The C6-2B glioma cell P2Y(AC) receptor is pharmacologically and molecularly identical to the platelet P2Y(12) receptor

Abstract

P2Y receptor activation in many cell types leads to phospholipase C activation and accumulation of inositol phosphates, while in blood platelets, C6-2B glioma cells, and in B10 microvascular endothelial cells a P2Y receptor subtype, which couples to inhibition of adenylyl cyclase, historically termed P2Y(AC), (P2T(AC) or P(2T) in platelets) has been identified. Recently, this receptor has been cloned and designated P2Y(12) in keeping with current P2 receptor nomenclature. Three selective P(2T) receptor antagonists, with a range of affinities, inhibited ADP-induced aggregation of washed human or rat platelets, in a concentration-dependent manner, with a rank order of antagonist potency (pIC(50), human: rat) of AR-C78511 (8.5 : 9.1)>AR-C69581 (6.2 : 6.0)>AR-C70300 (5.4 : 5.1). However, these compounds had no effect on ADP-induced platelet shape change. All three antagonists had no significant effect on the ADP-induced inositol phosphate formation in 1321N1 astrocytoma cells stably expressing the P2Y(1) receptor, when used at concentrations that inhibit platelet aggregation. These antagonists also blocked ADP-induced inhibition of adenylyl cyclase in rat platelets and C6-2B cells with identical rank orders of potency and overlapping concentration - response curves. RT - PCR and nucleotide sequence analyses revealed that the C6-2B cells express the P2Y(12) mRNA. These data demonstrate that the P2Y(AC) receptor in C6-2B cells is pharmacologically identical to the P2T(AC) receptor in rat platelets.

Figure 1

Chemical structures of the P_2T receptor antagonists used in the present study.

Figure 2

Effect of three AR-C compounds on ADP-induced (A) human and (B) rat platelet aggregation. Platelet aggregation studies were performed as described in the Methods in the presence or absence of different concentrations of AR-C78511, AR-C69581 or AR-C70300 (as marked) with varying concentrations of ADP.

Figure 3

Effect of P_2T receptor antagonists on ADP-induced aggregation of rat platelets. Rat blood was collected by cardiac puncture. Aspirin-treated washed platelets were stimulated with ADP in the presence or absence of AR-C78511, AR-C69581 or AR-70300 in a Chronolog lumi-aggregometer with stirring at 37°C. The additions are indicated by arrows.

Figure 4

Effect of A3P5PS on ADP-induced inositol phosphate formation in astrocytoma cells stably expressing the human P2Y₁ receptor. Effect of A3P5PS on ADP-induced inositol phosphate formation was determined at various ADP concentrations in the presence and absence of a single dose of 10 μM A3P5PS. The data are normalized to response by maximum concentration of ADP in the absence of the antagonist (taken as 100%).

Figure 5

Effect of P2Y₁ and P_2T receptor selective antagonists on ADP-induced inositol phosphate formation in astrocytoma cells stably expressing the human P2Y₁ receptor. Human P2Y₁ receptor was stably expressed in 1321N1 astrocytoma cells and ADP-induced total inositol phosphate formation was measured as described in the methods section. The measurements were made in confluent monolayers of cells in 24-well plates in triplicate. The antagonists, AR-C 78511 (1 μM), AR-C 69581 (1 μM), AR-C 70300 (300 μM), or A3P5P (1 mM), were added to the cells immediately before the addition of 10 μM ADP and incubated for 10 min at 37°C. The results were normalized to total inositol phosphate formation with 10 μM ADP (taken as 100%).

Figure 6

Effect of P_2T receptor antagonists on ADP-induced inhibition of adenylyl cyclase in (A) rat platelets and (B) C6-2B cells. Effect of varying concentrations of AR-C78511, AR-C69581 or AR-C70300 (as marked) on ADP (10 μM)-induced inhibition of forskolin (100 μM)-stimulated adenylyl cyclase activity in rat platelets and C6-2B cells was determined as described in the Methods section. The data are normalized to the maximum response obtained with each antagonist (taken as 100%).

Figure 7

RT–PCR analysis of RNA from C6-2B cells: PCR product electrophoresed on a 1.0% agarose gel and stained with ethidium bromide. PCR was carried out with C6-2B cell total RNA as described in the methods section. A: φX174 DNA/HaeIII marker; B: C6-2B cell RNA–PCR (control for Genomic DNA contamination); C: C6-2B cell RT–PCR.