(3)H]MRE 3008F20: a novel antagonist radioligand for the pharmacological and biochemical characterization of human A(3) adenosine receptors

The lack of a radiolabeled selective A(3) adenosine receptor antagonist is a major drawback for an adequate characterization of this receptor subtype. This paper describes the pharmacological and biochemical characterization of the tritiated form of a new potent A(3) adenosine receptor antagonist, the pyrazolo triazolo pyrimidine derivative [(3)H]5N-(4-methoxyphenylcarbamoyl)amino-8-propyl-2-(2-furyl )pyrazolo [4,3-e] -1,2,4- triazolo[1,5-c]pyrimidine ([(3)H]MRE 3008F20). [(3)H]MRE 3008F20 bound specifically to the human adenosine A(3) receptor expressed in CHO cells (hA(3)CHO), and saturation analysis revealed a single high affinity binding site, K(D) = 0.80 +/- 0.06 nM, with a B(max) = 300 +/- 33 fmol/mg protein. This new ligand displayed high selectivity (1294-, 165-, and 2471-fold) in binding assay to human A(3) versus A(1), A(2A), and A(2B) receptors, respectively, and binds to the rat A(3) receptors with a K(i) > 10 microM. The pharmacological profile of [(3)H]MRE 3008F20 binding to hA(3)CHO cells was evaluated using known adenosine receptor agonists and antagonists with a rank order of potency consistent with that typically found for interactions with the A(3) adenosine receptors. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency identical with that observed in binding experiments. Thermodynamic data indicated that [(3)H]MRE 3008F20 binding to hA(3)CHO is entropy- and enthalpy-driven in agreement with the typical behavior of other adenosine antagonists to A(1) and A(2A) receptors. These results show that [(3)H]MRE 3008F20 is the first antagonist radioligand with high affinity and selectivity for the human A(3) adenosine receptor and may be used to investigate the physiopathological role of A(3) adenosine receptors.     

Pharmacological and biochemical characterization of A3 adenosine receptors in Jurkat T cells

1. The present work was devoted to the study of A3 adenosine receptors in Jurkat cells, a human leukemia line. 2. The A3 subtype was found by means of RT-PCR experiments and characterized by using the new A3 adenosine receptor antagonist [3H]-MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with K(D) of 1.9+/-0.2 nM and B(max) of 1.3+/-0.1 pmol mg(-1) of protein. 3. The pharmacological profile of [3H]-MRE 3008F20 binding on Jurkat cells was established using typical adenosine ligands which displayed a rank order of potency typical of the A3 subtype. 4. Thermodynamic data indicated that [3H]-MRE 3008F20 binding to A3 subtype in Jurkat cells was entropy- and enthalpy-driven, according with that found in cells expressing the recombinant human A3 subtype. 5. In functional assays the high affinity A3 agonists Cl-IB-MECA and IB-MECA were able to inhibit cyclic AMP accumulation and stimulate Ca(2+) release from intracellular Ca(2+) pools followed by Ca(2+) influx. 6. The presence of the other adenosine subtypes was investigated in Jurkat cells. A1 receptors were characterized using [3H]-DPCPX binding with a K(D) of 0.9+/-0.1 nM and B(max) of 42+/-3 fmol mg(-1) of protein. A2A receptors were studied with [3H]-SCH 58261 binding and revealed a K(D) of 2.5+/-0.3 nM and a B(max) of 1.4+/-0.2 pmol mg(-1) of protein. 7. In conclusion, by means of the first antagonist radioligand [3H]-MRE 3008F20 we could demonstrate the existence of functional A3 receptors on Jurkat cells.     

Pharmacological and biochemical characterization of adenosine receptors in the human malignant melanoma A375 cell line.

