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.     

Adenosine receptors in post-mortem human brain.

1. Adenosine A2-like binding sites were characterized in post-mortem human brain membranes by examining several compounds for their ability to displace [3H]-CGS 21680 (2[p-(2 carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamido adenosine) binding. 2. Two A2-like binding sites were identified in the striatum. 3. The more abundant striatal site was similar to the A2a receptor previously described in rat striatum, both in its pharmacological profile and striatal localization. 4. The less abundant striatal site had a pharmacological profile similar to that of the binding site characterized in the other brain regions examined. This was intermediate in character between A1 and A2 and may represent another adenosine receptor subtype. 5. The co-purification of [3H]-CGS 21680 binding during immunoisolation of human striatal cholinergic membranes was used to assess the possible cholinergic localization of A2-like binding sites in the human striatum. Only the more abundant striatal site co-purified with cholinergic membranes. This suggests that this A2a-like site is present on cholinergic neurones in the human striatum.     

Adenosine A(2A) receptors in portal hypertension: their role in the abnormal response to adenosine of the cranial mesenteric artery in rabbits

1. Adenosine is a regulator of mesenteric vasodilation involved in auto-regulation and post-prandial hyperemia, but the adenosine receptor subtype involved in this relaxant effect is poorly characterized. We have now pharmacologically characterized this receptor in rabbit mesenteric arteries and investigated how this adenosine receptor response changes in portal hypertensive animals since the adenosine response is decreased. 2. The closest non-metabolisable adenosine analogue, 2-chloroadenosine (CADO), the mixed A(1)/A(2) receptor agonist, 5'-ethylcarboxamidoadenosine (NECA), and the selective A(2A) receptor agonist, 2-[4-(2-p-carbonyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680) (1 pM -- 1 mM) relaxed noradrenaline pre-contracted arteries with a rank order of potency of CGS 21680 (EC(50)=20 nM) > or = NECA (60 nM)>CADO (640 nM). 3. The selective A(2A) receptor antagonist, 4-(2-[7-amino-2-(2-furyl)-[1,2,4]-triazolo[2,3-a][1,3,5]-triazin-5-ylamino]ethyl)phenol (ZM 241385, 100 nM), shifted to the right the CADO concentration-response curve. 4. In portal hypertensive animals, there was mainly a decreased potency but also a decreased efficacy of all tested adenosine agonists compared to normal animals. Concomitantly, there was a decreased adenosine plasma level and a decreased binding density of [(3)H]-CGS 21680 and [(3)H]-ZM 241385 to mesenteric artery membranes from portal hypertensive compared to normal rabbits. 5. These results indicate that A(2A) receptor activation is required for the adenosine-induced mesenteric relaxation and that the decreased density of A(2A) receptors may contribute to the decreased relaxation induced by adenosine of mesenteric arteries in portal hypertensive animals.     

The selective adenosine A2A receptor antagonist SCH 58261 discriminates between two different binding sites for [3H]-CGS 21680 in the rat brain

We have used quantitative autoradiography to further examine two previously described binding sites for [³H]-CGS 21680 in cortical regions and in striatum, respectively. The striatal binding sites largely represent classical adenosine A_2A receptors whereas the cortical sites show characteristics that differ from those of recognised adenosine receptors. A recently developed non-xanthine A_2A receptor antagonist SCH 58261 displaced the binding of [³H]-CGS 21680 from the A_2A receptors in striatum with an estimated K_i value of 2.4 nM, but was more than 1000-fold less potent in displacing its binding from cortex. Conversely, the adenosine analogue 2-chloro-NECA was found to be some 10 times more potent in displacing CGS 21680 from the cortical binding sites than from A_2A receptors. The results provide additional evidence that CGS 21680 binds not only to classical A_2A receptors, but also to sites that differ from defined adenosine receptors. They also suggest that effects of CGS 21680 observed in the presence of SCH 58261 might reveal the functional significance (if any) of these sites.     

