title: 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.
authors.affiliation: Laboratory of Bioorganic Chemistry, National Inst. of Diabetes and Digestive Diseases and Kidney Diseases, NIH, Bethesda, MD 20892.
authors.forename: X D
authors.initials: XD
authors.lastname: Ji
authors.forename: G L
authors.initials: GL
authors.lastname: Stiles
authors.forename: P J
authors.initials: PJ
authors.lastname: van Galen
authors.forename: K A
authors.initials: KA
authors.lastname: Jacobson
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D000345:Affinity Labels; D000368:Aged; D003342:Corpus Striatum / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D008297:Male; D010627:Phenethylamines / Q000378:metabolism; D011869:Radioligand Assay; D011983:Receptors, Purinergic / Q000032:analysis*; D014970:Xanthines / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D010627:Phenethylamines; D011983:Receptors, Purinergic; D014970:Xanthines; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D006160:Guanosine Triphosphate; C051869:8-(4-((2-aminoethyl)aminocarbonylmethyloxy)phenyl)-1,3-dipropylxanthine; D000241:Adenosine