and inflammation. Here, we tested whether induction of cytoprotective sphingosine kinase (SK)-1 and sphingosine-1 phosphate (S1P) synthesis might be the mechanism of protection. A selective A1AR agonist (CCPA) increased the synthesis of S1P and selectively induced SK-1 in mouse kidney and HK-2 cells. This agonist failed to protect SK1-knockout but protected SK2-knockout mice against renal ischemia reperfusion injury indicating a critical role of SK1 in A1AR-mediated renal protection. Inhibition of SK prevented A1AR-mediated defense against necrosis and apoptosis in HK-2 cells. A selective S1P1R antagonist (W146) and global in vivo gene knockdown of S1P1Rs with small interfering RNA completely abolished the renal protection provided by CCPA. Mice selectively deficient in renal proximal tubule S1P1Rs (S1P1Rflox/flox PEPCKCre/−) were not protected against renal ischemia reperfusion injury by CCPA. Mechanistically, CCPA increased nuclear translocation of hypoxia inducible factor-1α in HK-2 cells and selective hypoxia inducible factor-1α inhibition blocked A1AR-mediated induction of SK1. Thus, proximal tubule SK-1 has a critical role in A1AR-mediated protection against renal ischemia reperfusion injury. PMID:22695326

  2. Synthesis and characterization of [76Br]-labeled high-affinity A3 adenosine receptor ligands for positron emission tomography.

    PubMed

    Kiesewetter, Dale O; Lang, Lixin; Ma, Ying; Bhattacharjee, Abesh Kumar; Gao, Zhan-Guo; Joshi, Bhalchandra V; Melman, Artem; de Castro, Sonia; Jacobson, Kenneth A

    2009-01-01

    Bromine-76-radiolabeled analogues of previously reported high-affinity A(3) adenosine receptor (A(3)AR) nucleoside ligands have been prepared as potential radiotracers for positron emission tomography. The radiosyntheses were accomplished by oxidative radiobromination on the N(6)-benzyl moiety of trimethyltin precursors. Biodistribution studies of the kinetics of uptake were conducted in awake rats. We prepared an agonist ligand {[(76)Br](1'S,2'R,3'S,4'R,5'S)-4'-{2-chloro-6-[(3-bromophenylmethyl)amino]purin-9-yl}-1'-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2',3'-diol (MRS3581)} in 59% radiochemical yield with a specific activity of 19.5 GBq/micromol and an antagonist ligand {[(76)Br](1'R,2'R,3'S,4'R,5'S)-4'-(6-(3-bromobenzylamino)-2-chloro-9H-purin-9-yl)bicyclo[3.1.0]hexane-2',3'-diol (MRS5147)} in 65% radiochemical yield with a specific activity of 22 GBq/micromol. The resultant products exhibited the expected high affinity (K(i) approximately 0.6 nM) and specific binding at the human A(3)AR in vitro. Biodistribution studies in the rat showed uptake in the organs of excretion and metabolism. The antagonist MRS5147 exhibited increasing uptake in testes, an organ that contains significant quantities of A(3)AR, over a 2-h time course, which suggests the presence of a specific A(3)AR retention mechanism. We were able to compare uptake of the [(76)Br]-labeled antagonist MRS5147 to [(76)Br]agonist MRS3581. The antagonist MRS5147 shows increasing uptake in the testes, an A(3)AR-rich tissue, suggesting that this ligand may have promise as a molecular imaging agent.

  3. Synthesis and Characterization of [76Br]-Labeled High Affinity A3 Adenosine Receptor Ligands for Positron Emission Tomography

    PubMed Central

    Kiesewetter, Dale O.; Lang, Lixin; Ma, Ying; Bhattacharjee, Abesh Kumar; Gao, Zhan-Guo; Joshi, Bhalchandra V.; Melman, Artem; de Castro, Sonia; Jacobson, Kenneth A.

    2009-01-01

    Introduction Bromine-76 radiolabeled analogues of previously reported high affinity A3 adenosine receptor (A3AR) nucleoside ligands have been prepared as potential radiotracers for Positron Emission Tomography (PET). Methods The radiosyntheses were accomplished by oxidative radiobromination on the N6-benzyl moiety of trimethyltin precursors. Biodistribution studies of the kinetics of uptake were conducted in awake rats. Results We prepared an agonist ligand {[76Br](1′R,2′R,3′S,4′R,5′S)-4-{2-chloro-6-[(3-bromophenylmethyl)amino]purin-9-yl}-1-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2,3-diol (MRS3581)} in 59% radiochemical yield (RCY) with a specific activity of 19.5 GBq/μmol and an antagonist ligand {[76Br](1R,2R,3S,4R,5S)-4-(6-(3-bromobenzylamino)-2-chloro-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol. (MRS5147)} in 65% RCY with a specific activity of 22 GBq/μmol). The resultant products exhibited the expected high affinity (Ki ~ 0.6 nM) and specific binding at the human A3AR in vitro. Biodistribution studies in the rat showed uptake in the organs of excretion and metabolism. The antagonist MRS5147 exhibited increasing uptake in testes, an organ that contains significant quantities of A3AR, over a 2 h time course, which suggests the presence of a specific A3AR retention mechanism. Conclusion We were able to compare uptake of the [76Br]labeled antagonist MRS5147 to [76Br]agonist MRS3581. The antagonist MRS5147 shows increasing uptake in the testes, an A3AR rich tissue, suggesting that this ligand may have promise as a molecular imaging agent. PMID:19181263

  4. Embryonic Caffeine Exposure Acts via A1 Adenosine Receptors to Alter Adult Cardiac Function and DNA Methylation in Mice

    PubMed Central

    Greenwood, Victoria; Xue, Huiling; Rivkees, Scott A.; Wendler, Christopher C.

