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1.  A role for central A3-adenosine receptors Mediation of behavioral depressant effects 
FEBS letters  1993;336(1):57-60.
The behavioral effects of a selective A3 adenosine receptor agonist 3-IB-MECA (N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine) in mice and the localization of radioligand binding sites in mouse brain were examined. Low levels of A3 adenosine receptors were detected in various regions of the mouse brain (hippocampus, cortex, cerebellum, striatum), using a radioiodinated, high-affinity Aragonist radioligand [125I]AB-MECA (N6-(3-iodo-4-aminobenzyl)-5′-N-methylcarboxamidoadenosine). Scatchard analysis in the cerebellum showed that the Kd value for binding to A3 receptors was 1.39 ± 0.04 nM with a Bmax of 14.8 ± 2.1 fmol/mg protein. 3-IB-MECA at 0.1 mg/kg i.p. was a locomotor depressant with> 50% reduction in activity. Although selective A1 or A2a antagonists reversed locomotor depression elicited by selective A1 or A2a agonists, respectively, the behavioral depressant effects of 3-IB-MECA were unaffected. 3-IB-MECA also caused scratching in mice, which was prevented by coadministration of the histamine antagonist cyproheptadine. The demonstration of a marked behavioral effect of A3 receptor activation suggests that the A3 receptor represents a potential new therapeutic target.
PMCID: PMC4287251  PMID: 8262217
Adenosine receptor; Xanthine; Locomotor activity; Histamine; Radioligand binding
2.  TRIFUNCTIONAL LIGANDS: A RADIOIODINATED HIGH AFFINITY ACYLATING ANTAGONIST FOR THE A1 ADENOSINE RECEPTOR 
Pharmacology communications  1992;1(2):145-154.
A new xanthine (adenosine antagonist) radioligand that binds covalently to A1-adenosine receptors was prepared and used as a receptor probe. BH-DITC-XAC was synthesized via a trifunctional aryl diisothiocyanate crosslinker. containing the p-hydroxyphenylpropionyl group for radioiodination. The xanthine competed against agonist or antagonist A1 receptor radioligands in bovine brain membranes with an IC50, of 40nM. 125I-BH-DITC-XAC, prepared directly by the chloramine T method and purified by HPLC. bound specifically to A1 receptors. This binding was inhibited in the presence of the adenosine agonists R-PIA, S-PIA. and NECA in a dose dependent manner and with the order of potency characteristic of bovine A1 receptors. Incubation of affinity purified bovine A1-receptors with 125I-BH-DITC-XAC (0.8 nM) for 2 hours resulted in the specific and clean labelling of a polypeptide band corresponding to MW 36,000, identical to that previously found for the A1 receptor.
PMCID: PMC4217529  PMID: 25374448
xanthines; adenosine receptors; affinity labeling; radioiodination
4.  A Binding Site Model and Structure-Activity Relationships for the Rat A3 Adenosine Receptor 
Molecular pharmacology  1994;45(6):1101-1111.
