15 results on '"Figler H"'
Search Results
2. 6-Aryl-8H-indeno[1,2-d]thiazol-2-ylamines: A<INF>1</INF> Adenosine Receptor Agonist Allosteric Enhancers Having Improved Potency
- Author
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Chordia, M. D., Zigler, M., Murphree, L. J., Figler, H., Macdonald, T. L., Olsson, R. A., and Linden, J.
- Abstract
Allosteric enhancers (AEs) of the A
1 adenosine receptor (A1 AR) have potential as drugs for treating neurological, cardiovascular, and renal diseases. This report describes the synthesis and evaluation of a series of 6-aryl-8H-indeno[1,2-d]thiazol-2-ylamines that exhibited AE activity at the A1 AR. Palladium-mediated condensation of arylboronic acids with 5-bromoindan-1-one generated arylindanones2a − aj for iodine-catalyzed condensation with thiourea, generating 2-aminothiazolium salts3a − aj . Binding studies using membranes from cells stably expressing human A1 ARs, A2A ARs, or A3 ARs evaluated AE activity and receptor subtype selectivity. The EC50 of the AE activities of compounds3m − o ,3x , and3ae were 2.2, 1.5, 0.9, 1.0, and 3.0 μM, respectively, substantially lower than that of the well characterized 2-amino-3-aroylthiophene (PD 81,723), >10 μM. The new compounds also have substantially higher maximal AE activity. These compounds had no AE activity at the A2A AR and only minimal activity at the A3 AR.- Published
- 2005
3. 2-Amino-3-benzoylthiophene Allosteric Enhancers of A<INF>1</INF> Adenosine Agonist Binding: New 3, 4-, and 5-Modifications
- Author
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Lutjens, H., Zickgraf, A., Figler, H., Linden, J., A., Olsson R., and Scammells, P. J.
- Abstract
2-Amino-3-aroylthiophenes are agonist allosteric enhancers (AE) at the A
1 adenosine receptor (A1 AR). Here we report the syntheses of three kinds of novel 2-aminothiophenes and assays of their AE activity at the human A1 AR (hA1 AR), namely, (1) 2-amino-4,5-diphenylthiophene-3-carboxylates,3a −h , (2) 2-amino-3-benzoyl-4,5-diphenylthiophenes,7a −p , and (3) 2-amino-5-bromo-3-benzoyl-4-phenylthiophenes,10a −h . An in vitro assay employing the A1 AR agonist [125I]ABA and membranes from CHO−K1 cells stably expressing the hA1 AR measured an index of AE activity, the ability of a candidate AE to stabilize the agonist-A1 AR-G protein ternary complex, scored as the percentage of ternary complex remaining after 10 min of dissociation initiated by CPX and GTPγS. The AE activity score of 2-amino-4,5-dimethyl-3-(3-trifluoromethylbenzoyl)thiophene (PD 81,723), which was 19%, served as a standard for comparison. Two 3-carboxythiophene 3-trifluoromethylbenzyl esters,3d (49%) and3f (63%), had substantial AE activity. The 3-(1-naphthoyl) substituent of7e (52%) also supported AE activity. Compounds in series 3 tended to be more potent,10a and10c having scores of 91 and 80%, respectively. The activity of 2-amino-5-bromo-3-ethoxycarbonyl-4-(3-nitrophenyl)thiophene,10h (26%), is an exception to the rule that a 3-ethoxycarbonyl substituent cannot support AE activity.- Published
- 2003
4. 2-Amino-3-aroyl-4,5-alkylthiophenes: Agonist Allosteric Enhancers at Human A<INF>1</INF> Adenosine Receptors
- Author
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Tranberg, C. E., Zickgraf, A., Giunta, B. N., Luetjens, H., Figler, H., Murphree, L. J., Falke, R., Fleischer, H., Linden, J., Scammells, P. J., and Olsson, R. A.