A2A adenosine receptors from rat striatum and rat pheochromocytoma PC12 cells: characterization with radioligand binding and by activation of adenylate cyclase.

Binding assays and assays of activation of adenylate cyclase with the agonists 5'-N-ethylcarboxyamidoadenosine (NECA) and CGS21680 have been used to compare adenosine receptors in rat pheochromocytoma PC12 cells and in rat striatum. The [3H]NECA binding showed two components, whereas [3H]CGS21680 bound to one component in both tissues. The Kd value for the high affinity site labeled with [3H]NECA in PC12 cell membranes (2.3 nM) was lower than that in striatum (6.5 nM). The [3H]CGS21680 binding site showed a Kd value of 6.7 nM and 11.3 nM in PC12 cells and striatum, respectively. In the presence of GTP the KD values of [3H]NECA and [3H]CGS21680 for the high affinity site were increased severalfold, whereas the low affinity sites for [3H]NECA were no longer detected with filtration assays. A comparison of the ability of a series of agonists and antagonists to inhibit high affinity binding of [3H]NECA to A2 receptors in PC12 cell and striatal membranes indicated that agonists had higher affinities and antagonists had lower affinities in PC12 cells, compared with affinities in striatal membranes. Analysis of activation of adenylate cyclase in PC12 cell membranes suggested that the dose-dependent stimulation by NECA involved two components, whereas CGS21680 stimulated via one component. The maximal stimulation by NECA significantly exceeded that caused by CGS21680. In intact PC12 cells, NECA caused a greater accumulation of AMP than did CGS21680, as was the case in membranes. In striatal membranes, NECA and CGS21680 showed similar maximal stimulations of adenylate cyclase. Both NECA and CGS21680 were more potent in PC12 cell membranes than in striatal membranes, in agreement with binding data. However, in contrast to binding data, antagonists were not less potent versus stimulation of adenylate cyclase by NECA or CGS21680 in PC12 cell membranes, compared with striatal membranes. In toto, the results suggest that A2A receptors in striatum are virtually identical to the A2A receptors in PC12 cells. But, in addition to an A2A receptor, it appears that a lower affinity functional receptor, probably an A2B receptor, is present in PC12 cells and PC12 cell membranes, whereas such a functional low affinity receptor is not detectable in striatal membrane.     

Further characterization of [3H]-CGS 21680 binding sites in the rat striatum and cortex.

1. The putative high affinity binding site for the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5'-N- ethylcarboxamidoadenosine (CGS 21680) in the rat cerebral cortex was characterized by use of a number of selective A1 and A2 adenosine receptor ligands, and compared to the characteristics of the more abundant striatal A2A receptor. 2. The binding of [3H]-CGS 21680 to cortical membranes was performed at pH 5.5, in order to increase the amount of specific binding. 3. Reduction of the pH from 7.4 to 5.5 increased the apparent affinity of the striatal binding side for both agonists and antagonists. The relative order of potencies of both groups of ligands were the same at both pH values, and were consistent with binding to the A2A receptor. There was no observable change in the Bmax, the values being 415 and 446 fmol mg-1 protein at pH 5.5 and 7.4 respectively. 4. The cortical binding site yielded a Bmax value of 117 fmol mg-1 protein. The relative order of potencies of the adenosine receptor ligands observed at this binding site were not the same as those observed in the striatum, exhibiting a profile with both A1 and A2 characteristics. 5. Further characterization of this cortical binding site in the presence of the A1 selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) revealed a more typical A2A profile. This indicated that under the conditions used there were two components of [3H]-CGS 21680 binding, approximately 20% of the A1 receptor and 80% to the A2A receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

(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.     