1. The present work characterizes, from a pharmacological and biochemical point of view, adenosine receptors in the human malignant melanoma A375 cell line. 2. Adenosine receptors were detected by RT - PCR experiments. A1 receptors were characterized using [3H]-DPCPX binding with a KD of 1.9+/-0.2 nM and Bmax of 23+/-7 fmol x mg(-1) of protein. A2A receptors were studied with [3H]-SCH 58261 binding and revealed a KD of 5.1+/-0.2 nM and a Bmax of 220+/-7 fmol x mg(-1) of protein. A3 receptors were studied with the new A3 adenosine receptor antagonist [3H]-MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with KD of 3.3+/-0.7 nM and Bmax of 291+/-50 fmol x mg(-1) of protein. 3. The pharmacological profile of radioligand binding on A375 cells was established using typical adenosine ligands which displayed a rank order of potency typical of the different adenosine receptor subtype. 4. Thermodynamic data indicated that radioligand binding to adenosine receptor subtypes in A375 cells was entropy- and enthalpy-driven. 5. In functional assays the high affinity A2A agonists HE-NECA, CGS 21680 and A2A - A2B agonist NECA were able to increase cyclic AMP accumulation in A375 cells whereas A3 agonists Cl-IB-MECA, IB-MECA and NECA were able to stimulate Ca2+ mobilization. In conclusion, all these data indicate, for the first time, that adenosine receptors with a pharmacological and biochemical profile typical of the A1, A2A, A2B and A3 receptor subtype are present on A375 melanoma cell line.     

Pharmacological characterization of novel adenosine ligands in recombinant and native human A2B receptors

The present study was designed to evaluate the effects of novel and recognised compounds at human recombinant A(2B) adenosine receptors expressed in Chinese hamster ovary (hA(2B)CHO), in human embryonic kidney 293 (hA(2B)HEK-293) and at endogenous A(2B) receptors in human mast cells (HMC-1). Saturation binding experiments performed using the new high affinity A(2B) adenosine radioligand [(3)H]-N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetra hydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide ([(3)H]-MRE 2029F20) revealed a single class of binding sites in hA(2B)CHO, hA(2B)HEK-293 and HMC-1 cells with K(D) (nM) of 1.65+/-0.18, 2.83+/-0.34, 2.62+/-0.27 and B(max) (fmol/mg protein) of 36+/-4, 475+/-50 and 128+/-15, respectively. The pharmacological profile of new compounds, determined in inhibition binding experiments in hA(2B)HEK-293 cells using [(3)H]-MRE 2029F20, showed a rank order of potency typical of the A(2B) receptors with K(i) values in the range 3.2-28nM. In functional assays, recognised agonists and antagonists were studied by evaluating their capability to modulate the cAMP production in hA(2B)CHO and in HMC-1 cells. Novel compounds were able to decrease NECA-stimulated cAMP production in hA(2B)CHO and in HMC-1 cells showing a high potency. New compounds were also able to inhibit cAMP levels in the absence of NECA and in the presence of forskolin stimulation in hA(2B)CHO and in HMC-1 cells. In HEK-293 cells MRE 2029F20 reduced cAMP basal levels with an IC(50) value of 2.9+/-0.3nM. These results suggest that novel compounds are antagonists with an inverse agonist activity in recombinant and native human A(2B) receptors.     

Demonstration of both A1 and A2 adenosine receptors in DDT1 MF-2 smooth muscle cells

Adenosine receptors of the A1 and A2 subtypes were characterized in membranes from DDT1 MF-2 smooth muscle cells. These cells possess a high density of A1 adenosine receptors (Bmax = 0.8-0.9 pmol/mg of protein), as measured by both agonist and antagonist radioligands. Agonists compete for [125I]N6-[2-(4-amino-3-iodophenyl)ethyl]-adenosine (A1 receptor-selective radioligand) binding with the following potency series: (R)-phenylisopropyladenosine [(R)-PIA] greater than 5'-N-ethylcarboxamide adenosine (NECA) greater than (S)-PIA, indicative of their interaction with A1 adenosine receptors. Agonist competition for [3H]8-(4-[[[(2-aminoethyl)amino]carbonyl)methyl)oxy]phenyl)-1, 3-dipropylxanthine [( 3H]XAC) (an antagonist radioligand for the A1 adenosine receptor) was described by a two-state model of 1.3 nM (high affinity state, KK) and 370 nM (low affinity state, KL), with 70% of the receptors in the high affinity state (RH). Addition of guanosine 5'-[beta, alpha-imido]triphosphate (100 microM) shifted the (R)-PIA competition curves to the right to lower affinities. Photoaffinity labeling with the agonist photoprobe [125I]N6-[2-(4-amino-3-iodophenyl) ethyl]adenosine indicates that the A1 adenosine receptor binding subunit is a Mr 38,000 protein. Adenosine receptor agonists [(R)-PIA, NECA, and (S)-PIA] inhibited isoproterenol-stimulated adenylate cyclase activity in DDT1 MF-2 cell membranes with IC50 values of 62, 538, and 750 nM, respectively. Inhibition of adenylate cyclase by (R)-PIA was attenuated by the A1 receptor antagonist XAC and following inactivation of Gi with pertussis toxin (100 ng/ml). Using a recently developed A2 adenosine receptor agonist radioligand 2-[4-(2-[( 4-aminophenyl]methylcarbonyl)ethyl) phenyl]ethylamino-5'-N-ethylcarboxamido adenosine (125I-PAPA-APEC), we have demonstrated the presence of A2 adenosine receptors in this cell line. Saturation curves with 125I-PAPA-APEC indicated the Bmax and Kd values to be 0.21 pmol/mg of protein and 4.0 nM, respectively. In competition experiments, NECA was more potent at inhibiting 125I-PAPA-APEC binding than (R)-PIA, with their respective IC50 values being 5.6 and 351 nM. The photolabeled A2 adenosine receptor migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an Mr of 42,000. Finally, adenosine receptor agonists stimulated adenylate cyclase activity by approximately 2-3 fold with the following potency series: PAPA-APEC greater than or equal to NECA greater than (R)-PIA, indicative of their interaction at A2 receptors. These data represent the first demonstration of the presence of both A1 and A2 receptors in a single cell line, DDT1 MF-2 smooth muscle cells.     