    2014-01-01

    Evidence indicates that disruption of normal prenatal development influences an individual's risk of developing obesity and cardiovascular disease as an adult. Thus, understanding how in utero exposure to chemical agents leads to increased susceptibility to adult diseases is a critical health related issue. Our aim was to determine whether adenosine A1 receptors (A1ARs) mediate the long-term effects of in utero caffeine exposure on cardiac function and whether these long-term effects are the result of changes in DNA methylation patterns in adult hearts. Pregnant A1AR knockout mice were treated with caffeine (20 mg/kg) or vehicle (0.09% NaCl) i.p. at embryonic day 8.5. This caffeine treatment results in serum levels equivalent to the consumption of 2–4 cups of coffee in humans. After dams gave birth, offspring were examined at 8–10 weeks of age. A1AR+/+ offspring treated in utero with caffeine were 10% heavier than vehicle controls. Using echocardiography, we observed altered cardiac function and morphology in adult mice exposed to caffeine in utero. Caffeine treatment decreased cardiac output by 11% and increased left ventricular wall thickness by 29% during diastole. Using DNA methylation arrays, we identified altered DNA methylation patterns in A1AR+/+ caffeine treated hearts, including 7719 differentially methylated regions (DMRs) within the genome and an overall decrease in DNA methylation of 26%. Analysis of genes associated with DMRs revealed that many are associated with cardiac hypertrophy. These data demonstrate that A1ARs mediate in utero caffeine effects on cardiac function and growth and that caffeine exposure leads to changes in DNA methylation. PMID:24475304

  5. Psychological Stress Activates the Inflammasome via Release of Adenosine Triphosphate and Stimulation of the Purinergic Type 2X7 Receptor.

    PubMed

    Iwata, Masaaki; Ota, Kristie T; Li, Xiao-Yuan; Sakaue, Fumika; Li, Nanxin; Dutheil, Sophie; Banasr, Mounira; Duric, Vanja; Yamanashi, Takehiko; Kaneko, Koichi; Rasmussen, Kurt; Glasebrook, Andrew; Koester, Anja; Song, Dekun; Jones, Kenneth A; Zorn, Stevin; Smagin, Gennady; Duman, Ronald S

    2016-07-01

    The mechanisms underlying stress-induced inflammation that contribute to major depressive disorder are unknown. We examine the role of the adenosine triphosphate (ATP)/purinergic type 2X7 receptor (P2X7R) pathway and the NLRP3 (nucleotide-binding, leucine-rich repeat, pyrin domain containing 3) inflammasome in interleukin (IL)-1β and depressive behavioral responses to stress. The influence of acute restraint stress on extracellular ATP, glutamate, IL-1β, and tumor necrosis factor alpha in hippocampus was determined by microdialysis, and the influence of acute restraint stress on the NLRP3 inflammasome was determined by western blot analysis. The influence of P2X7R antagonist administration on IL-1β and tumor necrosis factor alpha and on anxiety and depressive behaviors was determined in the chronic unpredictable stress rodent model. The role of the NLRP3 inflammasome was determined by analysis of Nlrp3 null mice. Acute restraint stress rapidly increased extracellular ATP, an endogenous agonist of P2X7R; the inflammatory cytokine IL-1β; and the active form of the NLRP3 inflammasome in the hippocampus. Administration of a P2X7R antagonist completely blocked the release of IL-1β and tumor necrosis factor alpha, another stress-induced cytokine, and activated NLRP3. Moreover, P2X7R antagonist administration reversed the anhedonic and anxiety behaviors caused by chronic unpredictable stress exposure, and deletion of the Nlrp3 gene rendered mice resistant to development of depressive behaviors caused by chronic unpredictable stress. These findings demonstrate that psychological "stress" is sensed by the innate immune system in the brain via the ATP/P2X7R-NLRP3 inflammasome cascade, and they identify novel therapeutic targets for the treatment of stress-related mood disorders and comorbid illnesses. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  6. Disruption of Adenosine 2A Receptor Exacerbates NAFLD through Increasing Inflammatory Responses and SREBP1c Activity.

    PubMed

    Cai, Yuli; Li, Honggui; Liu, Mengyang; Pei, Ya; Zheng, Juan; Zhou, Jing; Luo, Xianjun; Huang, Wenya; Ma, Linqiang; Yang, Qiuhua; Guo, Shaodong; Xiao, Xiaoqiu; Li, Qifu; Zeng, Tianshu; Meng, Fanyin; Francis, Heather; Glaser, Shannon; Chen, Lulu; Huo, Yuqing; Alpini, Gianfranco; Wu, Chaodong

    2018-01-09

    Adenosine 2A receptor (A 2A R) exerts protective roles in endotoxin- and/or ischemia-induced tissue damages. However, the role for A 2A R in non-alcoholic fatty liver disease (NAFLD) remains largely unknown. We sought to examine the effects of global and/or myeloid cell-specific A 2A R disruption on the aspects of obesity-associated NAFLD and to elucidate the underlying mechanisms. Global and/or myeloid cell-specific A 2A R-disrupted mice, as well as control mice were fed a high-fat diet (HFD) to induce NAFLD. Also, bone marrow-derived macrophages and primary mouse hepatocytes were examined for inflammatory and metabolic responses. Upon feeding an HFD, both global A 2A R-disrupted mice and myeloid cell-specific A 2A R-defcient mice revealed increased severity of HFD-induced hepatic steatosis and inflammation compared with their respective control mice. In in vitro experiments, A 2A R-deficient macrophages exhibited increased proinflammatory responses, and enhanced fat deposition of wild-type primary hepatocytes in macrophage-hepatocyte co-cultures. In primary hepatocytes, A 2A R deficiency increased the proinflammatory responses and enhanced the effect of palmitate on stimulating fat deposition. Moreover, A 2A R deficiency significantly increased sterol regulatory element-binding protein 1c (SREBP1c) abundance in livers of fasted mice and in hepatocytes upon nutrient deprivation. In the absence of A 2A R, SREBP1c transcription activity was significantly increased in mouse hepatocytes. Taken together, these results demonstrate that disruption of A 2A R in both macrophage and hepatocytes accounts for increased severity of NAFLD, likely through increasing inflammation and through elevating lipogenic events due to stimulation of SREBP1c expression and transcription activity. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

  7. Chronic Cerebral Ischemia Induces Downregulation of A1 Adenosine Receptors During White Matter Damage in Adult Mice.

    PubMed

    Cheng, Pengfei; Ren, Yifei; Bai, Shunjie; Wu, Yu; Xu, Yi; Pan, Junxi; Chen, Jin; Zhu, Xiaofeng; Qi, Zhiguo; Shao, Weihua; Tang, Weiju; Liu, Meiling; Xie, Peng; Huang, Wen

