Functional consequences of the A3AR specific agonist IB-MECA on WT-A3AR and Leu90Val-A3AR or Val171Ile-A3AR expressing cells. CHO cells co-transfected with either WT-A3AR or Leu90Val-A3AR or Val171Ile-A3AR and SERT and were stimulated using IB-MECA (1 μM). (A) Levels of cGMP production from WT-A3AR/SERT and Leu90Val-A3AR/SERT co-transfections were measured prior to and after IB-MECA stimulation. Leu90Val-A3AR/SERT cells display elevated basal cGMP production that persists over the entire 60 min time course experiment, revealing enhanced overall production of cGMP (1 tailed t-test P=0.049; n=3). Each of the three independent experiments contained a minimum of three internal replicates. (B) 60 min time-course of IB-MECA induced 5-HT uptake as measured as percent of control (wildtype; time = 0 min). CHO cells were co-transfected with either wildtype-A3AR and SERT or Leu90Val-A3AR and SERT, and 5-HT uptake was stimulated using IB-MECA (1 μM). Cells were incubated with [3H]5-HT (20 nM) for the indicated period. Over the time course (0-60 min) in Leu90Val-A3AR/SERT expressing cells, 5-HT uptake is elevated compared with the wildtype A3AR counterpart (1 tailed t-test, P=0.039; n=4). Each independent experiment contained a minimum of three internal replicates. (C) [3H]5-HT accumulation in cells co-transfected with A3AR and human SERT was measured, and indicate a failure of IB-MECA treatment of Val171Ile-A3AR/SERT to induce SERT dependent 5-HT re-uptake across all tested concentrations. Compared to the peak concentrations (0.8 μM and 1.0 μM) of IB-MECA induced 5-HT uptake in WT-A3AR/SERT, IBMECA induced 5-HT uptake was significantly reduced (for example, 1 μM IB-MECA Val171Ile: 101.8% ± 12.5 vs. WT: 136.3% ± 7.2; two-way ANOVA P=0.005; n=3-4). Significant (P <0.05) findings are indicated by an asterisk (*).