3H]MRS 1754, a selective antagonist radioligand for A(2B) adenosine receptors

MRS 1754 [N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide] is a selective antagonist ligand of A(2B) adenosine receptors. This is the least well-defined adenosine receptor subtype, and A(2B) antagonists have potential as antiasthmatic drugs. For use as a radioligand, MRS 1754, a p-cyanoanilide xanthine derivative, was tritiated on the propyl groups in a two-step reaction using a p-carboxamido precursor, which was dehydrated to the cyano species using trifluoroacetic anhydride. [3H]MRS 1754 (150 Ci/mmol) bound to recombinant human A(2B) adenosine receptors in membranes of stably transfected HEK-293 cells. Specific binding was saturable, competitive, and followed a one-site model, with a K(D) value of 1.13 +/- 0.12 nM and a B(max) value of 10.9 +/- 0.6 pmol/mg protein. Specific binding utilizing 0.7 nM [3H]MRS 1754 was > 70% of total binding. The affinity calculated from association and dissociation binding constants was 1.22 nM (N = 4). Binding to membranes expressing rat and human A(1) and A(3) adenosine receptors was not significant, and binding in membranes of HEK-293 cells expressing human A(2A) receptors was of low affinity (K(D) > 50 nM). The effects of cations and chelators were explored. Specific binding was constant over a pH range of 4.5 to 6.5, with reduced binding at higher pH. The pharmacological profile in competition experiments with [3H]MRS 1754 was consistent with the structure-activity relationship for agonists and antagonists at A(2B) receptors. The K(i) values of XAC (xanthine amine congener) and CPX (8-cyclopentyl-1,3-dipropylxanthine) were 16 and 55 nM, respectively. NECA (5'-N-ethylcarboxamidoadenosine) competed for [3H]MRS 1754 binding with a K(i) of 570 nM, similar to its potency in functional assays. Thus, [3H]MRS 1754 is suitable as a selective, high-affinity radioligand for A(2B) receptors.     

3H]HEMADO--a novel tritiated agonist selective for the human adenosine A3 receptor

Adenosine A(3) receptors are promising drug targets for a number of conditions like inflammatory diseases including asthma, ischemic injury or certain types of cancer. Consequently, intense efforts are dedicated to the development of selective A(3) agonists and antagonists. The only tritiated agonist that is available for radioligand binding is the nonselective [(3)H]5'-N-ethylcarboxamidoadenosine ([(3)H]NECA). Based on a recently characterized series of 2-substituted adenosine receptor agonists we developed a tritiated selective A(3) radioligand with high affinity. From this series 2-hexyn-1-yl-N(6)-methyladenosine (HEMADO) with a K(i)-value of 1.1 nM at the human A(3) subtype was chosen. HEMADO is 300-fold selective versus the A(1) subtype, and 1100-fold and more than 25,000-fold selective compared to the adenosine A(2A) and A(2B) receptors, respectively. The tritiated derivative [(3)H]HEMADO exhibited the same affinity as the unlabeled precursor. In concentrations up to 10 nM no specific binding to adenosine A(1), A(2A) or A(2B) receptors was observed confirming the high selectivity for adenosine A(3) receptors. Characterization of [(3)H]HEMADO in radioligand binding studies revealed reversible binding to the human adenosine A(3) subtype. In saturation binding studies for the A(3) subtype a K(D)-value of 1.1 nM was determined. Nonspecific binding at a radioligand concentration of 1 nM amounted to 1-2% of total binding. Competition binding with a panel of adenosine receptor ligands clearly confirmed the correct A(3) pharmacology of the binding site labeled by [(3)H]HEMADO. With [(3)H]HEMADO we present a tritiated agonist with high affinity and A(3)-selectivity and very low nonspecific binding. [(3)H]HEMADO is a useful tool for specific screening for A(3) receptor agonists and antagonists in improved radioligand binding assays with the human subtype.     