Binding of the prototypical adenosine A(2A) receptor agonist CGS 21680 to the cerebral cortex of adenosine A(1) and A(2A) receptor knockout mice

1. 2-p-(2-carboxyethylphenethylamino-5'-ethylcarboxamidoadenosine) (CGS 21680) is considered the reference compound to study adenosine A(2A) receptors. However, CGS 21680 binding in the cerebral cortex, where adenosine A(1) receptors are predominant, displays a mixed A(2A)/A(1) receptor pharmacology. We now use adenosine A(1) and A(2A) receptor knockout mice to investigate the characteristics of cortical [(3)H]CGS 21680 binding. 2. [(3)H]CGS 21680 binding to the cerebral cortex was strongly reduced in adenosine A(1) receptor knockout mice, but only slightly reduced in A(2A) receptor knockout mice compared with the corresponding wild-type littermates. 3. Another selective A(2A) receptor ligand, [(3)H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([(3)H]SCH 58261), displayed a saturable binding to mouse cortical membranes, albeit with a binding density 20 times lower than that of striatal membranes, and this [(3)H]SCH58261 binding was abolished in both striatal and cortical membranes of A(2A) receptor knockout mice and unchanged in A(1) receptor knockout mice. 4. The presence of A(2A) receptors in cortical neurons was further confirmed by Western blot in mouse cortical nerve terminal membranes. 5. It is concluded that, although A(2A) receptors are present in the cerebral cortex, the purportedly selective A(2A) receptor agonist [(3)H]CGS 21680 binds in the cerebral cortex to an entity that requires the presence of adenosine A(1) receptors. Thus, CGS 21680 should be used with care in all preparations where adenosine A(1) receptors out-number A(2A) receptors.     

Autoradiographic evidence for G-protein coupled A2-receptors in rat neostriatum using [3H]-CGS 21680 as a ligand

Summary Recently [³H]-CGS 21680 (2-[p-(2-carbonylethyl)-phenylethylamino]-5-N-ethylcarboxamidoadeno-sine) has been identified as a selective adenosine A₂-receptor agonist. In this study the binding of [³H]-CGS 21680 to 10 m sections of rat neostriatum was investigated with quantitative autoradiography. Specific, saturable binding was detectable, and Scatchard analysis of saturation experiments gave estimates for K _D and B _max of 1.7 nM and 322 fmol/mg protein, respectively. The rank order of potency for inhibition of [³H]-CGS 21680 binding was 5-N-ethylcarboxamidoadenosine (1.9 nM) > 2-chloroadenosine (18 nM) > R-N⁶-phenylisoprop-yladenosine (59 nM) > S-N⁶-phenylisoprophyladeno sine (460 nM) > 1,3-dipropyl-8-cyclopentylxanthine (700 nM). The binding of [³H]-CGS 21680 was sensitive to GTP, since 1 M GTP reduced binding to 4.7% of control. These data support the identity of CGS 21680 as an agonist at high affinity adenosine A₂-receptors and indicate these receptors in rat striatum are coupled to guanine nucleotide binding proteins. Send offprint requests to F. E. Parkinson at the above address     

Chemical modification and irreversible inhibition of striatal A2a adenosine receptors.

