Fig. 7.

Concentration–response curves for slowing the dissociation of the agonist [¹²⁵I]I-AB-MECA (A) and for increasing the dissociation of the antagonist [³H]PSB-11 (B) from human A₃ receptors by the amiloride analogues. [¹²⁵I]I-AB-MECA (1.0 nM) was pre-associated with CHO cell membranes (20 μg protein) for 60 min at 25° without additions (total binding) or in the presence of 3 μM Cl-IB-MECA (nonspecific binding). At the end of the incubation period, 3 μM Cl-IB-MECA was added simultaneously with vehicle or various concentrations of the test compounds. The incubation was terminated after an additional 45 min. Control specific binding at the end of the 60-min dissociation period was approximately 25% of the total binding. Data are mean values from three independent experiments performed in duplicate. The procedure for the experiments regarding [³H]PSB-11 was similar to that of [¹²⁵I]I-AB-MECA except that it was performed at 4°. MgCl₂ was present in both experiments. One hundred percent refers to the specific binding remaining after 45-min dissociation in the absence of added amiloride or amiloride analogues.