SUMMARY
A novel adenosine receptor, the A3 receptor, has recently been cloned. We have systematically investigated the hitherto largely unexplored structure-activity relationships (SARs) for binding at A3 receptors, using 125I-N6-2-(4-aminophenyl)ethyladenosine as a radioligand and membranes from Chinese hamster ovary cells stably transfected with the rat A3-cDNA. As is the case for A1 and A2a, receptors, substitutions at the N6 and 5′ positions of adenosine, the prototypic agonist ligand, may yield fairly potent compounds. However, the highest affinity and A3 selectivity is found for N6,5′-disubstituted compounds, in contrast to A1 and A2a receptors. Thus, N6-benzyladenosine-5′-N-ethylcarboxamide is highly potent (Ki, 6.8 nM) and moderately selective (13- and 14-fold versus A1 and A2a). The N6 region of the A3 receptor also appears to tolerate hydrophilic substitutions, in sharp contrast to the other subtypes. Potencies of N6,5′-disubstituted compounds in inhibition of adenylate cyclase via A3 receptors parallel their high affinity in the binding assay. None of the typical xanthine or nonxanthine (A1/A2) antagonists tested show any appreciable affinity for rat A3 receptors. 1,3-Dialkylxanthines did not antagonize the A3 agonist-induced inhibition of adenylate cyclase. A His residue in helix 6 that is absent in A3 receptors but present in A1/A2 receptors may be causal in this respect. In a molecular model for the rat A3 receptor, this mutation, together with an increased bulkiness of residues surrounding the ligand, make antagonist binding unfavorable when compared with a previously developed A1 receptor model. Second, this A3 receptor model predicted similarities with A1 and A2 receptors in the binding requirements for the ribose moiety and that xanthine-7-ribosides would bind to rat A3 receptors. This hypothesis was supported experimentally by the moderate affinity (Ki 6 μM) of 7-riboside of 1,3-dibutylxanthine, which appears to be a partial agonist at rat A3 receptors. The model presented here, which is consistent with the detailed SAR found in this study, may serve to suggest future chemical modification, site-directed mutagenesis, and SAR studies to further define essential characteristics of the ligand-receptor interaction and to develop even more potent and selective A3 receptor ligands.
PMCID: PMC3479652  PMID: 8022403
5.  Purification and Characterization of Bovine Cerebral Cortex A1 Adenosine Receptor1 
A1 adenosine receptors (A1AR) acting via the inhibitory guanine nucleotide binding protein inhibit adenylate cyclase activity in brain, cardiac, and adipose tissue. We now report the purification of the A1AR from bovine cerebral cortex. This A1AR is distinct from other A1ARs in that it displays an agonist potency series of N6-R- phenylisopropyladenosine (R-PIA) > N6-S-phenylisopropyladenosine > (S-PIA) > 5′-N-ethylcarboxamidoadenosine (NECA) compared to the traditional potency series of R-PIA > NECA > S-PIA.
The A1AR was solubilized in 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) and then purified by chromatography on an antagonist [xanthine amine congener (XAC)]-coupled Affi-Gel 10 followed by hydroxylapatite chromatography. Following purification, sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed a single protein of Mr 36,000 by silver staining, Na125I iodination with chloramine T and photoaffinity labeling with [125I]8-[4-[[[[2-(4-aminophenyl acetylamino) ethyl] carbonyl] methyl] oxy] - phenyl]-1,3-dipropylxanthine. This single protein displayed all the characteristics of the A1AR, including binding an antagonist radioligand ([3H]XAC) with high affinity (Kd = 0.7 nM) and in a saturable manner (Bmax > 4500 pmol/mg). Agonist competition curves demonstrated the expected bovine brain A1AR pharmacology: R-PIA > S-PIA > NECA. The overall yield from soluble preparation was 7%.
The glycoprotein nature of the purified A1AR was determined with endo- and exoglycosidases. Deglycosylation with endoglycosidase F increased the mobility of the A1AR from Mr 36,000 to Mr 32,000 in a single step. The A1AR was sensitive to neuraminidase but resistant to α-mannosidase, suggesting the single carbohydrate chain was of the complex type. This makes the bovine brain A1AR similar to rat brain and fat A1AR in terms of its carbohydrate chains yet the purified A1AR retains its unique agonist potency series observed in membranes.
PMCID: PMC3476062  PMID: 2275555
6.  Structure–Activity Relationships of 1,3-Dialkylxanthine Derivatives at Rat A3 Adenosine Receptors 
Journal of medicinal chemistry  1994;37(20):3373-3382.
