Agonist-induced desensitization of calcium responses and inositol phosphate accumulation in cells stably expressing P2Y₁ purinergic receptors. (A) IP accumulation was measured after the addition of ADP (1 nM-10 μM; 10 minutes) in cells stably expressing the P2Y₁ purinergic receptor (⬡) or vector (pcNEO) alone (○). Data are the mean ± SEM of 5 independent experiments, and results are expressed as fold IP accumulation over basal. ADP stimulated IP accumulation with an EC₅₀ of 14 ± 0.31 nM. (B) Cells either were not pretreated with ADP (control; ⬡) or were pretreated with ADP (1 μM; 5 [▿], 15 [○], or 30 minutes [▵]) and were incubated with apyrase (0.2 U/mL; 1 minute). Cells were then washed, and IP accumulation was measured after the addition of ADP (1 μM; 0-30 minutes). Data are mean ± SEM of 4 independent experiments, and results are expressed as fold IP accumulation over basal. (C) Cells stably transfected with P2Y₁ purinergic receptor or vector alone (pcNEO; ▴) were either not pretreated (Control; ⬡) or were pretreated with agonist (ADP; 0.1 μM[▿], 1 μM[○], or 10 μM[▵] as indicated; 2 minutes). Peak calcium responses were measured on the addition of ADP (1 nM-100 μM). Values represent the mean ± SEM of 4 independent experiments. Results are expressed as the difference between basal resting and maximal response (peak rise) in cytosolic calcium concentration ([Ca²⁺]_i nM), as assessed by Fura-2AM in intact cells. Prestimulation with ADP (0.1-1 μM) caused a shift in the dose-response curve for agonist-induced calcium mobilization (EC₅₀ of 59.7 ± 15.3, 100.3 ± 11.3, 300 ± 4.9, and 384 ± 18.7 nM for ADP-induced calcium mobilization before and after 0.1, 1, and 10 μM ADP pretreatment, respectively).