Generation of P2Y1R agonist-sensitive adenylyl cyclase inhibition of A1R. (A and B) Concentration-dependent reduction of maximal FSK (10 μM)-stimulated intracellular cAMP accumulation by CPA (A) or ADPβS (B) in A1R/P2Y1R-transfected cells. The attenuation was blocked by the PTX pretreatment (100 ng/ml, 16 h). Dotted line, cells expressing HA-A1R alone; solid line, cells coexpressing HA-A1R and Myc-P2Y1R; circles, nontreated cells; diamonds, PTX-pretreated cells. The 100% values of cAMP for the cells transfected with HA-A1R and HA-A1R plus Myc-P2Y1R were 72 ± 14 and 67 ± 19 pmol/105 cells, respectively (mean ± SEM, n = 5). Estimated IC50 values are shown in the text. (C) Pretreatment of cells with A1R antagonist DPCPX, but not P2Y1R antagonist MRS2179, significantly inhibited maximal ADPβS-induced adenylyl cyclase attenuation in the A1R/P2Y1R-transfected cells. (Upper) HA-A1R transfected cells. (Lower) HA-A1R/Myc-P2Y1R cotransfected cells. The 100% values of cAMP for the cells transfected with HA-A1R and HA-A1R/Myc-P2Y1R were 70 ± 12 and 71 ± 17 pmol/105 cells, respectively (mean ± SEM, n = 5). Data represent the means ± SEM of the percentage of FSK-induced cAMP accumulation values. Results from 3–5 independent experiments performed in duplicate are shown. ***, P < 0.01, Student's t test.