Figure 1.

Pharmacology of radioligand [³H]18 in A₃AR binding saturation, inhibition, and kinetic experiments. Specific binding saturation (A,C,E) and total (●) and nonspecific (■) binding (B,D,F) at three species homologues (human, mouse, rat) of A₃AR expressed in HEK293 cells using 100 μM 23 to define nonspecific binding. Data shown are representative experiments performed in duplicate; the K_d values from four independent experiments are listed in the text. Association (G, k₁ = 0.297 min^–1) and dissociation (I, k_–1 = 0.209 min^–1; t_1/2 = 3.32 min) kinetics of [³H]18 (1 nM) at the hA₃AR. (H,J) Binding inhibition at hA₃AR and comparison of [³H]18 (1 nM) with the agonist radioligand [¹²⁵I]3 (0.1 nM) as a tracer. Competing ligands were agonists (A₃-selective 1, nonselective 23) and hA₃-selective antagonists (N-[9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-c]quinazolin-5-yl]benzeneacetamide, MRS1220 24; 1,4-dihydro-2-methyl-6-phenyl-4-(phenylethynyl)-3,5-pyridinedicarboxylic acid 3-ethyl-5-[(3-nitrophenyl)methyl] ester, MRS1334 25).¹³K_i values (nM) for the inhibition of antagonist [³H]18 binding: 24, 3.24; 25, 6.49; 23, 44.1; 1, 0.96. K_i values (nM) for the inhibition of agonist [¹²⁵I]3 binding: 24, 2.97; 25, 10.6; 23, 19.6; 1, 0.37. Corresponding published K_i values for the inhibition of [¹²⁵I]3 binding: 24, 0.96 ± 0.32; 25, 4.58 ± 0.89; 23, 26; 1, 1.74 ± 0.36.^13,27,29