The ligand recognition site of A2a-adenosine receptors in rabbit striatal membranes was probed using non-site-directed labeling reagents and specific affinity labels. Exposure of membranes to diethylpyrocarbonate at a concentration of 2.5 mM, followed by washing, was found to inhibit the binding of [3H]CGS 21680 and [3H]xanthine amine congener to A2a receptors, by 86 and 30%, respectively. Protection from diethylpyrocarbonate inactivation by an adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine, and an antagonist, theophylline, suggested the presence of two histidyl residues on the receptor, one associated with agonist binding and the other with antagonist binding. Binding of [3H]CGS 21680 or [3H]xanthine amine congener was partially restored after incubation with 250 mM hydroxylamine, further supporting histidine as the modification site. Preincubation with disulfide-reactive reagents, dithiothreitol or sodium dithionite, at greater than 5 mM inhibited radioligand binding, indicating the presence of essential disulfide bridges in A2a receptors, whereas the concentration of mercaptoethanol required to inhibit binding was greater than 50 mM. A number of isothiocyanate-bearing affinity labels derived from the A2a-selective agonist 2-[(2-aminoethylamino) carbonylethylphenylethylamino]-5'-N- ethylcarboxamidoadenosine (APEC) were synthesized and found to inhibit A2a receptor binding in rabbit and bovine striatal membranes. Binding to rabbit A1 receptors was not inhibited. Preincubation with the affinity label 4-isothiocyanatophenylaminothiocarbonyl-APEC (100 nM) diminished the Bmax for [3H]CGS 21680 binding by 71%, and the Kd was unaffected, suggesting a direct modification of the ligand binding site. Reversal of 4-isothiocyanatophenylaminothiocarbonyl-APEC inhibition of [3H]CGS 21680 binding with hydroxylamine suggested that the site of modification by the isothiocyanate is a cysteine residue. A bromoacetyl derivative of APEC was ineffective as an affinity label at submicromolar concentrations.     

Binding of the radioligand [3H]-SCH 58261, a new non-xanthine A2A adenosine receptor antagonist,to rat striatal membranes.

1. The present study describes the binding to rat striatal A2A adenosine receptors of the new potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazol o [1,5-c] pyrimidine, [3H]-SCH 58261. 2. [3H]-SCH 58261 specific binding to rat striatal membranes ( > 90%) was saturable, reversible and dependent upon protein concentration. Saturation experiments revealed that [3H]-SCH 58261 labelled a single class of recognition sites with high affinity (Kd = 0.70 nM) and limited capacity (apparent Bmax = 971 fmol mg-1 of protein). The presence of 100 microM GTP in the incubation mixture did not modify [3H]-SCH 58261 binding parameters. 3. Competition experiments showed that [3H]-SCH 58261 binding is consistent with the labelling of A2A striatal receptors. Adenosine receptor agonists competed with the binding of 0.2 nM [3H]-SCH 58261 with the following order of potency: 2-hexynyl-5''-N-ethyl carboxamidoadenosine (2HE-NECA) > 5''-N-ethylcarboxamidoadenosine (NECA) > 2-[4-(2-carboxyethyl)-phenethylamino]-5''-N-ethylcarboxamidoadenosi ne (CGS 21680) > 2-phenylaminoadenosine (CV 1808) > R-N6-phenylisopropyladenosine (R-PIA) > N6-cyclohexyladenosine (CHA) = 2-chloro-N6-cyclopentyladenosine (CCPA) > S-N6-phenylisopropyladenosine (S-PIA). 4. Adenosine antagonists inhibited [3H]-SCH 58261 binding with the following order: 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo[1,5-c] quinazoline (CGS 15943) > 5-amino-8-(4-fluorobenzyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine (8FB-PTP) = SCH 58261 > xanthine amine congener (XAC) = (E,18%-Z,82%)7-methyl-8-(3,4-dimethoxystyryl)-1,3-dipropylxanthine (KF 17837S) > 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > or = 8-phenyltheophylline (8-PT). 5. The Ki values for adenosine antagonists were similar to those labelled with the A2A agonist [3H]-CGS 21680. Affinities of agonists were generally lower. The A1-selective agonist, R-PIA, was found to be about 9 fold more potent than its stereoisomer, S-PIA, thus showing the stereoselectivity of [3H]-SCH 58261 binding. Except for 8-PT, the adenosine agonists and antagonists examined inhibited [3H]-SCH 58261 binding with Hill coefficients not significantly different from unity. 6. The present results indicate that [3H]-SCH 58261 is the first non-xanthine adenosine antagonist radioligand which directly labels A2A striatal receptors. High receptor affinity, good selectivity and very low non-specific binding make [3H]-SCH 58261 an excellent probe for studying the A2A adenosine receptor subtype in mammalian brain.     