Radioligand binding and functional responses of ligands for human recombinant adenosine A(3) receptors

The binding and functional properties of adenosine receptor ligands were compared in Chinese hamster ovary cells transfected with human adenosine A(3) receptors. Inhibition of [(125)I]-aminobenzyl-5'-N-methylcarboamidoadenosine ([(125)I]-AB-MECA) binding by adenosine receptor ligands was examined in membrane preparations. Inhibition of forskolin-induced cAMP accumulation by agonists was measured using a cAMP enzyme immunoassay. The rank order of agonist potency for both assays was N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > (-)-N(6)-[(R)-phenylisopropyl] adenosine (R-PIA) > 4-aminobenzyl-5'-N-methylcarboxamidoadenosine (AB-MECA) > N(6)-cyclopentyl adenosine (CPA) > adenosine. The radioligand binding rank order of antagonist potency was N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS1220) > 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) > 8-phenyltheophylline (8-PT) > 8-(p-sulfophenyl)-theophylline (8-SPT). MRS1220 competitively inhibited the effect of IB-MECA on cAMP production, with a K(B) value of 0.35 nm. These data are characteristic of adenosine A(3) receptors. The absence of Mg(2+) and presence of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) significantly reduced agonist binding inhibition potency, indicating binding to high- and low-affinity states. The IB-MECA, NECA and R-PIA IC(50) values were greater for the cAMP assay than for radioligand binding, suggesting an efficient stimulus-response transduction pathway.     

Alteration of A(3) adenosine receptors in human neutrophils and low frequency electromagnetic fields

The present study was designed to evaluate the binding and functional characterization of A(3) adenosine receptors in human neutrophils exposed to low frequency, low energy, pulsing electromagnetic fields (PEMFs). Great interest has grown concerning the use of PEMF in the clinical practice for therapeutic purposes strictly correlated with inflammatory conditions. Saturation experiments performed using the high affinity and selective A(3) adenosine antagonist 5N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([3H]-MRE 3008F20) revealed a single class of binding sites with similar affinity in control and in PEMF treated human neutrophils (K(D)=2.36+/-0.16 and 2.45+/-0.15 nM, respectively). PEMFs treatment revealed that the receptor density was statistically increased (P<0.01) (B(max)=451+/-18 and 736+/-25fmolmg(-1) protein, respectively). Thermodynamic data indicated that [3H]-MRE 3008F20 binding in control and in PEMF-treated human neutrophils was entropy and enthalpy driven. Competition of radioligand binding by the high affinity A(3) receptor agonists, N(6)-(3-iodo-benzyl)-2-chloro-adenosine-5'-N-methyluronamide (Cl-IB-MECA) and N(6)-(3-iodo-benzyl)adenosine-5'-N-methyl-uronamide (IB-MECA), in the absence of PEMFs revealed high and low affinity values similar to those found in the presence of PEMFs. In both experimental conditions, the addition of GTP 100 microM shifted the competition binding curves of the agonists from a biphasic to a monophasic shape. In functional assays Cl-IB-MECA and IB-MECA were able to inhibit cyclic AMP accumulation and their potencies were statistically increased after exposure to PEMFs. These results indicate in human neutrophils treated with PEMFs the presence of significant alterations in the A(3) adenosine receptor density and functionality.     

