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Characterization of human striatal A2-adenosine receptors using radioligand binding and photoaffinity labeling.

ABSTRACT: The adenosine agonist [3H]CGS21680 (2-[4-[[2-carboxyethyl]phenyl]ethylamino]-5'- N-ethylcarboxamidoadenosine) bound to A2 receptors in human striatal membranes with a Kd of 17.8 +/- 1.1 nM and a Bmax of 313 +/- 10 fmol/mg protein. The addition of 100 microM GTP diminished both the affinity of agonist radioligand for A2 adenosine binding sites and the total binding, resulting in Kd and Bmax values of 28.6 +/- 1.0 nM and 185 +/- 22 fmol/mg of protein. Adenosine ligands competed for [3H]CGS21680 with the expected potency order. The adenosine antagonist [3H]XAC (8-[4-[[[[(2-aminoethyl)-amino]carbonyl]methyl] oxy]phenyl]-1,3-dipropylxanthine), although A1-selective in the rat, binds to human striatal A2 receptors with high affinity. 25 nM CPX (8-cyclopentyl-1,3-dipropylxanthine), an A1-selective antagonist, was added to the incubation medium and effectively eliminated 91% of [3H]XAC (1 nM) binding to human A1 receptors, yet preserved 90% of binding to A2 receptors. [3H]XAC exhibited saturable, specific binding (50% of total) to A2 sites with a Kd of 2.98 +/- 0.54 nM and a Bmax of 0.71 +/- 0.23 pmol/mg protein (25 degrees C, non-specific binding defined with 100 microM NECA). The potency order for antagonists against 1 nM [3H]XAC was CGS15943A greater than XAC approximately PD115,119 greater than PAPA-XAC greater than CPX greater than HTQZ approximately XCC approximately CP-66,713 greater than theophylline approximately caffeine, indicative of an A2-type binding site. A2a-receptors were found to be present in the human cortex, albeit at a much lower density than in the striatum. Photoaffinity labeling using 125I-PAPA-APEC revealed a molecular weight of 45K, but proteolytic cleavage was observed, resulting in fragments of MW 43K and 37K. In the absence of proteolytic inhibitors the 37K fragment, which still bound 125I-PAPA-APEC, was predominant.

SUBMITTER: Ji XD

PROVIDER: S-EPMC3429337 | biostudies-literature | 1992

REPOSITORIES: biostudies-literature

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Characterization of human striatal A2-adenosine receptors using radioligand binding and photoaffinity labeling.

Ji X D XD Stiles G L GL van Galen P J PJ Jacobson K A KA

Journal of receptor research 19920101 2

The adenosine agonist [3H]CGS21680 (2-[4-[[2-carboxyethyl]phenyl]ethylamino]-5'- N-ethylcarboxamidoadenosine) bound to A2 receptors in human striatal membranes with a Kd of 17.8 +/- 1.1 nM and a Bmax of 313 +/- 10 fmol/mg protein. The addition of 100 microM GTP diminished both the affinity of agonist radioligand for A2 adenosine binding sites and the total binding, resulting in Kd and Bmax values of 28.6 +/- 1.0 nM and 185 +/- 22 fmol/mg of protein. Adenosine ligands competed for [3H]CGS21680 wi ...[more]

PMID: 1583620

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Project description: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.

Dataset Information

Characterization of human striatal A2-adenosine receptors using radioligand binding and photoaffinity labeling.

Publications

Characterization of human striatal A2-adenosine receptors using radioligand binding and photoaffinity labeling.

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