- Abstract
2-Amino-3-benzoylthiophenes are allosteric enhancers (AE) of agonist activity at the A
1 adenosine receptor. The present report describes syntheses and assays of the AE activity at the human A1 AR (hA1 AR) of a panel of compounds consisting of nine 2-amino-3-aroylthiophenes (3a −i ), eight 2-amino-3-benzoyl-4,5-dimethylthiophenes (12a −h ), three 3-aroyl-2-carboxy-4,5-dimethylthiophenes (15a −c ), 10 2-amino-3-benzoyl-5,6-dihydro-4H-cyclopenta[b]thiophenes (17a −j ), 14 2-amino-3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophenes (18a −n ), and 15 2-amino-3-benzoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophenes (19a −o ). An in vitro assay employing the A1 AR agonist [125I]ABA and membranes from CHO-K1 cells stably expressing the hA1 AR measured, as an index of AE activity, the ability of a candidate AE to stabilize the agonist-A1 AR-G protein ternary complex. Compounds3a −i had little or no AE activity, and compounds12a −h had only modest activity, evidence that AE activity depended absolutely on the presence of at least a methyl group at C-4 and C-5. Compounds17a −c lacked AE activity, suggesting the 2-amino group is essential. Polymethylene bridges linked thiophene C-4 and C-5 of compounds17a −j ,18a −n , and19a −o . AE activity increased with the size of the -(CH2 )n - bridge, n = 3 < n = 4 < n = 5. The 3-carbethoxy substituents of 17a ,18a , and19a did not support AE activity, but a 3-aroyl group did. Bulky (or hydrophobic) substituents at the meta and para positions of the 3-benzoyl group and also 3-naphthoyl groups greatly enhanced activity. Thus, the hA1 AR contains an allosteric binding site able to accommodate 3-aroyl substituents that are bulky and/or hydrophobic but not necessarily planar. A second region in the allosteric binding site interacts constructively with alkyl substituents at thiophene C-4 and/or C-5.- Published
- 2002
5. Multivalent site-specific phage modification enhances the binding affinity of receptor ligands.
- Author
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Beech J, Saleh L, Frentzel J, Figler H, Corrêa IR Jr, Baker B, Ramspacher C, Marshall M, Dasa S, Linden J, Noren CJ, and Kelly KA
- Subjects
- Animals, Binding Sites physiology, CHO Cells, Cricetinae, Cricetulus, Humans, Ligands, Protein Binding physiology, Bacteriophage M13 metabolism, Receptor, Adenosine A1 metabolism
- Abstract
High-throughput screening of combinatorial chemical libraries is a powerful approach for identifying targeted molecules. The display of combinatorial peptide libraries on the surface of bacteriophages offers a rapid, economical way to screen billions of peptides for specific binding properties and has impacted fields ranging from cancer to vaccine development. As a modification to this approach, we have previously created a system that enables site-specific insertion of selenocysteine (Sec) residues into peptides displayed pentavalently on M13 phage as pIII coat protein fusions. In this study, we show the utility of selectively derivatizing these Sec residues through the primary amine of small molecules that target a G protein-coupled receptor, the adenosine A1 receptor, leaving the other coat proteins, including the major coat protein pVIII, unmodified. We further demonstrate that modified Sec-phage with multivalent bound agonist binds to cells and elicits downstream signaling with orders of magnitude greater potency than that of unconjugated agonist. Our results provide proof of concept of a system that can create hybrid small molecule-containing peptide libraries and open up new possibilities for phage-drug therapies.