1,3-Dialkylxanthine analogues containing carboxylic acid and other charged groups on 8-position substituents were synthesized. These derivatives were examined for affinity in radioligand binding assays at rat brain A3 adenosine receptors stably expressed in CHO cells using the new radioligand [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide), and at rat brain A1 and A2a receptors using [3H]PIA and [3H]CGS 21680, respectively. A synthetic strategy for introducing multiple carboxylate groups at the 8-position using iminodiacetic acid derivatives was explored. The presence of a sulfonate, a carboxylate, or multiple carboxylate groups did not result in a significant enhancement of affinity at rat A3 receptors, although as previously observed an anionic group tended to diminish potency at A1 and A2a receptors. The rat A3 receptor affinity was not highly dependent on the distance of a carboxylate group from the xanthine pharmacophore. 2-Thio vs 2-oxo substitution favored A3 potency, and 8-alkyl vs 8-aryl substitution favored A3 selectivity, although few derivatives were truly selective for rat A3 receptors. 1,3-Dimethyl-8-(3-carboxypropyl)-2-thioxanthine was 7-fold selective for A3 vs A2a receptors. 1,3,7-Trimethyl-8-(trans-2-carboxyvinyl)xanthine was somewhat selective for A3 vs A1 receptors. For 8-arylxanthines affinity at A3 receptors was enhanced by 1,3-dialkyl substituents, in the order dibutyl > dipropyl > diallyl.
PMCID: PMC3471218  PMID: 7932565
7.  2-Substitution of N6-Benzyladenosine-5′-uronamides Enhances Selectivity for A3 Adenosine Receptors 
Journal of medicinal chemistry  1994;37(21):3614-3621.
Adenosine derivatives bearing an N6-(3-iodobenzyl) group, reported to enhance the affinity of adenosine-5′-uronamide analogues as agonists at A3 adenosine receptors (J. Med. Chem. 1994, 37, 636–646), were synthesized starting from methyl β-d-ribofuranoside in 10 steps. Binding affinities at A1 and A2a receptors in rat brain membranes and at cloned rat A3 receptors from stably transfected CHO cells were compared. N6-(3-Iodobenzyl)adenosine was 2-fold selective for A3 vs A1 or A2a receptors; thus it is the first monosubstituted adenosine analogue having any A3 selectivity. The effects of 2-substitution in combination with modifications at the N6- and 5′-positions were explored. 2-Chloro-N6-(3-iodobenzyl)adenosine had a Ki value of 1.4 nM and moderate selectivity for A3 receptors. 2-Chloro-N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide, which displayed a Ki value of 0.33 nM, was selective for A3 vs A1 and A2a receptors by 2500- and 1400-fold, respectively. It was 46,000-fold selective for A3 receptors vs the Na+-independent adenosine transporter, as indicated in displacement of [3H]N6-(4-nitrobenzyl)-thioinosine binding in rat brain membranes. In a functional assay in CHO cells, it inhibited adenylate cyclase via rat A3 receptors with an IC50 of 67 nM. 2-(Methylthio)-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide and 2-(methylamino)-N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide were less potent, but nearly as selective for A3 receptors. Thus, 2-substitution (both small and sterically bulky) is well-tolerated at A3 receptors, and its A3 affinity-enhancing effects are additive with effects of uronamides at the 5′-position and a 3-iodobenzyl group at the N6-position.
PMCID: PMC3468333  PMID: 7932588
8.  Cloning, Expression, and Characterization of the Unique Bovine A1 Adenosine Receptor 
The Journal of biological chemistry  1992;267(15):10764-10770.
