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Pharmacological and biochemical characterization of purified A 2a adenosine receptors in human platelet membranes by [ 3 H]‐CGS 21680 binding
Author(s) -
Varani Katia,
Gessi Stefania,
Dalpiaz Alessandro,
Borea Pier Andrea
Publication year - 1996
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1996.tb15341.x
Subject(s) - cgs 21680 , adenosine , adenylyl cyclase , agonist , chemistry , receptor , biochemistry , adenosine receptor , adenosine a2a receptor
1 The binding properties of human platelet A 2a adenosine receptors, assayed with the A 2a ‐selective agonist, [ 3 H]‐2‐[ p ‐(2‐carboxyethyl)‐phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine ([ 3 H]‐CGS 21680), are masked by a non‐receptorial component, the adenotin site. In order to separate A 2a receptors from adenotin sites, human platelet membranes were solubilized with 1% 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulphonate (CHAPS). The soluble platelet extract was precipitated with polyethylene glycol (PEG) and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. 2 The present paper describes the binding characteristics of the selective A 2a agonist, [ 3 H]‐CGS 21680, to this purified platelet membrane preparation. In addition, receptor affinity and potency of several adenosine agonists and antagonists were determined in binding and adenylyl cyclase studies. 3 Saturation experiments revealed a single class of binding site with K d and B max values of 285 nM and 2.07 pmol mg −1 of protein respectively. Adenosine receptor ligands competed for the binding of 50 nM [ 3 H]‐CGS 21680 to purified protein, showing a rank order of potency consistent with that typically found for interactions with the A 2a adenosine receptors. In the adenylyl cyclase assay the compounds examined exhibited a rank order of potency very close to that observed in binding experiments. 4 Thermodynamic data indicated that [ 3 H]‐CGS 21680 binding to the purified receptor is totally entropy‐driven in agreement with results obtained in rat striatal A 2a adenosine receptors. 5 It is concluded that in the purified platelet membranes there is a CGS 21680 binding site showing the characteristic properties of the A 2a receptor. This makes it possible to use this compound for reliable radioligand binding studies on the A 2a adenosine receptor of human platelets.