    2015-11-01

    The role of A1 adenosine receptors (A1ARs) in the white matter under chronic cerebral ischemic conditions remains unclear. Here, we used right unilateral common carotid artery occlusion (rUCCAO) to construct a chronic cerebral ischemic mouse model. A1AR expression and proteolipid protein (PLP, a marker of white matter myelination) in the corpus callosum were observed by immunoreaction and immunohistochemistry, respectively. Pro-inflammatory interleukin-1β (IL-1β) and anti-inflammatory interleukin-10 (IL-10) levels were determined by ELISA. The Morris water maze test was employed to detect cognitive impairment. A1AR expression significantly decreased in the rUCCAO group as compared with the sham control group on weeks 2, 4, and 6, respectively. IL-10 levels in the rUCCAO group significantly declined on week 6, while there was no significant change in IL-1β expression. PLP expression significantly decreased in the rUCCAO group on weeks 2, 4, and 6. Moreover, latency time for the Morris water maze test significantly increased in the rUCCAO group on weeks 4 and 6, while the number of platform location crossing significantly decreased in the rUCCAO group on weeks 2, 4, and 6. In conclusion, this study provides the first evidence that chronic cerebral ischemia appears to induce A1AR downregulation and inhibition of IL-10 production, which may play key roles in the neuropathological mechanisms of ischemic white matter lesions. These data will facilitate future studies in formulating effective therapeutic strategies for ischemic white matter lesions.

  8. Genetic Deletion of the Adenosine A2A Receptor Confers Postnatal Development of Relative Myopia in Mice

    PubMed Central

    Zhou, Xiangtian; Huang, Qinzhu; An, Jianhong; Lu, Runxia; Qin, Xiaoyi; Jiang, Liqin; Li, Yuan; Wang, Jianhua; Chen, Jiangfan; Qu, Jia

    2010-01-01

    Purpose. To critically evaluate whether the adenosine A2A receptor (A2AR) plays a role in postnatal refractive development in mice. Methods. Custom-built biometric systems specifically designed for mice were used to assess the development of relative myopia by examining refraction and biometrics in A2AR knockout (KO) mice and wild-type (WT) littermates between postnatal days (P)28 and P56. Ocular dimensions were measured by customized optical coherence tomography (OCT), refractive state by eccentric infrared photorefraction (EIR), and corneal radius of curvature by modified keratometry. Scleral collagen diameter and density were examined by electron microscopy on P35. The effect of A2AR activation on collagen mRNA expression and on soluble collagen production was examined in cultured human scleral fibroblasts by real-time RT-PCR and a collagen assay kit. Results. Compared with WT littermates, the A2AR KO mice displayed relative myopia (average difference, 5.1 D between P28 and P35) and associated increases in VC depth and axial length from P28 to P56. Furthermore, the myopic shift in A2AR KO mice was associated with ultrastructural changes in the sclera: Electron microscopy revealed denser collagen fibrils with reduced diameter in A2AR KO compared with WT. Last, A2AR activation induced expression of mRNAs for collagens I, III, and V and increased production of soluble collagen in cultured human scleral fibroblasts. Conclusions. Genetic deletion of the A2AR promotes development of relative myopia with increased axial length and altered scleral collagen fiber structure during postnatal development in mice. Thus, the A2AR may be important in normal refractive development. PMID:20484596

  9. METABOTROPIC GLUTAMATE TYPE 5, DOPAMINE D2 AND ADENOSINE A2A RECEPTORS FORM HIGHER-ORDER OLIGOMERS IN LIVING CELLS

    PubMed Central

    Cabello, Nuria; Gandía, Jorge; Bertarelli, Daniela C. G.; Watanabe, Masahiko; Lluís, Carme; Franco, Rafael; Ferré, Sergi; Luján, Rafael; Ciruela, Francisco

    2009-01-01

    G protein-coupled receptors are known to form homo- and heteromers at the plasma membrane, but the stoichiometry of these receptor oligomers are relatively unknown. Here, by using bimolecular fluorescence complementation, we visualized for the first time the occurrence of heterodimers of metabotropic glutamate mGlu5 receptors (mGlu5R) and dopamine D2 receptors (D2R) in living cells. Furthermore, the combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques, as well as the sequential resonance energy transfer (SRET) technique, allowed us to detect the occurrence receptor oligomers containing more than two protomers, mGlu5R, D2R and adenosine A2A receptor (A2AR). Interestingly, by using high-resolution immunoelectron microscopy we could confirm that the three receptors co-distribute within the extrasynaptic plasma membrane of the same dendritic spines of asymmetrical, putative glutamatergic, striatal synapses. Also, co-immunoprecipitation experiments in native tissue demonstrated the existence of an association of mGlu5R, D2R and A2AR in rat striatum homogenates. Overall, these results provide new insights into the molecular composition of G protein-coupled receptor oligomers in general and the mGlu5R/D2R/A2AR oligomer in particular, a receptor oligomer that might constitute an important target for the treatment of some neuropsychiatric disorders. PMID:19344374

  10. Caffeine reverses age-related deficits in olfactory discrimination and social recognition memory in rats. Involvement of adenosine A1 and A2A receptors.

    PubMed

    Prediger, Rui D S; Batista, Luciano C; Takahashi, Reinaldo N

    2005-06-01

    Caffeine, a non-selective adenosine receptor antagonist, has been suggested as a potential drug to counteract age-related cognitive decline since critical changes in adenosinergic neurotransmission occur with aging. In the present study, olfactory discrimination and short-term social memory of 3, 6, 12 and 18 month-old rats were assessed with the olfactory discrimination and social recognition tasks, respectively. The actions of caffeine (3.0, 10.0 and 30.0 mg/kg, i.p.), the A1 receptor antagonist DPCPX (1.0 and 3.0 mg/kg, i.p.) and the A2A receptor antagonist ZM241385 (0.5 and 1.0 mg/kg, i.p.) in relation to age-related effects on olfactory functions were also studied. The 12 and 18 month-old rats exhibited significantly impaired performance in both models, demonstrating deficits in their odor discrimination and in their ability to recognize a juvenile rat after a short period of time. Acute treatment with caffeine or ZM241385, but not with DPCPX, reversed these age-related olfactory deficits. The present results suggest the participation of adenosine receptors in the control of olfactory functions and confirm the potential of caffeine for the treatment of aged-related cognitive decline.