Effects of adenosine A(2A) receptor stimulation in vivo on dopamine D3 receptor agonist binding in the rat brain

To investigate if adenosine A2A receptor stimulation in vivo modulates dopamine D3 receptor binding, we analyzed the effects of 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxyamidoade nosine (CGS 21680) on the binding properties of the selective D3 receptor agonist [N-propyl-2,3,-3H]4aR,10bR-(+)-trans-3,4,4a,10b-tetrahydro-4 -n-propyl2H,5H-[1]benzopyrano[4,3-b]1,4-oxazin-9-ol ([3H]PD 128907) in the rat forebrain using quantitative autoradiography. Intraperitoneally administered CGS 21680 (0.1-3 mg/kg) increased the Kd and Bmax values of [3H]PD 128907 binding in the islands of Calleja and in subregions of the caudate-putamen. These results suggest that stimulation of adenosine A2A receptors in vivo causes alterations in the binding characteristics of dopamine D3 receptors in the basal ganglia, and that this effect may relate to the neuroleptic-like effect of adenosine A2A receptor agonists.     

Human breast cancer cell line MDA-MB-231 expresses endogenous A2B adenosine receptors mediating a Ca2+ signal

1 Two human breast cancer cell lines, MCF-7 and MDA-MB-231, were screened for the presence of functionally significant adenosine receptor subtypes. 2 MCF-7 cells did not contain adenosine receptors as judged by the lack of an effect of nonselective agonists on adenylyl cyclase activity or intracellular Ca(2+) levels. MDA-MB-231 cells showed both a stimulation of adenylyl cyclase and a PLC-dependent increase in intracellular Ca(2+) in response to nonselective adenosine receptor agonists. 3 Both adenosine-mediated responses in MDA-MB-231 cells were observed with the nonselective agonists 5'-N-ethylcarboxamidoadenosine (NECA) and 2-(3-hydroxy-3-phenyl)propyn-1-yladenosine-5'-N-ethyluronamide (PHPNECA), but no responses were observed with agonists selective for A(1), A(2A) or A(3) adenosine receptors. The Ca(2+) signal was antagonized by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and the nonselective antagonist 9-ethyl-8-furyladenine (ANR 152), but not by A(2A) or A(3) selective compounds. 4 In radioligand binding with [2-(3)H](4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol) ([(3)H]ZM 241385), a specific binding site with a K(D) value of 87 nM and a B(max) value of 1600 fmol mg(-1) membrane protein was identified in membranes from MDA-MB-231 cells. 5 The pharmacological characteristics provide evidence for the expression of an A(2B) adenosine receptor in MDA-MB-231 cells, which not only mediates a stimulation of adenylyl cyclase but also couples to a PLC-dependent Ca(2+) signal, most likely via G(q/11). The A(2B) receptor in such cancer cells may serve as a target to control cell growth and proliferation. 6 The selective expression of high levels of endogenous A(2B) receptors coupled to two signaling pathways make MDA-MB-231 cells a suitable model for this human adenosine receptor subtype.     

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.     

Effects of N-ethylmaleimide on adenosine receptors of rat fat cells and human platelets

