Figure 4. Pharmacological Characterization of Two Neoagonists Indicates Selective Interaction with Neoceptors Derived from the A_2AAR.

Binding (A and B) and functional (C and D) effects of two adenosine derivatives, the hydrazide derivative 7 (A) and the N⁶-(2-methyl-benzyl)-5′-aminoethyluronamide derivative 14 (B) at WT (■) and mutant A_2AARs (T88D [▲], N181D [◆], and Q89D [▼]) transiently expressed in COS-7 cells. In the binding experiments, cell membranes (10–20 μg protein) were incubated with the radiolabeled antagonist [³H]ZM241385 (2.0 nM) in duplicate, together with increasing concentrations of the competing nucleoside, in a final volume of 0.4 ml Tris-HCl buffer (50 mM, pH 7.4) at 25°C for 120 min. Results were from a representative experiment performed in duplicate. The K_i values listed in Table 1 were from at least three separate experiments. In the functional experiments, cells expressing WT or mutant receptors were then treated with agonist 7 (C) or 14 (D) in the presence of rolipram (10 μM) and adenosine deaminase (3 units/ml) and incubated at 37°C for 1 hr. cAMP accumulation was determined using a competitive protein binding method [29]. The EC₅₀ values (n = 3) determined for stimulation of cAMP formation were (nM, mean ± SEM): WT, 826 ± 138; T88D, 2970 ± 980; Q89D, 5.1 ± 0.8; N181D, 120 ± 22 for 7; and WT, 5800 ± 1230; T88D, 12,600 ± 3200; Q89D, 58 ± 12; N181D, 52 ± 6 for 14.