  11. Behavioural and biochemical responses to morphine associated with its motivational properties are altered in adenosine A2A receptor knockout mice

    PubMed Central

    Castañé, A; Wells, L; Soria, G; Hourani, S; Ledent, C; Kitchen, I; Opacka-Juffry, J; Maldonado, R; Valverde, O

    2008-01-01

    Background and purpose: The purinergic system through the A2A adenosine receptor regulates addiction induced by different drugs of abuse. The aim of the present study was to investigate the specific role of A2A adenosine receptors (A2ARs) in the behavioural and neurochemical responses to morphine associated with its motivational properties. Experimental approach: Mice lacking A2ARs (A2A knockout (KO) mice) and wild-type littermates were used to evaluate behavioural responses induced by morphine. Antinociception was assessed using the tail-immersion and the hot-plate tests. Place-conditioning paradigms were used to evaluate the rewarding effects of morphine and the dysphoric responses of morphine withdrawal. Microdialysis studies were carried out to evaluate changes in the extracellular levels of dopamine in the nucleus accumbens of A2A KO mice after morphine administration. Key results: The acute administration of morphine induced a similar enhancement of locomotor activity and antinociceptive responses in both genotypes. However, the rewarding effects induced by morphine were completely blocked in A2A KO mice. Also, naloxone did not induce place aversion in animals lacking the A2ARs. Conclusions and implications: Our findings demonstrate that the rewarding and aversive effects associated with morphine abstinence were abolished in A2A KO mice, supporting a differential role of the A2A adenosine receptor in the somatic and motivational effects of morphine addiction. This study provides evidence for the role of A2ARs as general modulators of the motivational properties of drugs of abuse. Pharmacological manipulation of these receptors may represent a new target in the management of drug addiction. PMID:18660831

  12. A 2A adenosine receptor regulates glia proliferation and pain after peripheral nerve injury.

    PubMed

    Bura, S Andreea; Nadal, Xavier; Ledent, Catherine; Maldonado, Rafael; Valverde, Olga

    2008-11-15

    Peripheral nerve injury produces a persistent neuropathic pain state characterized by spontaneous pain, allodynia and hyperalgesia. In this study, we evaluated the possible involvement of A 2ARs in the development of neuropathic pain and the expression of microglia and astrocytes in the spinal cord after sciatic nerve injury. For this purpose, partial ligation of the sciatic nerve was performed in A 2A knockout mice and wild-type littermates. The development of mechanical and thermal allodynia, as well as thermal hyperalgesia was evaluated by using the von Frey filament model, the cold-plate test and the plantar test, respectively. In wild-type animals, sciatic nerve injury led to a neuropathic pain syndrome that was revealed in these three nociceptive behavioural tests. However, a significant decrease of the mechanical allodynia and a suppression of thermal hyperalgesia and allodynia were observed in A 2AR deficient mice. The expression of microglia and astrocytes was enhanced in wild-type mice exposed to sciatic nerve injury and this response was attenuated in knockout animals. Taken together, our results demonstrate the involvement of A 2ARs in the control of neuropathic pain and propose this receptor as an interesting target for the development of new drugs for the management of this clinical syndrome.

  13. Adenosine A{sub 2A} receptor-dependent proliferation of pulmonary endothelial cells is mediated through calcium mobilization, PI3-kinase and ERK1/2 pathways

    SciTech Connect

    Ahmad, Aftab, E-mail: Aftab.Ahmad@UCDenver.edu; Schaack, Jerome B.; White, Carl W.

    2013-05-10

    Highlights: •A{sub 2A} receptor-induced pulmonary endothelial growth is mediated by PI3K and ERK1/2. •Cytosolic calcium mobilization is also critical for pulmonary endothelial growth. •Effectors of A{sub 2A} receptor, like tyrosine kinases and cAMP increase PI3K/Akt signaling. •Activation of A{sub 2A} receptor can contribute to vascular remodeling. -- Abstract: Hypoxia and HIF-2α-dependent A{sub 2A} receptor expression and activation increase proliferation of human lung microvascular endothelial cells (HLMVECs). This study was undertaken to investigate the signaling mechanisms that mediate the proliferative effects of A{sub 2A} receptor. A{sub 2A} receptor-mediated proliferation of HLMVECs was inhibited by intracellular calcium chelation, and by specific inhibitorsmore » of ERK1/2 and PI3-kinase (PI3K). The adenosine A{sub 2A} receptor agonist CGS21680 caused intracellular calcium mobilization in controls and, to a greater extent, in A{sub 2A} receptor-overexpressing HLMVECs. Adenoviral-mediated A{sub 2A} receptor overexpression as well as receptor activation by CGS21680 caused increased PI3K activity and Akt phosphorylation. Cells overexpressing A{sub 2A} receptor also manifested enhanced ERK1/2 phosphorylation upon CGS21680 treatment. A{sub 2A} receptor activation also caused enhanced cAMP production. Likewise, treatment with 8Br-cAMP increased PI3K activity. Hence A{sub 2A} receptor-mediated cAMP production and PI3K and Akt phosphorylation are potential mediators of the A{sub 2A}-mediated proliferative response of HLMVECs. Cytosolic calcium mobilization and ERK1/2 phosphorylation are other critical effectors of HLMVEC proliferation and growth. These studies underscore the importance of adenosine A{sub 2A} receptor in activation of survival and proliferative pathways in pulmonary endothelial cells that are mediated through PI3K/Akt and ERK1/2 pathways.« less

  14. Adenosine A1-receptor blockade impairs the ability of rat pups to autoresuscitate from primary apnea during repeated exposure to hypoxia