N-Ethylmaleimide (NEM) differentially modified Ri adenosine receptors in rat fat cells and Ra adenosine receptors in human platelets. Pretreatment of rat fat cell membranes with NEM inhibited the binding of the agonist (-)N6-phenylisopropyl[3H]adenosine [( 3H]PIA), but did not affect the binding of the antagonist 1,3-diethyl-8-[3H]phenylxanthine [( 3H]DPX). The IC50-value for inhibition of [3H]PIA binding was 0.067 mM. Saturation of [3H]PIA binding revealed that NEM converts the high affinity form of the Ri receptor into a low affinity form. NEM also decreased the potency of agonists to displace [3H]DPX binding, as shown by a 74-fold shift of the Ki-value for (-)PIA, whereas antagonist-induced displacement remained unchanged. In addition, low concentrations of NEM (0.01-0.1 mM) attenuated the (-)PIA-induced inhibition of adenylate cyclase activity of rat fat cells. At higher concentrations (0.1-1 mM) NEM reduced basal and stimulated adenylate cyclase activities in rat fat cells and human platelets, presumably by inactivation of the catalytic unit. Radioligand binding of 5'-N-ethylcarboxamido[3H]-adenosine [( 3H]NECA) to Ra adenosine receptors of human platelet membranes was not changed by NEM at low radioligand concentrations. Saturation analysis of [3H]-NECA binding showed that NEM led to an apparent increase of agonist affinity with a concomitant decrease in total [3H]NECA binding sites. These results suggest that NEM reduces the affinity of Ri adenosine receptors, probably by affecting the inhibitory guanine nucleotide binding protein (Ni), whereas [3H]NECA binding sites are inversely affected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guanine nucleoside diphosphate and triphosphate modulate [3H]CGS 21680 binding to A2 adenosine receptor in rat striatal membranes

In striatum and several other tissue, a guanine nucleotide binding protein (G_s) couples A₂ adenosine receptor to activation of adenylyl cyclase. We have examined the effect of guanine nucleoside diphosphate and triphosphate on [³H]CGS 21680 binding to A_2A adenosine receptors of rat striatum. Both GDP and GTP inhibited specific [³H]CGS 21680 binding to rat striatal membranes by 50% at about 0.1 mM. GMP was inhibitory only at higher concentrations, and the estimated IC₅₀ value was greater than 1mM. The nonhydrolyzable analog of GTP, GPP (NH)p, was as potent as GTP with an IC₅₀ value of approximately 86 μM. These results suggest that the regulation of A_2a adenosine receptor binding properties by guanine nucleotides is independent of G_s activation, since inhibition of agonist binding is achieved by addition of agonist binding is achieved by addition of both guanine nucleoside diphosphate and triphosphate © 1993 Wiley-Liss, Inc.     

Effects of propentofylline on adenosine A1 and A2 receptors and nitrobenzylthioinosine-sensitive nucleoside transporters: quantitative autoradiographic analysis.

Previous studies have demonstrated that the xanthine compound, propentofylline, has beneficial effects in models of cerebral ischemia and can enhance some and exhibit other effects of adenosine. We investigated the in vitro effects of propentofylline and its hydroxy metabolite, A72,0287, on the binding of [3H]cyclohexyladenosine ([3H]CHA), [3H]2-[p-(2-carbonyl-ethyl)-phenylethyl-amino]-5'-N- ethylcarboxamido adenosine ([3H]CGS 21680) and [3H]nitrobenzylthioinosine ([3H]NBMPR) to adenosine A1 and A2 receptors and NBMPR-sensitive nucleoside transporters, respectively, in 10-microns coronal rat brain sections. Both xanthines had micromolar affinity for each of these sites with approximately 10-fold lower affinity for A2 receptors than for A1 receptors and [3H]NBMPR binding sites. Saturation analysis of [3H]CHA or [3H]CGS 21680 binding in the presence of increasing concentrations of propentofylline produced significant increases in KD values without affecting Bmax values; thus propentofylline is a competitive inhibitor at A1 and A2 receptors. The effects on A2 receptors apparently require higher concentrations (Ki approximately 200 microM) than the effects on A1 receptors (Ki approximately 20 microM). Propentofylline was also found to be a competitive inhibitor of [3H]NBMPR binding. Therefore we conclude that propentofylline interacts with adenosine-responsive systems to increase interstitial adenosine concentrations and to selectively inhibit A1 receptors.     