- Published
- 2015
- Full Text
- View/download PDF
6. The second extracellular loop of the adenosine A1 receptor mediates activity of allosteric enhancers.
- Author
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Kennedy DP, McRobb FM, Leonhardt SA, Purdy M, Figler H, Marshall MA, Chordia M, Figler R, Linden J, Abagyan R, and Yeager M
- Subjects
- Allosteric Regulation, Animals, Binding Sites, Dogs, HEK293 Cells, Humans, Models, Molecular, Molecular Docking Simulation, Mutagenesis, Site-Directed, Species Specificity, Structure-Activity Relationship, Receptor, Adenosine A1 chemistry, Receptor, Adenosine A1 physiology
- Abstract
Allosteric enhancers of the adenosine A1 receptor amplify signaling by orthosteric agonists. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied by an agonist, thereby conferring specificity to stressed or injured tissues that produce adenosine. To explore the mechanism of allosteric enhancer activity, we examined their action on several A1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants, and (4) site-specific mutants. These findings were combined with homology modeling of the A1 receptor and in silico screening of an allosteric enhancer library. The binding modes of known docked allosteric enhancers correlated with the known structure-activity relationship, suggesting that these allosteric enhancers bind to a pocket formed by the second extracellular loop, flanked by residues S150 and M162. We propose a model in which this vestibule controls the entry and efflux of agonists from the orthosteric site and agonist binding elicits a conformational change that enables allosteric enhancer binding. This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists.
- Published
- 2014
- Full Text
- View/download PDF
7. Synthesis and evaluation of new N6-substituted adenosine-5'-N-methylcarboxamides as A3 adenosine receptor agonists.
- Author
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Devine SM, Gregg A, Figler H, McIntosh K, Urmaliya V, Linden J, Pouton CW, White PJ, Bottle SE, and Scammells PJ
- Subjects
- Adenosine chemical synthesis, Adenosine chemistry, Adenosine pharmacology, Animals, Binding Sites, Cell Line, Combinatorial Chemistry Techniques, Drug Design, Molecular Structure, Myocytes, Cardiac cytology, Myocytes, Cardiac drug effects, Rats, Structure-Activity Relationship, Adenosine analogs & derivatives, Adenosine A3 Receptor Agonists, Cardiotonic Agents chemical synthesis, Cardiotonic Agents chemistry, Cardiotonic Agents pharmacology
- Abstract
A number of N(6)-substituted adenosine-5'-N-methylcarboxamides were synthesised and their pharmacology, in terms of their receptor affinity, selectivity and cardioprotective effects, were explored. The first series of compounds, 4a-4f and 5a-5f, showed modest receptor affinity for the A(3)AR with K(i) values in the low to mid muM range. However, the incorporation of a 4-(2-aminoethyl)-2,6-di-tert-butylphenol group in the N(6)-position (in compounds 4g and 5g) significantly improved the affinity with K(i) values of 30 and 9 nM, respectively. Improvements in affinity, as well as selectivity were seen when a functionalized linker was introduced. The N(6)-phenyl series, compounds 7a-7d, demonstrated low to mid nanomolar receptor affinities (K(i)=2.3-45.0 nM), with 7b displaying 109-fold selectivity for the A(3)AR (vs A(1)). The N(6)-benzyl series 9a-9c also proved to be potent and selective A(3)AR agonists and the longer chain length linker 13 was tolerated at the A(3)AR without abrogation of affinity or selectivity. Cardioprotection was demonstrated by a simulated ischaemia cell culture assay, whereby 7b, 7c, 9a, 9b and 9c all showed cardioprotective effects at 100 nM comparable or better than the benchmark A(3)AR agonist IB-MECA, but which were indistinguishable by statistical analysis. For example, compound 9c reduced cell death by 68.0+/-3.6%., ((c) 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
- Full Text
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8. 3- and 6-Substituted 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines as A1 adenosine receptor allosteric modulators and antagonists.