The bovine brain A1 adenosine receptor (A1AR) is distinct from other A1ARs in that it displays the unique agonist potency series of N6-R-phenylisopropyladenosine (R-PIA) > N6-S-phenylisopropyladenosine (S-PIA) > 5′-N-ethylcarboxamidoadenosinea nd has a 5–10-fold higher affinity for both agonists and antagonists. The cDNA for this receptor has been cloned from a size-selected (2–4-kb) bovine brain library and sequenced. The 2.0-kb cDNA encodes a protein of 326 amino acid residues with a molecular mass of 36,570 daltons. The amino acid sequence fits well into the seven-transmembrane domain motif typical of G protein-coupled receptors. Northern analysis in bovine tissue using the full length cDNA demonstrates mRNAs of 3.4 and 5.7 kb with a tissue distribution consistent with A1AR binding. Subcloning of the cDNA in a pCMV5 expression vector with subsequent transfection into both COS7 and Chinese hamster ovary cells revealed a fully functional A1AR which could inhibit adenylylcyclase and retained the unique pharmacologic properties of the bovine brain A1AR
The A1AR was found to have a single histidine residue in each of transmembrane domains 6 and 7. Histidine residues have been postulated by biochemical studies to be important for ligand binding. Mutation of His-278 to Leu-278 (seventh transmembrane domain) dramatically decreased both agonist and antagonist binding by >90%. In contrast, mutation of His-251 to Leu-251 decreased antagonist affinity and the number of receptors recognized by an antagonist radioligand. In contrast, agonist affinity was not perturbed but the number of receptors detected by an agonist radioligand was also reduced. These data suggest that both histidines are important for both agonist and antagonist binding, but His-278 appears critical for ligand binding to occur.
PMCID: PMC3463104  PMID: 1587851
9.  Search for New Purine- and Ribose-Modified Adenosine Analogues as Selective Agonists and Antagonists at Adenosine Receptors† 
Journal of medicinal chemistry  1995;38(7):1174-1188.
The binding affinities at rat A1, A2a, and A3 adenosine receptors of a wide range of derivatives of adenosine have been determined. Sites of modification include the purine moiety (1-, 3-, and 7-deaza; halo, alkyne, and amino substitutions at the 2- and 8-positions; and N6-CH2-ring, -hydrazino, and -hydroxylamino) and the ribose moiety (2′-, 3′-, and 5′-deoxy; 2′- and 3′-O-methyl; 2′-deoxy 2′-fluoro; 6′-thio; 5′-uronamide; carbocyclic; 4′- or 3′-methyl; and inversion of configuration). (−)- and (+)-5′-Noraristeromycin were 48- and 21-fold selective, respectively, for A2a vs A1 receptors. 2-Chloro-6′-thioadenosine displayed a Ki value of 20 nM at A2a receptors (15-fold selective vs A1). 2-Chloroadenin-9-yl(β-L-2′-deoxy-6′-thiolyxofuranoside) displayed a Ki value of 8 μM at A1 receptors and appeared to be an antagonist, on the basis of the absence of a GTP-induced shift in binding vs a radiolabeled antagonist (8-cyclopentyl-1,3-dipropylxanthine). 2-Chloro-2′-deoxyadenosine and 2-chloroadenin-9-yl(β-D-6′-thioarabinoside) were putative partial agonists at A1 receptors, with Ki values of 7.4 and 5.4 μM, respectively. The A2a selective agonist 2-(1-hexynyl)-5′-(N-ethylcarbamoyl)adenosine displayed a Ki value of 26 nM at A3 receptors. The 4′-methyl substitution of adenosine was poorly tolerated, yet when combined with other favorable modifications, potency was restored. Thus, N6-benzyl-4′-methyladenosine-5′-(N-methyluronamide) displayed a Ki value of 604 nM at A3 receptors and was 103- and 88-fold selective vs A1 and A2a receptors, respectively. This compound was a full agonist in the A3-mediated inhibition of adenylate cyclase in transfected CHO cells. The carbocyclic analogue of N6-(3-iodobenzyl)adenosine-5′-(N-methyluronamide) was 2-fold selective for A3 vs A1 receptors and was nearly inactive at A2a receptors.
PMCID: PMC3457658  PMID: 7707320
10.  Molecular Characterization of A1 and A2a Adenosine Receptors 
Drug development research  2004;28(3):226-231.