Use of the triazolotriazine [3H]ZM 241385 as a radioligand at recombinant human A2B adenosine receptors

Radiolabeled ZM 241385 (4-(2-[7-amino-2- ?furyl??1,2,4?triazolo?2,3-a??1,3,5?triazin-5-ylaminoethyl)p henol), has previously been used as a high affinity radioligand for the labeling of A2A adenosine receptors in cell membranes. Another subtype, the A2B receptor, is the least well-defined subtype of adenosine receptors and lacks selective pharmacological probes. In the present study, we have used [3H]ZM 241385 as a radioligand to label recombinant human A2B adenosine receptors in HEK-293 cell membranes, that do not express A2A adenosine receptors, and found that the pharmacological profile is consistent with the SAR of A2B receptors. Saturable, specific binding (Kd 33.6 nM, Bmax 4.48 pmol/mg protein) that was best described by a one-site model was found, and specific binding was approximately 75% of total binding. [3H]ZM 241385 binding was displaceable by a large number of compounds known to interact with A2B receptors; thus, this method has promise as a tool in the search for agonists and antagonists selective for this subtype. Xanthine analogs, which are antagonists, proved to be the most potent displacers. The Ki of XAC, xanthine amine congener, was 12.3 nM, while CPX (8-cyclopentyl-1,3-dipropylxanthine) was less potent. The non-selective triazoloquinazoline antagonist CGS 15943 (Ki 16.4 nM), which is similar in structure to ZM 241385, was slightly less potent than XAC. The non-xanthine A2B-antagonist alloxazine displaced [3H]ZM 241385-binding with a Ki of 462 nM, similar to its affinity in functional assays. Adenosine derivatives known to activate this receptor subtype, such as NECA (5'-N-ethylcarboxamidoadenosine) and R-PIA (N6-phenylisopropyladenosine), were considerably less potent than the 8-substituted xanthines examined.     

The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine to adenosine A1 receptors in rat smooth muscle preparations.

1. The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), an antagonist radioligand selective for adenosine A1 receptors, was studied in rat duodenum, colon muscularis mucosae and longitudinal muscle, urinary bladder and vasa deferentia. 2. [3H]-DPCPX bound with high affinity to a single site in all membrane preparations studied with the exception of the rat urinary bladder in which no specific binding was detected. The affinity (Kd) of the binding site for [3H]-DPCPX was similar in all membrane preparations, the colon longitudinal muscle (1.18 +/- 0.47 nM), colon muscularis mucosae (0.84 +/- 0.15 nM), duodenum (1.59 +/- 0.18 nM) and vasa deferentia (0.93 +/- 0.17 nM). The density of [3H]-DPCPX binding sites was similar in the duodenum (38.8 +/- 4 fmol mg-1 protein), muscularis mucosae (43 +/- 3.5 fmol mg-1 protein) and vasa deferentia (43.3 +/- 12.2 fmol mg-1 protein), but in the longitudinal muscle 6-7 fold more binding sites (295 +/- 70 fmol mg-1 protein) were identified. 3. Inhibition studies using DPCPX (0.1-100 nM), N6-cyclopentyladenosine (CPA) (0.1-100 nM), 5'-N-ethylcarboxamidoadenosine (NECA) (2 nM-10 microM) and (R)-N6-phenylisopropyladenosine (R-PIA) (1 nM-1 microM) to displace the binding of [3H]-DPCPX at a concentration around the Kd value (1 nM), demonstrated an order of potency of displacement in all tissues of DPCPX > or = CPA > R-PIA > NECA. This potency order is characteristic of an A1 receptor, indicating that [3H]-DPCPX binds to adenosine A1 receptors in the rat duodenum, colon and vasa deferentia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of adenosine binding sites in bovine testicular tissue using 8-cyclopentyl-1,3-[3H]dipropylxanthine

The existence of specific adenosine binding sites in bovine testicular tissue was evaluated using the novel antagonist radioligand 8-cyclopentyl-1,3-[3H]dipropylxanthine ([3H]DPCPX). Saturation analysis revealed specific binding that was saturable at approximately 1 nM. Scatchard analysis indicated a single class of binding sites with a KD = 0.26 nM and a Bmax = 0.37 pmol/mg protein. Affinity profiles suggest an A1 subtype recognition site that is different from the classical A1 adenosine receptor. The results presented should prove useful in subsequent studies concerning heterogeneity among adenosine receptors and also aid in discerning the role of adenosine in reproduction.     