- Author
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Aurelio L, Valant C, Figler H, Flynn BL, Linden J, Sexton PM, Christopoulos A, and Scammells PJ
- Subjects
- Adenosine A1 Receptor Antagonists, Allosteric Regulation, Animals, CHO Cells, Cricetinae, Cricetulus, Magnetic Resonance Spectroscopy, Pyridines chemistry, Receptor, Adenosine A1 metabolism, Spectrometry, Mass, Electrospray Ionization, Structure-Activity Relationship, Pyridines pharmacology, Receptor, Adenosine A1 drug effects
- Abstract
A series of 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines were prepared and evaluated as potential allosteric modulators at the A(1) adenosine receptor. The structure-activity relationships of the 3- and 6-positions of a series of 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines were explored. Despite finding that 3- and 6-substituted 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines possess the ability to recognize an allosteric site on the agonist-occupied A(1)AR at relatively high concentrations, the structural modifications we have performed on this scaffold favor the expression of orthosteric antagonist properties over allosteric properties. This research has identified 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines as novel class of orthosteric antagonist of the A(1)AR and highlighted the close relationship between structural elements governing allosteric modulation and orthosteric antagonism of agonist function at the A(1)AR.
- Published
- 2009
- Full Text
- View/download PDF
9. 2-aminothienopyridazines as novel adenosine A1 receptor allosteric modulators and antagonists.
- Author
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Ferguson GN, Valant C, Horne J, Figler H, Flynn BL, Linden J, Chalmers DK, Sexton PM, Christopoulos A, and Scammells PJ
- Subjects
- Allosteric Regulation drug effects, Allosteric Site drug effects, Dose-Response Relationship, Drug, Humans, Kinetics, Molecular Structure, Pyridazines chemical synthesis, Pyridazines chemistry, Receptor, Adenosine A1 chemistry, Small Molecule Libraries, Stereoisomerism, Structure-Activity Relationship, Thiophenes chemical synthesis, Thiophenes chemistry, Adenosine A1 Receptor Agonists, Adenosine A1 Receptor Antagonists, Pyridazines pharmacology, Thiophenes pharmacology
- Abstract
A pharmacophore-based screen identified 32 compounds including ethyl 5-amino-3-(4- tert-butylphenyl)-4-oxo-3,4-dihydrothieno[3,4- d]pyridazine-1-carboxylate ( 8) as a new allosteric modulator of the adenosine A1 receptor (A1AR). On the basis of this lead, various derivatives were prepared and evaluated for activity at the human A 1AR. A number of the test compounds allosterically stabilized agonist-receptor-G protein ternary complexes in dissociation kinetic assays, but were found to be more potent as antagonists in subsequent functional assays of ERK1/2 phosphorylation. Additional experiments on the most potent antagonist, 13b, investigating A1AR-mediated [(35)S]GTPgammaS binding and [(3)H]CCPA equilibrium binding confirmed its antagonistic mode of action and also identified inverse agonism. This study has thus identified a new class of A1AR antagonists that can also recognize the receptor's allosteric site with lower potency.
- Published
- 2008
- Full Text
- View/download PDF
10. 5-Substituted 2-aminothiophenes as A1 adenosine receptor allosteric enhancers.
- Author
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Aurelio L, Figler H, Flynn BL, Linden J, and Scammells PJ
- Subjects
- Allosteric Regulation, Amination, Aniline Compounds chemical synthesis, Animals, CHO Cells, Carboxylic Acids chemical synthesis, Carboxylic Acids chemistry, Carboxylic Acids pharmacology, Cricetinae, Cricetulus, Humans, Molecular Structure, Receptor, Adenosine A1 metabolism, Structure-Activity Relationship, Adenosine A1 Receptor Antagonists, Aniline Compounds chemistry, Aniline Compounds pharmacology
- Abstract
Two series of 5-substituted 2-amino-4-(3-trifluoromethylphenyl)thiophenes were prepared and evaluated as allosteric enhancers at the A(1) adenosine receptor (A(1)AR). In the 3-benzoyl series, a 5-phenyl group was found to confer the greatest potency (9a: ED(50)=2.1 microM, AE score=18%). However, the analogue with no 5-substituent (6b: ED(50)=15.8 microM, AE score=77%) proved to be the most efficacious. In the 3-ethoxycarbonyl series, the 5-(4-chlorophenyl) analogue was clearly the most potent and efficacious (9l: ED(50)=6.6 microM, AE score=57%). The antagonist activity of all compounds was measured using a [(3)H]CPX competitive binding assay.