Direct autoradiographic localization of adenosine A2 receptors in the rat brain using the A2-selective agonist, [3H]CGS 21680

The regional distribution of adenosine A2 receptors in the rat brain was determined using the A2-selective agonist ligand [3H](2-p-carboxyethyl)phenylamino)-5'-N-carboxamidoadenosine (CGS 21680) by quantitative receptor autoradiography. [3H]CGS 21680 binding was highly localized in the striatal region of the rat brain with the greatest density of binding found in the caudate-putamen, nucleus accumbens and olfactory tubercle. Additionally, lower levels of binding were also found in the globus pallidus. No significant amounts of [3H]CGS 21,680 binding were detected in other brain regions. This localization of brain adenosine A2 receptors was markedly different from the known regional distribution of A1 receptors which are highly concentrated in cerebellum, hippocampus, thalamus and cortex. The present results provide further evidence for a specific contribution of adenosine in the modulation of central neurotransmission.     

Experimental hypothyroidism modifies specific binding of A1 and A2A analogues to adenosine receptors in the rat kidney

1 Binding kinetic studies with the adenosine analogues [3H]CPA (0.250-50 nm) and [3H]CGS21680 (0.1-100 nm) were performed in renal tissue from control (NL) and thyroidectomised (HTX) rats. We propose that the low renal adenosine content reported in hypothyroid rats may induce changes in the density and/or affinity of adenosine receptor, distributed in the cortex (C), outer medulla (OM), and inner medulla (IM) of the kidney. 2 [3H]CPA and [3H]CGS21680 binding saturation isotherms were fitted by nonlinear regression analysis and evaluated by Furchgott's method. These results revealed high (KH) and low (KL) affinity (KD) sites for both compounds. As expected, a heterogeneous pattern was observed for Bmax and KD values. 3 Bound [3H]CPA and [3H]CGS21680 were displaced by increasing concentrations of nonlabelled DPCPX and NECA, respectively, indicating the presence of A1 and A2A adenosine receptors distributed in the renal segments studied. 4 The relative intrinsic efficacy (epsilon) for [3H]CPA and [3H]CGS21680 showed extreme values (far from 1.0), 0.5 in IM NL and 2.70 in IM HTX for [3H]CGS21680. 5 Our results indicate that A2A adenosine receptor is predominant in IM from HTX, but A1 receptors are expressed preferentially in C in NL. 6 We conclude that the changes observed in number, affinity, and epsilon for the A2A receptor in IM from HTX might be responsible from alterations in medullary function, that is, incapacity for urine concentration as observed in the hypothyroid kidney.     

Binding of adenosine receptor ligands to brain of adenosine receptor knock-out mice: evidence that CGS 21680 binds to A1 receptors in hippocampus

The adenosine receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-5-N-ethylcarboxamidoadenosine (CGS 21680) is generally considered to be a selective adenosine A_2A receptor ligand. However, the compound has previously been shown to exhibit binding characteristics that are not compatible with adenosine A_2A receptor binding, at least in brain regions other than the striatum. We have examined binding of [³H]CGS 21680 and of antagonist radioligands with high selectivity for adenosine A₁ or A_2A receptors to hippocampus and striatum of mice lacking either adenosine A₁ (A1R^(–/–)) or A_2A (A2AR^(–/–)) receptors. Both receptor autoradiography and membrane binding techniques were used for this purpose and gave similar results. There were no significant changes in the binding of the A₁ receptor antagonist [³H]DPCPX in mice lacking A_2A receptors, or in the binding of the A_2A receptor antagonists [³H]SCH 58261 and [³H]ZM 241385 in mice lacking A₁ receptors. Furthermore, [³H]CGS 21680 binding in striatum was abolished in the A2AR^(–/–), and essentially unaffected in striatum from mice lacking A₁ receptors. In hippocampus, however, binding of [³H]CGS 21680 remained in the A2AR^(–/–), whereas binding was virtually abolished in the A1R^(–/–). There were no adaptive alterations in A_2A receptor expression in this region in A1R^(–/–) mice. Thus, most of the [³H]CGS 21680 binding in hippocampus is dependent on the presence of adenosine A₁ receptors, but not on A_2A receptors, indicating a novel binding site or novel binding mode.