Agonist radioligand interactions with the solubilized porcine atrial A1 adenosine receptor

Porcine atrial adenosine receptors have been solubilized using a detergent system consisting of digitonin and sodium cholate and characterized with the agonist radioligand N6[125I]hydroxyphenylisopropyl) adenosine [125I]HPIA. 125IHPIA labeled an apparently homogeneous population of solubilized recognition sites with a Bmax of 88 +/- 4 fmol/mg of protein and a KD of 1.4 +/- 0.1 nM. Solubilization resulted in a 2.5-fold enrichment of adenosine receptor specific activity and an enhanced signal to noise ratio over that observed for porcine atrial membrane preparations. Solubilized cardiac adenosine receptors were relatively stable and exhibited many of the properties of membrane-bound receptors. The rank order potency of adenosine receptor agonists inhibiting the binding of [125I]HPIA was consistent with the labeling of a solubilized A1 adenosine receptor. Association rate experiments suggested that the interaction of [125I]HPIA with solubilized cardiac adenosine receptors was consistent with that of a simple bimolecular reaction. The dissociation constant calculated from kinetic data (0.73 nM) was in good agreement with that determined by equilibrium binding measurements (1.4 nM). The interaction of cardiac A1 adenosine receptors and guanine nucleotide binding protein(s) G protein(s) was retained in this detergent system. Addition of guanosine-5'-O-(3-thio)triphosphate to an equilibrated mixture of solubilized cardiac adenosine receptors and [125I]HPIA resulted in a rapid and complete dissociation of [125I]HPIA. This dissociation was resolved into two kinetic phases, which appear to arise from two populations of independent, noninterconvertible receptor-G protein complexes that display differing sensitivities to guanine nucleotides. The A1 adenosine receptor-G protein complex solubilized in digitonin/cholate appears to provide an excellent system by which agonist radioligand-receptor-G protein interactions can be further studied.     

(3)H]ZM241385--an antagonist radioligand for adenosine A(2A) receptors in rat brain

Binding of the novel adenosine A(2A) receptor-selective antagonist radioligand [2-(3)H]-4-(2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5,)triazin-5-yl amino]ethyl)phenol ([(3)H]ZM241385) was examined using particulate preparations and frozen sections of rat brain. In membranes from the rat striatum, binding was saturable, reversible and temperature-dependent. Analysis of saturation isotherms indicated that [(3)H]ZM241385 bound with high affinity (K(d) of 0.84 nM), high density (1680 fmol mg protein(-1)) and with a high proportion of specific binding (93% at 1 nM radioligand). Examination of competition profiles indicated that [(3)H]ZM241385 bound to sites with an A(2A) adenosine receptor-like rank order. The presence of guanosine 5'-(3-thio)-triphosphate failed to alter either [(3)H]ZM241385 binding or agonist competition for [(3)H]ZM241385 binding. Autoradiographic analysis of [(3)H]ZM241385 binding to frozen sections of rat brain indicated specific binding to the rat striatum of similar affinity (K(d) of 0.43 nM) and susceptibility to adenosine receptor ligands. At 2 nM [(3)H]ZM241385, specific binding comprised 95+/-1% total binding. In the hippocampus and frontal cortex, binding of [(3)H]ZM241385 failed to saturate and was of lower density. Taken together, these results indicate that [(3)H]ZM241385 should prove to be a useful radioligand in the characterisation of adenosine A(2A) receptors.     

Characterization of adenosine receptors in brush-border membranes from pig kidney.

1. The adenosine receptors from pig kidney proximal tubules have been studied in membrane vesicle preparations derived from either luminal (brush-border membranes-BBM-) or basolateral (BL) sides. There was a substantial amount of A2-like NECA binding in both preparations, but the A1 subtype of adenosine receptors was not found in either BBM or BL membranes. The use of [3H]-CGS21680 which is a more specific ligand for A2a receptors revealed true adenosine receptors in the BBM. 2. The kinetic parameters for [3H]-CGS21680 binding to pig renal BBM were: Bmax = 1.48 pmol mg-1 protein and Kd = 150 nM. In the presence of Gpp(NH)p the affinity decreased (Kd = 220 nM), whereas the addition of Mg2+ induced a marked increase in affinity (Kd = 83 nM). These equilibrium constants are higher than those found for the A2a adenosine receptors present in pig brain striatal membranes (Kd = 12 nM), and are close to those found in rat renal BBM (Kd = 90 nM). 3. The order of potency of agonist and antagonists was not consistent with the presence of either A1 or A2 receptors, but it was very similar to the agonist order of potency for the A3 receptor subtype. Furthermore, the blockade of the [3H]-CGS21680 binding by both cholera and pertussis toxin further supports the view that the subtypes present in BBM are neither A1 nor A2. 4. Overall the results suggest the presence in BBM of an A3 receptor, or of a new subtype of adenosine receptor, which is linked to G proteins sensitive to both cholera and pertussis toxins.     