    PubMed Central

    Fewell, James E; Lun, Rongzhi

    2015-01-01

    Failure of gasping to bring about autoresuscitation from hypoxia-induced apnea has been suggested to play a role in sudden unexpected infant death. Little is known, however, about factors that influence the ability of gasping to restore life during severe hypoxia in newborns. Given that adenosine modulates cardiac function during hypoxia-induced apnea and that cardiac dysfunction plays a role in mediating autoresuscitation failure, the present experiments were carried out on 34, 5- to 6-, and 10- to 11-day-old rat pups to investigate their ability to autoresuscitate from hypoxia-induced apnea during repeated exposure to hypoxia after adenosine A1-receptor blockade. Each pup was placed into a temperature-controlled chamber regulated to 37 ± 1°C and repeatedly exposed to an anoxic gas mixture (97% N2 and 3% CO2) until the occurrence of autoresuscitation failure. One group was studied following administration of the selective adenosine A1-receptor antagonist 8-Cyclopentyl-1,3,-dipropylxanthine (DPCPX) and one group was studied following vehicle. DPCPX significantly attenuated bradycardia during hypoxia-induced apnea and impaired the ability of both age groups of pups to autoresuscitate during repeated exposure to hypoxia (5–6 days tolerated – vehicle 17 ± 4 vs. DPCPX 10 ± 2 hypoxia exposures [P < 0.05]; 10–11 days tolerated – vehicle 10 ± 2 vs. DPCPX 7 ± 2 hypoxia exposures [P < 0.05]). Death in all pups resulted from the inability of gasping to restore cardiovascular function during hypoxia-induced apnea although the mechanism of cardiovascular dysfunction/failure was influenced and the occurrence hastened by DPCPX. Thus, our data provide evidence that adenosine acting via adenosine A1-receptors enhances the ability of rat pups to tolerate repeated exposure to severe hypoxia during early postnatal maturation. PMID:26272732

  15. 2’,3’-cAMP, 3’-AMP, 2’-AMP and Adenosine Inhibit TNF-α and CXCL10 Production From Activated Primary Murine Microglia via A2A Receptors

    PubMed Central

    Newell, Elizabeth A.; Exo, Jennifer L.; Verrier, Jonathan D.; Jackson, Travis C.; Gillespie, Delbert G.; Janesko-Feldman, Keri; Kochanek, Patrick M.

    2014-01-01

    Background Some cells, tissues and organs release 2’,3’-cAMP (a positional isomer of 3’,5’-cAMP) and convert extracellular 2’,3’-cAMP to 2’-AMP plus 3’-AMP and convert these AMPs to adenosine (called the extracellular 2’,3’-cAMP-adenosine pathway). Recent studies show that microglia have an extracellular 2’,3’-cAMP-adenosine pathway. The goal of the present study was to investigate whether the extracellular 2’,3’-cAMP-adenosine pathway could have functional consequences on the production of cytokines/chemokines by activated microglia. Methods Experiments were conducted in cultures of primary murine microglia. In the first experiment, the effect of 2’,3’-cAMP, 3’-AMP, 2’-AMP and adenosine on LPS-induced TNF-α and CXCL10 production was determined. In the next experiment, the first protocol was replicated but with the addition of 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX) (0.1 µM; antagonist of adenosine receptors). The last experiment compared the ability of 2-chloro-N6-cyclopentyladenosine (CCPA) (10 µM; selective A1 agonist), 5’-N-ethylcarboxamide adenosine (NECA) (10 µM; agonist for all adenosine receptor subtypes) and CGS21680 (10 µM; selective A2A agonist) to inhibit LPS-induced TNF-α and CXCL10 production. Results 1) 2’,3’-cAMP, 3’-AMP, 2’-AMP and adenosine similarly inhibited LPS-induced TNF-α and CXCL10 production; 2) DPSPX nearly eliminated the inhibitory effects of 2’,3’-cAMP, 3’-AMP, 2’-AMP and adenosine on LPS-induced TNF-α and CXCL10 production; 3) CCPA did not affect LPS-induced TNF-α and CXCL10; 4) NECA and CGS21680 similarly inhibited LPS-induced TNF-α and CXCL10 production. Conclusions 2’,3’-cAMP and its metabolites (3’-AMP, 2’-AMP and adenosine) inhibit LPS-induced TNF-α and CXCL10 production via A2A-receptor activation. Adenosine and its precursors, via A2A receptors, likely suppress TNF-α and CXCL10 production by activated microglia in brain diseases. PMID:25451117

  16. 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine inhibit TNF-α and CXCL10 production from activated primary murine microglia via A2A receptors.

    PubMed

    Newell, Elizabeth A; Exo, Jennifer L; Verrier, Jonathan D; Jackson, Travis C; Gillespie, Delbert G; Janesko-Feldman, Keri; Kochanek, Patrick M; Jackson, Edwin K

    2015-01-12

    Some cells, tissues and organs release 2',3'-cAMP (a positional isomer of 3',5'-cAMP) and convert extracellular 2',3'-cAMP to 2'-AMP plus 3'-AMP and convert these AMPs to adenosine (called the extracellular 2',3'-cAMP-adenosine pathway). Recent studies show that microglia have an extracellular 2',3'-cAMP-adenosine pathway. The goal of the present study was to investigate whether the extracellular 2',3'-cAMP-adenosine pathway could have functional consequences on the production of cytokines/chemokines by activated microglia. Experiments were conducted in cultures of primary murine microglia. In the first experiment, the effect of 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine on LPS-induced TNF-α and CXCL10 production was determined. In the next experiment, the first protocol was replicated but with the addition of 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX) (0.1 μM; antagonist of adenosine receptors). The last experiment compared the ability of 2-chloro-N(6)-cyclopentyladenosine (CCPA) (10 μM; selective A1 agonist), 5'-N-ethylcarboxamide adenosine (NECA) (10 μM; agonist for all adenosine receptor subtypes) and CGS21680 (10 μM; selective A2A agonist) to inhibit LPS-induced TNF-α and CXCL10 production. (1) 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine similarly inhibited LPS-induced TNF-α and CXCL10 production; (2) DPSPX nearly eliminated the inhibitory effects of 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine on LPS-induced TNF-α and CXCL10 production; (3) CCPA did not affect LPS-induced TNF-α and CXCL10; (4) NECA and CGS21680 similarly inhibited LPS-induced TNF-α and CXCL10 production. 2',3'-cAMP and its metabolites (3'-AMP, 2'-AMP and adenosine) inhibit LPS-induced TNF-α and CXCL10 production via A2A-receptor activation. Adenosine and its precursors, via A2A receptors, likely suppress TNF-α and CXCL10 production by activated microglia in brain diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Adenosine A(2A) receptor antagonism reverses the effects of dopamine receptor antagonism on instrumental output and effort-related choice in the rat: implications for studies of psychomotor slowing.