Detailed amino acid sequence analyses of A1 and A2a adenosine receptors were assembled by analogy to other G-protein-coupled receptors and correlated with pharmacological observations. Sites for phosphorylation, palmitoylation, and sodium binding have been proposed. Striatal A2a receptors from human and other species were photoaffinity-labeled using the selective, radioiodinated agonist PAPA-APEC. Selective chemical affinity labels for A1 and A2a receptors have been introduced. For example, an isothiocyanate, p-DITC-APEC (100 nM), irreversibly diminished the Bmax for [3H]CGS 21680 (2-[4-[(2-carboxyethyl) phenyl] ethylamino]-5’-N-ethylcarboxamidoadenosine) binding in rabbit striatal membranes by 71% (Kd unaffected), suggesting a direct modification of the ligand binding site. Novel trifunctional affinity labels have been designed. Rabbit and human A2a receptors were characterized using [3H]XAC binding in the presence of 50 or 25 nM CPX (8-cyclopentyl-l,3-dipropylxanthine), respectively. The inhibition of A2 radioligand binding by the histidyl-modifying reagent diethylpyrocarbonate suggested the involvement of His residues in interactions with adenosine agonists and antagonists. Properties of transiently expressed mutants of bovine A1 receptors in which either His251 or His278 residues have been substituted with Leu suggest that both histidines are important in binding.
doi:10.1002/ddr.430280307
PMCID: PMC3446818  PMID: 23002320
affinity labeling; sequence analysis; xanthines; chemical modification; mutagenesis
11.  Structure–Activity Relationships of 9-Alkyladenine and Ribose-Modified Adenosine Derivatives at Rat A3 Adenosine Receptors† 
Journal of medicinal chemistry  1995;38(10):1720-1735.
9-Alkyladenine derivatives and ribose-modified N6-benzyladenosine derivatives were synthesized in an effort to identify selective ligands for the rat A3 adenosine receptor and leads for the development of antagonists. The derivatives contained structural features previously determined to be important for A3 selectivity in adenosine derivatives, such as an N6-(3-iodobenzyl) moiety, and were further substituted at the 2-position with halo, amino, or thio groups. Affinity was determined in radioligand binding assays at rat brain A3 receptors stably expressed in Chinese hamster ovary (CHO) cells, using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5′-(N-methyluronamide)), and at rat brain A1 and A2a receptors using [3H]-N6-PIA ((R)-N6-phenylisopropyladenosine) and [3H]CGS 21680 (2-[[[4-(2-carboxyethyl)-phenyl]ethyl]amino]-5′-(N-ethylcarbamoyl)adenosine), respectively. A series of N6-(3-iodobenzyl) 2-amino derivatives indicated that a small 2-alkylamino group, e.g., methylamino, was favored at A3 receptors. N6-(3-Iodobenzyl)-9-methyl-2-(methylthio)adenine was 61-fold more potent than the corresponding 2-methoxy ether at A3 receptors and of comparable affinity at A1 and A2a receptors, resulting in a 3–6-fold selectivity for A3 receptors. A pair of chiral N6-(3-iodobenzyl) 9-(2,3-dihydroxypropyl) derivatives showed stereoselectivity, with the R-enantiomer favored at A3 receptors by 5.7-fold. 2-Chloro-9-(β-d-erythrofuranosyl)-N6-(3-iodobenzyl)adenine had a Ki value at A3 receptors of 0.28 µM. 2-Chloro-9-[2-amino-2,3-dideoxy-β-d-5-(methylcarbamoyl)-arabinofuranosyl]-N6-(3-iodobenzyl)adenine was moderately selective for A1 and A3 vs A2a receptors. A 3′-deoxy analogue of a highly A3-selective adenosine derivative retained selectivity in binding and was a full agonist in the inhibition of adenylyl cyclase mediated via cloned rat A3 receptors expressed in CHO cells. The 3′-OH and 4′-CH2OH groups of adenosine are not required for activation at A3 receptors. A number of 2′,3′-dideoxyadenosines and 9-acyclic-substituted adenines appear to inhibit adenylyl cyclase at the allosteric “P” site.
PMCID: PMC3445626  PMID: 7752196
12.  Role of the Second Extracellular Loop of Adenosine Receptors in Agonist and Antagonist Binding 
The Journal of biological chemistry  1994;269(40):24692-24698.