Labeling of A1 adenosine receptors in porcine atria with the antagonist radioligand 8-cyclopentyl-1,3-[3H]dipropylxanthine

We have used the antagonist radioligand 8-cyclopentyl-1,3-[3H]dipropylxanthine to label adenosine recognition sites in porcine atrial membranes. 8-Cyclopentyl-1,3-[3H]dipropylxanthine bound saturably, reversibly and with high affinity to an apparently homogeneous population of recognition sites with a Bmax of 32.0 +/- 0.9 fmol/mg protein and a KD of 0.394 +/- 0.049 nM. Prototypic adenosine receptor agonists inhibited the specific binding of 8-cyclopentyl-1,3-[3H]dipropylxanthine in a manner consistent with the labeling of an A1 adenosine receptor. 8-Cyclopentyl-1,3-[3H]dipropylxanthine appears to be a valuable antagonist radioligand for the characterization of cardiac adenosine receptors.     

Increase in the vasorelaxant potency of K(ATP) channel opening drugs by adenosine A(1) and A(2) receptors in the pig coronary artery

Myograph recording from ring segments of pig small coronary arteries was used to investigate the effects of adenosine receptor activation on the vasorelaxant potency of ATP-sensitive K(+) channel opening drugs. Receptor activation with 2-chloroadenosine (2-CA, 300 nM) increased the potency of both nicorandil and levcromakalim, shifting the pEC(50)s from 4.68+/-0.03 to 5.05+/-0.04 and from 6.34+/-0.06 to 6.72+/-0.06, respectively (P<0.05 in each case). Experiments with selective adenosine receptor agonists (2-chloro-N(6)-cyclopentyladenosine (CCPA), 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680)) and antagonists (8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), 4-(2-[7-amino-2-(2-furyl)[1,2, 4]triazolo[2,3-a] [1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385)) suggest that both A(1) and A(2a) receptors can increase the potency of nicorandil, while that of levcromakalim is increased only by A(2) receptors. Adenosine receptor activation did not affect the potency of pinacidil. Thus, adenosine receptor activation can increase the potency of some K(+) channel opening drugs to relax coronary arteries, but the details of the interaction with adenosine receptors depend on the particular drug.     

2-Substituted N-ethylcarboxamidoadenosine derivatives as high-affinity agonists at human A3 adenosine receptors

A number of 2-substituted 5'-N-ethylcarboxamidoadenosine (NECA) derivatives was investigated for their affinity and selectivity at human A3 adenosine receptors. The compounds were tested in radioligand competition studies and modulation of adenylyl cyclase activity on membranes from CHO cell lines stably transfected with the four human adenosine receptor subtypes. In binding studies the most potent compound, 2-(3-hydroxy-3-phenyl)propyn-1-yl-NECA (PHPNECA), exhibited a subnanomolar affinity for A3 adenosine receptors with a Ki value of 0.4 nM. As opposed to the limited A3 selectivity of PHPNECA, a 100-fold selectivity compared to both A1 and A2A receptors was found for 2-(2-phenyl)ethynyl-NECA (PENECA; Ki 6 nM). The EC50 values for activation of adenylyl cyclase via A2A adenosine receptors were in good agreement with the respective Ki values from binding experiments. In contrast, IC50 values for A1 and A3 receptor-mediated inhibition of adenylyl cyclase were shifted to higher values compared to the respective affinities determined in radioligand competition studies. Similar discrepancies between binding and functional data have been observed for the inhibitory A1 adenosine receptor in previous studies. Therefore, the same A3 selectivity of PENECA compared to A1 receptors was found in binding and adenylyl cyclase inhibition whereas the selectivity compared to A2A receptors that was detected in ligand binding was obscured in the functional assay. The series of compounds presented in this study identifies 2-substitution of the purine system as a promising target for the development of A3-selective high-affinity ligands.