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3420 N. Broad Street, Department of Physiology, Temple University Medical School, Philadelphia, PA 19140, U.S.A.; E-mail: email@example.com.
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 P2YAC, (P2TAC or P2T in platelets) has been identified. Recently, this receptor has been cloned and designated P2Y12 in keeping with current P2 receptor nomenclature.
Three selective P2T 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 (pIC50, 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 P2Y1 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 P2Y12 mRNA.
These data demonstrate that the P2YAC receptor in C6-2B cells is pharmacologically identical to the P2TAC receptor in rat platelets.
P2T, or P2Y12, platelet ADP receptor coupled to inhibition of adenylyl cyclase
platelet ADP receptor coupled to stimulation of phospholipase C
ATP and ADP are released from several sources in the body, including purinergic nerve endings, platelets, chromaffin cells, and endothelial cells (Gordon, 1986). The nucleotides elicit several physiological responses through activation of specific cell surface receptors known as P2 receptors (Burnstock, 1978). The P2 receptors are divided into P2X ligand gated channels, and P2Y receptors coupled to heterotrimeric G proteins (Fredholm et al., 1997). To date, several members of the P2X family and the P2Y family have been cloned (Fredholm et al., 1997). Several other P2 receptors have been characterized, but not cloned, including the P2Y receptors coupled to adenylyl cyclase in platelets (Kunapuli, 1998), C6 rat glioma cells (Boyer et al., 1993; Planet et al., 1989), and B10 microvascular endothelial cells (Feolde et al., 1995). In C6 cells and B10 cells, P2Y receptor stimulation does not lead to phospholipase C (PLC) activation (Boyer et al., 1993; Feolde et al., 1995).
In the platelet, we proposed a three-receptor model for activation of platelets by ADP, that includes a receptor coupled to inhibition of adenylyl cyclase (P2TAC), a receptor coupled to activation of phospholipase C (P2Y1), and a ligand gated channel (P2X1) (Daniel et al., 1998; Jin et al., 1998). Several laboratories have independently confirmed this model, but the ADP receptor coupled to adenylyl cyclase has been designated as P2T (Fagura et al., 1998) P2cyc (Hechler et al., 1998a), P2YADP (Jantzen et al., 1999), P2YAC (Fabre et al., 1999; Geiger et al., 1998), and P2Y? (Cattaneo & Gachet, 1999). We have demonstrated that ADP-induced platelet shape change is exclusively mediated by the P2Y1 receptor and that P2TAC receptor antagonists have no effect on this event. Furthermore, ADP-induced platelet aggregation is mediated by concomitant signalling from both the P2Y1 and the P2TAC receptors and hence either P2Y1 receptor antagonists or the P2TAC receptor antagonists can abolish ADP-induced platelet aggregation (Jin & Kunapuli, 1998). Recently, the ADP receptor coupled to adenylyl cyclase has been cloned and designated P2Y12 (Hollopeter et al., 2001). In addition, an orphan receptor in the brain was recently shown to be an ADP receptor that couples to Gi signalling (Zhang et al., 2001) and to have a primary sequence identical to that of the P2Y12 receptor. We have also designated the C6-2B cell P2Y receptor coupled to the inhibition of adenylyl cyclase, P2YAC, and speculated that the platelet and C6-2B cell receptors will be identified as the same subtype (Jin et al., 1998). In this manuscript, P2TAC indicates the adenylyl cyclase coupled platelet ADP receptor subtype, and P2YAC is used for the C6-cell P2Y receptor coupled to the inhibition of adenylyl cyclase. Previous studies have shown that AR-C66096 and related compounds are competitive antagonists at the P2 receptor subtype, which mediates ADP-induced inhibition of adenylyl cyclase in platelets (Humphries et al., 1995). Since the discovery of these compounds pre-dates description of the 3-receptor model, the term P2T receptor antagonist remains in common usage for these potent, selective antagonists at the P2TAC receptor.
