Fig. 6.
Recovery of A2aAR function after long term desensitization. CHO-A2aAR cells were either exposed for 24 hr at 37° to 0.3 unit/ml ADA in the absence or presence of 10 μM NECA or exposed for 24 hr to 10 μM NECA and 0.3 unit/ml ADA before extensive washing with PBS, addition of agonist-free medium containing 0.3 unit/ml ADA, and further incubation at 37° for the indicated times. Membranes were then prepared simultaneously for assay of adenylyl cyclase activity, as described in Experimental Procedures. A, Time course of recovery of the 10 μM NECA-stimulated adenylyl cyclase response in isolated CHO-A2aAR cell membranes after 24-hr desensitization. Data are pooled from four experiments, with each data point representing the mean ± standard error of three determinations. B, Dose-response curves for NECA-stimulated adenylyl cyclase activity in membranes from ADA-treated controls (○), 24-hr desensitized cells (●), or 24-hr desensitized cells allowed to recover in agonist-free medium for 16 hr (□). The EC50 values for NECA in this experiment were 0.28 μM (control), 0.56 μM (desensitized), and 0.33 μM (resensitized). This experiment is one of two performed, which produced identical results. C, Resensitization detected by immunoblotting. CHO-A2aAR cells were treated as described in B and subjected to immunoblotting with affinity-purified TP/2 as described in Experimental Procedures. In this experiment, agonist treatment down-regulated receptors to 28% of the control level. Subsequent recovery in the absence of agonist increased receptor levels to 110% of the control value. This is one of three immunoblots performed.