- Published
- 2008
- Full Text
- View/download PDF
11. Dual acting antioxidant A1 adenosine receptor agonists.
- Author
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Gregg A, Bottle SE, Devine SM, Figler H, Linden J, White P, Pouton CW, Urmaliya V, and Scammells PJ
- Subjects
- Adenosine chemical synthesis, Adenosine pharmacology, Animals, Binding Sites, Cardiotonic Agents chemical synthesis, Cardiotonic Agents pharmacology, Cell Line, Indicators and Reagents, Myocardial Ischemia drug therapy, Myocardial Ischemia pathology, Myocardial Reperfusion Injury drug therapy, Myocardial Reperfusion Injury pathology, Myocytes, Cardiac drug effects, Rats, Structure-Activity Relationship, Xanthines pharmacology, Adenosine analogs & derivatives, Adenosine A1 Receptor Agonists, Antioxidants chemical synthesis, Antioxidants pharmacology, Isoindoles chemical synthesis, Isoindoles pharmacology, Pyrrolidines chemical synthesis, Pyrrolidines pharmacology
- Abstract
Herein we report the synthesis and biological evaluation of some potent and selective A(1) adenosine receptor agonists, which incorporate a functionalised linker attached to an antioxidant moiety. N(6)-(2,2,5,5-Tetramethylpyrrolidin-1-yloxyl-3-ylmethyl)adenosine (VCP28, 2e) proved to be an agonist with high affinity (K(i)=50nM) and good selectivity (A(3)/A(1) > or = 400) for the A(1) adenosine receptor. N(6)-[4-[2-[1,1,3,3-Tetramethylisoindolin-2-yloxyl-5-amido]ethyl]phenyl]adenosine (VCP102, 5a) has higher binding affinity (K(i)=7 nM), but lower selectivity (A(3)/A(1)= approximately 3). All compounds bind weakly (K(i)>1 microM) to A(2A) and A(2B) receptors. The combination of A(1) agonist activity and antioxidant activity has the potential to produce cardioprotective effects.
- Published
- 2007
- Full Text
- View/download PDF
12. 2-Aminothiophene-3-carboxylates and carboxamides as adenosine A1 receptor allosteric enhancers.
- Author
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Nikolakopoulos G, Figler H, Linden J, and Scammells PJ
- Subjects
- Allosteric Regulation drug effects, Amides chemical synthesis, Amides chemistry, Carboxylic Acids chemical synthesis, Carboxylic Acids chemistry, Dose-Response Relationship, Drug, Humans, Molecular Structure, Structure-Activity Relationship, Thiophenes chemical synthesis, Thiophenes chemistry, Amides pharmacology, Carboxylic Acids pharmacology, Receptor, Adenosine A1 drug effects, Thiophenes pharmacology
- Abstract
Three series of 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene and 2-amino-5,6,7,8-tetrahydrocyclohepta[b]thiophenes with 3-carboxylates and carboxamides have been prepared using the Gewald synthesis and evaluated as A(1)AR allosteric enhancers. The structure-activity relationships of these classes of compound are described. A number of compounds, notably 7b, are more potent and efficacious than PD81,723 (1).
- Published
- 2006
- Full Text
- View/download PDF
13. Allosteric enhancers of A1 adenosine receptors increase receptor-G protein coupling and counteract Guanine nucleotide effects on agonist binding.