    PubMed

    Farrar, Andrew M; Pereira, Mariana; Velasco, Francisco; Hockemeyer, Jörg; Müller, Christa E; Salamone, John D

    2007-04-01

    Organisms frequently make effort-related decisions based upon assessments of motivational value and response costs. Energy-related dysfunctions such as psychomotor slowing and apathy are critically involved in some clinical syndromes. Dopamine (DA), particularly in the nucleus accumbens, regulates effort-related processes. Dopamine antagonism and accumbens dopamine depletions cause rats performing on choice tasks to reallocate their behavior away from food-reinforced tasks that have high response requirements. There is evidence of a functional interaction between DA and adenosine A(2A) receptors in the neostriatum and nucleus accumbens. The present experiments were conducted to determine if adenosine A(2A) receptor antagonism could reverse the effects of dopamine receptor antagonism on instrumental behavior and effort-related choice. The adenosine A(2A) receptor antagonist MSX-3 was investigated for its ability to reverse the effects of the dopamine receptor antagonist haloperidol (0.1 mg/kg) on fixed ratio 5 instrumental lever-pressing and on response allocation using a concurrent lever-pressing/chow-feeding choice task. Haloperidol significantly suppressed fixed ratio 5 responding, and with rats responding on the concurrent choice task, it altered choice behavior, significantly reducing lever-pressing for food and increasing chow intake. Injections of MSX-3 (0.5-2.0 mg/kg) produced a dose-related attenuation of the effects of 0.1 mg/kg haloperidol on both tasks. The high dose of MSX-3, when administered in the absence of haloperidol, did not significantly affect responding on either task. Adenosine and dopamine systems interact to regulate instrumental behavior and effort-related processes, which may have implications for the treatment of psychiatric symptoms such as psychomotor slowing or anergia.

  18. The Corticostriatal Adenosine A2A Receptor Controls Maintenance and Retrieval of Spatial Working Memory.

    PubMed

    Li, Zhihui; Chen, Xingjun; Wang, Tao; Gao, Ying; Li, Fei; Chen, Long; Xue, Jin; He, Yan; Li, Yan; Guo, Wei; Zheng, Wu; Zhang, Liping; Ye, Fenfen; Ren, Xiangpeng; Feng, Yue; Chan, Piu; Chen, Jiang-Fan

    2018-03-15

    Working memory (WM) taps into multiple executive processes including encoding, maintenance, and retrieval of information, but the molecular and circuit modulation of these WM processes remains undefined due to the lack of methods to control G protein-coupled receptor signaling with temporal resolution of seconds. By coupling optogenetic control of the adenosine A 2A receptor (A 2A R) signaling, the Cre-loxP-mediated focal A 2A R knockdown with a delayed non-match-to-place (DNMTP) task, we investigated the effect of optogenetic activation and focal knockdown of A 2A Rs in the dorsomedial striatum (n = 8 to 14 per group) and medial prefrontal cortex (n = 16 to 22 per group) on distinct executive processes of spatial WM. We also evaluated the therapeutic effect of the A 2A R antagonist KW6002 on delayed match-to-sample/place tasks in 6 normal and 6 MPTP-treated cynomolgus monkeys. Optogenetic activation of striatopallidal A 2A Rs in the dorsomedial striatum selectively at the delay and choice (not sample) phases impaired DNMTP performance. Optogenetic activation of A 2A Rs in the medial prefrontal cortex selectively at the delay (not sample or choice) phase improved DNMTP performance. The corticostriatal A 2A R control of spatial WM was specific for a novel but not well-trained DNMTP task. Focal dorsomedial striatum A 2A R knockdown or KW6002 improved DNMTP performance in mice. Last, KW6002 improved spatial WM in delayed match-to-sample and delayed match-to-place tasks of normal and dopamine-depleted cynomolgus monkeys. The A 2A Rs in striatopallidal and medial prefrontal cortex neurons exert distinctive control of WM maintenance and retrieval to achieve cognitive stability and flexibility. The procognitive effect of KW6002 in nonhuman primates provides the preclinical data to translate A 2A R antagonists for improving cognitive impairments in Parkinson's disease. Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  19. The Heat Shock Cognate Protein hsc73 Assembles with A1 Adenosine Receptors To Form Functional Modules in the Cell Membrane

    PubMed Central

    Sarrió, Sara; Casadó, Vicent; Escriche, Marisol; Ciruela, Francisco; Mallol, Josefa; Canela, Enric I.; Lluis, Carmen; Franco, Rafael

    2000-01-01

    A1 adenosine receptors (A1Rs) are G protein-coupled heptaspanning receptors that interact at the outer face of the plasma membrane with cell surface ecto-adenosine deaminase (ecto-ADA). By affinity chromatography the heat shock cognate protein hsc73 was identified as a cytosolic component able to interact with the third intracellular loop of the receptor. As demonstrated by surface plasmon resonance, purified A1Rs interact specifically with hsc73 with a dissociation constant in the nanomolar range (0.5 ± 0.1 nM). The interaction between hsc73 and A1R led to a marked reduction in the binding of the ligands and prevented activation of G proteins, as deduced from 35S-labeled guanosine-5′-O-(3-thio)triphosphate binding assays. Interestingly this effect was stronger than that exerted by guanine nucleotide analogs, which uncouple receptors from G proteins, and was completely prevented by ADA. As assessed by immunoprecipitation a high percentage of A1Rs in cell lysates are coupled to hsc73. A relatively high level of colocalization between A1R and hsc73 was detected in DDT1MF-2 cells by means of confocal microscopy, and no similar results were obtained for other G protein-coupled receptors. Colocalization between hsc73 and A1R was detected in specific regions of rat cerebellum and in the body of cortical neurons but not in dendrites or synapses. Remarkably, agonist-induced receptor internalization leads to the endocytosis of A1Rs by two qualitatively different vesicle types, one in which A1R and hsc73 colocalize and another in which hsc73 is absent. These results open the interesting possibility that signaling via G protein-coupled receptors may be regulated by heat shock proteins. PMID:10866672

  20. Dopamine depletion shifts behavior from activity based reinforcers to more sedentary ones and adenosine receptor antagonism reverses that shift: Relation to ventral striatum DARPP32 phosphorylation patterns.