Adenosine receptor (AR) agonists and antagonists are ~100-fold and 100,000-fold, respectively, more potent at the bovine A1AR as compared to the rat A3AR. To determine regions of ARs involved in ligand recognition, chimeric receptors composed of bovine A1AR and rat A3AR sequence were constructed and their ligand binding properties examined following expression in COS-7 cells. Substitutions oft he second extracellular loop or a region encompassing transmembrane domains 6 and 7 of the A1AR into the A3AR resulted in enhanced affinities of both agonists and antagonists compared to wild-type A3AR. The region of the second extracellular loop of the A1AR responsible for this effect was identified as the distal eleven amino acids of the loop. Replacement of this segment of the A3AR with that of the A1AR in combination with the regions encompassing transmembrane domains 6 and 7 resulted in a 50,000-fold increase in the Kd for antagonist radioligand, [3H]1,3-dipropyl-8-cyclopentylxanthine. Agonist affinity at this chimeric was over 100-fold greater than that displayed by wild-type A3AR. Thus, multiple regions of ARs including a segment of the second extracellular loop are involved in ligand recognition, and considerable overlap exists in structural features required for agonist and antagonist binding.
PMCID: PMC3437327  PMID: 7929142
13.  Pharmacological Characterization of Novel A3 Adenosine Receptor-selective Antagonists 
Neuropharmacology  1997;36(9):1157-1165.
Summary
The effects of putative A3 adenosine receptor antagonists of three diverse chemical classes (the flavonoid MRS 1067, the 6-phenyl-1,4-dihydropyridines MRS 1097 and MRS 1191, and the triazoloquinazo-line MRS 1220) were characterized in receptor binding and functional assays. MRS1067, MRS 1191 and MRS 1220 were found to be competitive in saturation binding studies using the agonist radioligand [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide) at cloned human brain A3 receptors expressed in HEK-293 cells. Antagonism was demonstrated in functional assays consisting of agonist-induced inhibition of adenylate cyclase and the stimulation of binding of [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP-γ-S) to the associated G-proteins. MRS 1220 and MRS 1191, with KB values of 1.7 and 92 nM, respectively, proved to be highly selective for human A3 receptor vs human A1 receptor-mediated effects on adenylate cyclase. In addition, MRS 1220 reversed the effect of A3 agonist-elicited inhibition of tumor necrosis factor-α formation in the human macrophage U-937 cell line, with an IC50 value of 0.3 μM. Published by Elsevier Science Ltd.
PMCID: PMC3433714  PMID: 9364471
Dihydropyridine; flavonoid; triazoloquinazoline; adenylate cyclase; tumor necrosis factor; guanine nucleotides; adenosine A3 receptor; adenosine
14.  Effect of simulated microgravity on oxidation-sensitive gene expression in PC12 cells 
Oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). A baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. The objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (PC12)] under simulated micro-g conditions. The PC12 cell line is well characterized in its response to oxygen, and is widely recognized as a sensitive model for studying the responses of oxygen-sensitive molecular and cellular processes. This study uses the rotating wall vessel bioreactor (RWV) designed at NASA to simulate micro-g. Gene expression in PC12 cells in response to micro-g was analyzed by DNA microarray technology. The microarray analysis of PC12 cells cultured for 4 days under simulated micro-g under standardized oxygen environment conditions revealed more than 100 genes whose expression levels were changed at least twofold (up-regulation of 65 genes and down-regulation of 39 genes) compared with those from cells in the unit gravity (unit-g) control. This study observed that genes involved in the oxidoreductase activity category were most significantly differentially expressed under micro-g conditions. Also, known oxidation-sensitive transcription factors such as hypoxia-inducible factor-2α, c-myc, and the peroxisome proliferator-activated receptor-γ were changed significantly. Our initial results from the gene expression microarray studies may provide a context in which to evaluate the effect of varying oxygen environments on the background of differential gene regulation of biological processes under variable gravity conditions.
doi:10.1016/j.asr.2006.02.059
PMCID: PMC2600499  PMID: 19081771
Rotating wall vessel; Simulated microgravity; Gene expression; Microarray

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