- Author
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Figler H, Olsson RA, and Linden J
- Subjects
- Allosteric Regulation drug effects, Allosteric Regulation physiology, Animals, Binding Sites drug effects, Binding Sites physiology, CHO Cells, Cricetinae, Dose-Response Relationship, Drug, Adenosine A1 Receptor Agonists, Guanine Nucleotides metabolism, Receptor, Adenosine A1 metabolism, Receptors, G-Protein-Coupled metabolism
- Abstract
Endogenous ligands of G protein-coupled receptors bind to orthosteric sites that are topologically distinct from allosteric sites. Certain aminothiophenes such as (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluromethyl)-phenyl]-methanone (PD81,723) and 2-amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-yl)-biphenyl-4-yl-methanone (ATL525) are positive allosteric regulators, or enhancers, of the human A1 adenosine receptor (A1AR). In equilibrium binding assays, 125I-N6-aminobenzyladenosine (125I-ABA) binds to two affinity states of A1AR with KD-high (0.33 microM) and KD-low ( approximately 10 nM). Enhancers have little effect on KD-high but convert all A1AR binding sites to the high-affinity state. Enhancers decrease the potency of guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) as an inhibitor of agonist binding by 100-fold and increase agonist-stimulated guanine nucleotide exchange. The association of 125I-ABA to high-affinity receptors on Chinese hamster ovary (CHO)-hA1 membranes does not follow theoretical single-site association kinetics but is approximated by a bi-exponential equation with t1/2 values of 1.85 and 12.8 min. Allosteric enhancers selectively increase the number of slow binding sites, possibly by stabilizing newly formed receptor-G protein complexes. A new rapid assay method scores enhancer activity on a scale from 0 to 100 based on their ability to prevent the rapid dissociation of 125I-ABA from A1AR in response to GTPgammaS. Compared with PD81,723, ATL525 (100 microM) scores higher (27 versus 79) and has less antagonist activity. ATL525 functionally enhances A1 signaling to inhibit cAMP accumulation in CHO-hA1 cells. These data suggest that simultaneously binding orthosteric and allosteric enhancer ligands convert the A1AR from partly to fully coupled to G proteins and prevents rapid uncoupling upon binding of GTPgammaS.
- Published
- 2003
- Full Text
- View/download PDF
14. Gene dose effect reveals no Gs-coupled A2A adenosine receptor reserve in murine T-lymphocytes: studies of cells from A2A-receptor-gene-deficient mice.
- Author
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Armstrong JM, Chen JF, Schwarzschild MA, Apasov S, Smith PT, Caldwell C, Chen P, Figler H, Sullivan G, Fink S, Linden J, and Sitkovsky M
- Subjects
- Animals, Mice, Mice, Inbred C57BL, Mice, Inbred DBA, Mice, Knockout, GTP-Binding Protein alpha Subunits, Gs metabolism, Gene Dosage, Receptors, Purinergic P1 metabolism, T-Lymphocytes metabolism
- Abstract
Agonist binding to extracellular A2A adenosine receptors (A2ARs) inhibits the activation of virtually all tested functions of T-cells and can induce apoptosis in thymocytes. The evaluation of levels of expression of these immunosuppressive receptors is expected to clarify whether the absence of spare A2ARs (no 'receptor reserve') might be one of the mechanisms of attenuation of the effects of extracellular adenosine on T-cells. A2A transcript is found in T-cells and functional receptors can be demonstrated, but the density of receptor on T-cells is too low to be detected by radioligand binding. Studies of direct radioligand binding to murine brain with the selective A2AR agonist [3H]CGS21680 (2-(4-[(2-carboxyethyl)-phenyl]ethylamino)-5'-N-ethylcarboxamidoadenosine) established that striata levels of A2AR are virtually absent from A2A knock-out mice. Mice that are heterozygous (A2AR+/-) for the A2AR express significantly decreased levels of A2AR. To test for the presence of spare receptors in T-cells we took advantage of this gene dose effect and examined whether the decrease in the number of receptors in thymocytes from A2AR+/- mice was proportionately reflected in a decrease in the functional cAMP response of T-cells to adenosine. cAMP accumulation and apoptosis induced by adenosine and by A2AR agonist are of a lower magnitude in T-cells from A2AR+/- heterozygous mice than in T-cells from A2AR+/+ littermate control mice. These results indicate that there is no A2AR reserve in murine T-cells. Strongly decreased adenosine-triggered cAMP increases were detected in thymocytes from A2AR-/- mice, suggesting that A2B adenosine receptors cannot fully compensate for the loss of A2ARs in murine T-cells. We conclude that the number of A2ARs is the limiting factor in determining the maximal cAMP response of T-lymphocytes to extracellular adenosine, thereby minimizing the immunosuppressive effects of extracellular adenosine.