    PubMed

    López-Cruz, Laura; San Miguel, Noemí; Carratalá-Ros, Carla; Monferrer, Lidón; Salamone, John D; Correa, Mercè

    2018-02-02

    The mesolimbic dopamine (DA) system plays a critical role in behavioral activation and effort-based decision-making. DA depletion produces anergia (shifts to low effort options) in animals tested on effort-based decision-making tasks. Caffeine, the most consumed stimulant in the world, acts as an adenosine A 1 /A 2A receptor antagonist, and in striatal areas DA D 1 and D 2 receptors are co-localized with adenosine A 1 and A 2A receptors respectively. In the present work, we evaluated the effect of caffeine on anergia induced by the VMAT-2 inhibitor tetrabenazine (TBZ), which depletes DA. Anergia was evaluated in a three-chamber T-maze task in which animals can chose between running on a wheel (RW) vs. sedentary activities such as consuming sucrose or sniffing a neutral odor. TBZ-caffeine interactions in ventral striatum were evaluated using DARPP-32 phosphorylation patterns as an intracellular marker of DA-adenosine receptor interaction. In the T-maze, control mice spent more time running and much less consuming sucrose or sniffing. TBZ (4.0 mg/kg) reduced ventral striatal DA tissue levels as measured by HPLC, and also shifted preferences in the T-maze, reducing selection of the reinforcer that involved vigorous activity (RW), but increasing consumption of a reinforcer that required little effort (sucrose), at doses that had no effect on independent measures of appetite or locomotion in a RW. Caffeine at doses that had no effect on their own reversed the effects of TBZ on T-maze performance, and also suppressed TBZ-induced pDARPP-32(Thr34) expression as measured by western blot, suggesting a role for D 2 -A 2A interactions. These results support the idea that DA depletion produces anergia, but does not affect the primary motivational effects of sucrose. Caffeine, possibly by acting on A 2A receptors in ventral striatum, reversed the DA depletion effects. It is possible that caffeine, like selective adenosine A2A antagonists, could have some therapeutic benefit

  1. Adenosine Stimulate Proliferation and Migration in Triple Negative Breast Cancer Cells.

    PubMed

    Fernandez-Gallardo, Miriam; González-Ramírez, Ricardo; Sandoval, Alejandro; Felix, Ricardo; Monjaraz, Eduardo

    2016-01-01

    Emerging evidence suggests that the adenosine (Ado) receptors may play crucial roles in tumor progression. Here, we show that Ado increases proliferation and migration in a triple negative breast cancer model, the MDA-MB 231 cell line. The use of specific agonists and antagonists evidenced that these effects depend on the activation of the A2B receptor, which then triggers an intracellular response mediated by the adenylate cyclase/PKA/cAMP signaling pathway. Ado also increases the expression of NaV1.5 channels, a potential biomarker in breast cancer. Together, these data suggest important roles of the A2B receptors and NaV1.5 channels in the Ado-induced increase in proliferation and migration of the MDA-MB 231 cells.

  2. A(2a) adenosine receptor mediates PKA-dependent glutamate release from synaptic-like vesicles and Ca(2+) efflux from an IP(3)- and ryanodine-insensitive intracellular calcium store in astrocytes.

    PubMed

    Kanno, Takeshi; Nishizaki, Tomoyuki

    2012-01-01

    The mechanism underlying transmitter release from astrocytes is not fully understood. The present study examined A(2a) adenosine receptor-mediated glutamate release and intracellular Ca(2+) rise in cultured rat hippocampal astrocytes. Intracellular amino acids were measured with HPLC. Glutamate release from astrocytes and intracellular Ca(2+) mobilizations were monitored in the NADH imaging, FM1-43 imaging, and fura-2 imaging. The siRNA to silence the A(2a) adenosine receptor-targeted gene was constructed and transfected into cells. Glutamate was condensed in 'synaptic-like vesicle' fractions. In the NADH imaging, CGS21680, an agonist of A2a adenosine receptors, increased NADH fluorescent signals, that reflects glutamate release, and the effect was inhibited by DMPX, an inhibitor of A(2a) adenosine receptors, H-89, a PKA inhibitor, vesicular transport inhibitors, or botulinum toxin-A, an exocytosis inhibitor. In the FM1-43 imaging to see vesicular recycling, CGS21680 decreased FM1-43 fluorescent signals, that was also prevented by DMPX, H-89, vesicular transport inhibitors, or botulinum toxin-A. CGS21680 increased intracellular Ca(2+) concentrations both in Ca(2+)-containing and -free extracellular solution. The Ca(2+) rise was inhibited by DMPX, H-89, or the vesicular transport inhibitor brefeldin A, but it was not affected by inhibitors for phospholipase C, IP(3) receptor, and ryanodine receptor. CGS21680-induced glutamate release and intracellular Ca(2+) rise were prevented by knocking-down A(2a) adenosine receptor. The results of the present study show that A(2a) adenosine receptor/PKA promotes glutamate release from synaptic-like vesicles and stimulates Ca(2+) efflux from an IP(3)- and ryanodine-insensitive intracellular calcium store. Copyright © 2012 S. Karger AG, Basel.

  3. Adenosine transporter ENT1 regulates the acquisition of goal-directed behavior and ethanol drinking through A2A receptor in the dorsomedial striatum.

    PubMed

    Nam, Hyung Wook; Hinton, David J; Kang, Na Young; Kim, Taehyun; Lee, Moonnoh R; Oliveros, Alfredo; Adams, Chelsea; Ruby, Christina L; Choi, Doo-Sup

    2013-03-06

    Adenosine signaling has been implicated in the pathophysiology of many psychiatric disorders including alcoholism. Striatal adenosine A2A receptors (A2AR) play an essential role in both ethanol drinking and the shift from goal-directed action to habitual behavior. However, direct evidence for a role of striatal A2AR signaling in ethanol drinking and habit development has not been established. In the present study, we found that decreased A2AR-mediated CREB activity in the dorsomedial striatum (DMS) enhanced initial behavioral acquisition of goal-directed behaviors and the vulnerability to progress to excessive ethanol drinking during operant conditioning in mice lacking ethanol-sensitive adenosine transporter ENT1 (ENT1(-/-)). Using mice expressing β-galactosidase (lacZ) under the control of seven repeated CRE sites in both genotypes (CRE-lacZ/ENT1(+/+) mice and CRE-lacZ/ENT1(-/-) mice) and the dominant-negative form of CREB, we found that reduced CREB activity in the DMS was causally associated with decreased A2AR signaling and increased goal-directed ethanol drinking. Finally, we have demonstrated that the A2AR antagonist ZM241385 dampened protein kinase A activity-mediated signaling in the DMS and promoted excessive ethanol drinking in ENT1(+/+) mice, but not in ENT1(-/-) mice. Our results indicate that A2AR-mediated CREB signaling in the DMS is a key determinant in enhancing the development of goal-directed ethanol drinking in mice.

