Agonist-induced activation and desensitization of P2Y₁₂ purinergic receptors in 1321N1 cells. (A) cAMP accumulation (10 minutes) in the absence (basal; □) or presence of 1 μM forskolin (forskolin; ▨) ± ADP (10 nM; forsk/ADP; ▪) was assessed under 5 different conditions: (1) in the absence of pretreatment (untreated), (2) after pretreatment with the P2Y₁₂ receptor antagonist AR-C69931MX (1 μM; 5 minutes), (3) after pretreatment with the P2Y receptor agonist ADPβS (1 mM; 5 minutes), (4) after pretreatment with apyrase (0.2 U/mL; 180 seconds; + apyrase), and (5) after treatment with ADP (10 nM; 5 minutes) and subsequent treatment with apyrase (+ ADP/apyrase). Data represent mean ± SEM of 4 independent experiments and are expressed as pmol cAMP per well. (B) Concentration-dependent inhibition of forskolin (1 μM; 10 minutes)–stimulated adenylyl cyclase activity by P2Y₁₂ purinergic receptor activation after pretreatment with ADP (10 nM; 5 minutes) (○) or vehicle alone (⬡). Values represent mean ± SEM of 4 independent experiments. Data are expressed as a percentage of reduction in maximal forskolin response. ADP pretreatment induced a significant (P < .05; 2-way analysis of variance [ANOVA]) reduction in P2Y₁₂ purinergic receptor activity with a 30-fold shift in the dose-response curve (ADP-induced inhibition of forskolin-stimulated adenylyl cyclase had an EC₅₀ of 2.5 ± 0.9 pM and 76.7 ± 4.2 pM before and after ADP pretreatment respectively). (C) Time dependence of desensitization of P2Y₁₂ purinergic receptor activity. ADP pretreatment induced a rapid and significant reduction in P2Y₁₂ purinergic receptor activity. Values represent mean ± SEM of 4 independent experiments. Data are expressed as a percentage of reduction in maximal forskolin response. (D) Cells stably transfected with P2Y₁₂ purinergic receptor construct were transiently transfected with either pcDNA3-^K220RGRK2 (GRK2-DNM), pcDNA3-K215RGRK6 (GRK6-DNM), vector alone (pcDNA3), or scrambled, GRK2-, or GRK6-specific siRNA constructs. Cells were incubated in the absence or presence of the PKC inhibitor GF109203X (GF; 2 μM) for 15 minutes before experimentation. Agonist (ADP; 10 nM)–dependent inhibition of forskolin (1 μM; 10 minutes)–stimulated adenylyl cyclase activity by P2Y₁₂ purinergic receptor activation after pretreatment with ADP (10 nM; 5 minutes, ▪) or vehicle alone (□) was subsequently determined. Values represent the mean ± SEM of 4 independent experiments and are expressed as a percentage of inhibition of forskolin-stimulated adenylyl cyclase. ^*Statistical significance at P < .05 for data compared with respective nonpretreated agonist-induced inhibition of forskolin-stimulated controls (Mann-Whitney U test).