- Published
- 2001
- Full Text
- View/download PDF
15. Characterization of human A(2B) adenosine receptors: radioligand binding, western blotting, and coupling to G(q) in human embryonic kidney 293 cells and HMC-1 mast cells.
- Author
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Linden J, Thai T, Figler H, Jin X, and Robeva AS
- Subjects
- Animals, Anti-Asthmatic Agents pharmacology, Blotting, Western, CHO Cells, Calcium metabolism, Cell Degranulation drug effects, Cells, Cultured, Cricetinae, Cyclic AMP metabolism, Humans, Kidney cytology, Kidney metabolism, Mast Cells metabolism, Purinergic P1 Receptor Agonists, Radioligand Assay, Receptor, Adenosine A2B, Receptors, Purinergic P1 genetics, Theophylline pharmacology, Type C Phospholipases metabolism, Xanthines metabolism, Xanthines pharmacology, Xanthines therapeutic use, GTP-Binding Proteins metabolism, Receptors, Purinergic P1 metabolism
- Abstract
Recombinant human A(2B) adenosine receptors (A(2B)ARs) and receptors extended on the amino terminus with hexahistidine and the FLAG epitope, DYKDDDDK (H/F-A(2B)) were stably overexpressed (to >20,000 fmol/mg protein) in human embryonic kidney 293 cells (HEK-A(2B)). By Western blotting, the H/F-A(2B) receptor runs as a 34.8-kDa glycoprotein. Pharmacological properties of A(2B)ARs were characterized with (125)I-3-aminobenzyl-8-phenyl-(4-oxyacetic acid)-1-propylxanthine (K(D), 36 nM). In competition binding assays, the affinity of agonists is reduced by substitution on either the N(6)- or the C-2 position of the adenine ring, whereas 5'-substitutions increase affinity, resulting in the potency order: 5'-N-ethylcarboxamidoadenosine (NECA) >> N(6)-aminobenzyl-NECA approximately 2-chloroadenosine > 2-[4-(2-carboxyethyl)phenethylamino]-NECA (CGS21680) > N(6)-aminobenzyladenosine. The A(2B)AR is potently blocked by the A(2A)-selective 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; K(I), 32 nM for A(2B), 1.4 nM for A(2A)) and the A(1) selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (K(I), 50.5 nM for A(2B); 2.5 nM for A(1)). The K(I) values for the antiasthmatic xanthines, theophylline (7.8 microM) and enprofylline (6.4 microM), are below their therapeutic plasma concentrations (20 to 50 microM), and agree with K(I) determinations for inhibition of NECA-stimulated cAMP accumulation in HEK-A(2B) cells. NECA or N(6)-(2-iodo)benzyl-5'-N-methylcarboxamidodoadenosine (IB-MECA) stimulate inositol trisphosphates and calcium accumulation in HEK-A(2B) or HEK-A(3) cells, respectively, but only the A(3) response is prevented by pertussis toxin. In human HMC-1 mast cells, A(2B)AR activation stimulates calcium mobilization and cAMP accumulation. We conclude that HEK-A(2B) cells and HMC-1 mast cells possess A(2B)AR glycoproteins that are coupled to both G(q/11) and G(s).
- Published
- 1999
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