  4. Administration of caffeine inhibited adenosine receptor agonist-induced decreases in motor performance, thermoregulation, and brain neurotransmitter release in exercising rats.

    PubMed

    Zheng, Xinyan; Hasegawa, Hiroshi

    2016-01-01

    We examined the effects of an adenosine receptor agonist on caffeine-induced changes in thermoregulation, neurotransmitter release in the preoptic area and anterior hypothalamus, and endurance exercise performance in rats. One hour before the start of exercise, rats were intraperitoneally injected with either saline alone (SAL), 10 mg kg(-1) caffeine and saline (CAF), a non-selective adenosine receptor agonist (5'-N-ethylcarboxamidoadenosine [NECA]: 0.5 mg kg(-1)) and saline (NECA), or the combination of caffeine and NECA (CAF+NECA). Rats ran until fatigue on the treadmill with a 5% grade at a speed of 18 m min(-1) at 23 °C. Compared to the SAL group, the run time to fatigue (RTTF) was significantly increased by 52% following caffeine administration and significantly decreased by 65% following NECA injection (SAL: 91 ± 14.1 min; CAF: 137 ± 25.8 min; NECA: 31 ± 13.7 min; CAF+NECA: 85 ± 11.8 min; p<0.05). NECA decreased the core body temperature (Tcore), oxygen consumption, which is an index of heat production, tail skin temperature, which is an index of heat loss, and extracellular dopamine (DA) release at rest and during exercise. Furthermore, caffeine injection inhibited the NECA-induced decreases in the RTTF, Tcore, heat production, heat loss, and extracellular DA release. Neither caffeine nor NECA affected extracellular noradrenaline or serotonin release. These results support the findings of previous studies showing improved endurance performance and overrides in body limitations after caffeine administration, and imply that the ergogenic effects of caffeine may be associated with the adenosine receptor blockade-induced increases in brain DA release. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Adenosine Deaminase That Acts on RNA 3 (ADAR3) Binding to Glutamate Receptor Subunit B Pre-mRNA Inhibits RNA Editing in Glioblastoma.

    PubMed

    Oakes, Eimile; Anderson, Ashley; Cohen-Gadol, Aaron; Hundley, Heather A

    2017-03-10

    RNA editing is a cellular process that precisely alters nucleotide sequences, thus regulating gene expression and generating protein diversity. Over 60% of human transcripts undergo adenosine to inosine RNA editing, and editing is required for normal development and proper neuronal function of animals. Editing of one adenosine in the transcript encoding the glutamate receptor subunit B, glutamate receptor ionotropic AMPA 2 (GRIA2), modifies a codon, replacing the genomically encoded glutamine (Q) with arginine (R); thus this editing site is referred to as the Q/R site. Editing at the Q/R site of GRIA2 is essential, and reduced editing of GRIA2 transcripts has been observed in patients suffering from glioblastoma. In glioblastoma, incorporation of unedited GRIA2 subunits leads to a calcium-permeable glutamate receptor, which can promote cell migration and tumor invasion. In this study, we identify adenosine deaminase that acts on RNA 3 (ADAR3) as an important regulator of Q/R site editing, investigate its mode of action, and detect elevated ADAR3 expression in glioblastoma tumors compared with adjacent brain tissue. Overexpression of ADAR3 in astrocyte and astrocytoma cell lines inhibits RNA editing at the Q/R site of GRIA2 Furthermore, the double-stranded RNA binding domains of ADAR3 are required for repression of RNA editing. As the Q/R site of GRIA2 is specifically edited by ADAR2, we suggest that ADAR3 directly competes with ADAR2 for binding to GRIA2 transcript, inhibiting RNA editing, as evidenced by the direct binding of ADAR3 to the GRIA2 pre-mRNA. Finally, we provide evidence that both ADAR2 and ADAR3 expression contributes to the relative level of GRIA2 editing in tumors from patients suffering from glioblastoma. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Evaluation of antidepressant-like and anxiolytic-like activity of purinedione-derivatives with affinity for adenosine A2A receptors in mice.

    PubMed

    Dziubina, Anna; Szmyd, Karina; Zygmunt, Małgorzata; Sapa, Jacek; Dudek, Magdalena; Filipek, Barbara; Drabczyńska, Anna; Załuski, Michał; Pytka, Karolina; Kieć-Kononowicz, Katarzyna

    2016-12-01

    It has recently been suggested that the adenosine A 2A receptor plays a role in several animal models of depression. Additionally, A 2A antagonists have reversed behavioral deficits and exhibited a profile similar to classical antidepressants. In the present study, imidazo- and pyrimido[2,1-f]purinedione derivatives (KD 66, KD 167, KD 206) with affinity to A 2A receptors but poor A 1 affinity were evaluated for their antidepressant- and anxiolytic-like activity. The activity of these derivatives was tested using a tail suspension and forced swim test, two widely-used behavioral paradigms for the evaluation of antidepressant-like activity. In turn, the anxiolytic activity was evaluated using the four-plate test. The results showed the antidepressant-like activity of pyrimido- and imidazopurinedione derivatives (i.e. KD 66, KD 167 and KD 206) in acute and chronic behavioral tests in mice. KD 66 revealed an anxiolytic-like effect, while KD 167 increased anxiety behaviors. KD 206 had no effect on anxiety. Furthermore, none of the tested compounds increased locomotor activity. Available data support the proposition that the examined compounds with adenosine A 2A receptor affinity may be an interesting target for the development of antidepressant and/or anxiolytic agents. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  7. Decreases in yeast expression yields of the human adenosine A2a receptor are a result of translational or post-translational events.