Selective antagonists are preferable to agonist potency orders in pharmacologically defining a receptor subtype. This is particularly relevant in the P2 receptor area given the complexities in analysing such data, which can be confounded by the presence of impurities (Leon et al., 1997). In addition, the agonist potencies and agonist/antagonist profiles appear to depend on the receptor number in a heterologous expression system. For example, when P2Y1 receptor is expressed in astrocytoma cells, ATP was found to be a partial agonist (Palmer et al., 1998), whereas ATP is an antagonist at the P2Y1 receptor in platelets (Mills, 1996), where it is constitutively expressed, and in Jurkat cells, upon heterologous stable expression (Hechler et al., 1998b). This difference could be due to the variations in the receptor number expressed in different cells. Furthermore, any ectonucleotidases present on a cell could potentially complicate the agonist/antagonist profile. In the present study we have investigated the effects of three selective P2T receptor antagonists on both aggregation of rat platelets and on ADP-mediated inhibition of adenylyl cyclase in rat platelets and C6-2B cells. In addition, we demonstrate that the C6-2B cells express the P2Y12 mRNA. The findings indicate that the P2YAC receptor is pharmacologically similar to the P2Y12 receptor on platelets.
Myo-[2-3H] inositol, [3H]-adenine, and [14C]-cyclic AMP were from NEN Life Science Products (Boston, MA, U.S.A.). Fibrinogen, PGI2, ADP, A2P5P, A3P5P and A3P5PS were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). AR-C70300, AR-C69581 and AR-C78511 were all synthesized by the Department of Medicinal Chemistry, AstraZeneca R&D Charnwood, Loughborough, U.K.
1321N1 astrocytoma cells and C6-2B rat glioma cells were a gift from T.K. Harden, Department of Pharmacology, University of North Carolina, Chapel Hill, NC, U.S.A. and were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% foetal calf serum, 1% penicillin/streptomycin at 37°C with 5% CO2. The P2Y1 receptor transfected cells were grown in the same medium supplemented with 500 μg ml−1 G418. Experiments were carried out in confluent cultures 2 days after plating out in 12-well plates.
Stable expression of human P2Y1 receptor in 1321N1 astrocytoma cells
The expression construct (pcDNA3 – HP2Y1) containing the short form of the P2Y1 receptor cDNA (Ayyanathan et al., 1996) was used to transfect 1321N1 astrocytoma cells using lipofectamine as described previously (Akbar et al., 1996). 1321N1 astrocytoma cells were also transfected with pcDNA3 to serve as a control. The medium was replaced after 6 h with fresh medium containing 0.5 mg ml−1 G418. Stable transfectants were selected on medium containing 0.5 mg ml−1 G418 and screened for the expression of the P2Y1 receptor by second messenger (inositol phosphate; InsP) generation. One clone (HP2Y1-1), out of six clones selected, was chosen for further characterization.
Measurements of inositol phosphates (InsP)
The inositol phosphates were measured essentially as described (Filtz et al., 1994; Kunapuli et al., 1997). Confluent cultures of HP2Y1-1 cells in 12-well plates were labelled with 1 μCi ml−1 of myo-[2-3H]-inositol in inositol-free DMEM for 24 h. Labelled cells were washed once, the medium was replaced with 890 μl of [3H]-inositol-free 20 mM HEPES-buffered Eagle's medium, pH 7.4, and the cells were incubated at 37°C for 30 min before proceeding. This step helps to reduce background levels of [3H]-inositol phosphates, so that agonist-stimulated accumulation could be detected more easily. After incubation for 30 min at 37°C, 10 μl of 1 M LiCl were added to a final concentration of 10 mM and the incubation continued for an additional 10 min. The cells were stimulated with 100 μl of ADP at a final concentration of 10 μM for 15 min and the reaction was terminated by aspiration of the medium, addition of 0.75 ml of 10 mM formic acid and incubation at room temperature for 30 min. The solution containing the extracted InsP was neutralized and diluted with 3 ml of 10 mM NH4OH (yielding a final pH of 8 – 9) and then applied directly to a column containing 0.7 ml of the anion exchange resin, AG 1-X8. The column was washed with 4 ml of 40 mM ammonium formate, pH 5.0, to remove the free inositol and the glyceroinositol. Total InsPs were eluted with 4 ml of 2 M ammonium formate, pH 5.0. One ml of the eluate was counted with 9 ml of scintillation fluid. Results presented are an average of three – five independent experiments.
Cyclic AMP measurements and aggregation on chronologue
Rats (Sprague-Dawley) were anaesthetized with Isoflurane, an inhalation anaesthetic. Entry was gained into the abdominal cavity through a transverse incision inferior to the diaphragm. The heart was exposed by cutting through the sternum to the clavicle. Syringes (1 ml) prepared with 100 μl of ACD (acid/citrate/dextrose) buffer were used to draw blood from the heart. Platelet rich plasma was acquired by stepped centrifugation of the whole blood, for maximal platelet content. Platelet rich plasma (PRP) was aspirinated (1 mM) for 1 h at 37°C. Platelets were isolated from plasma by centrifugation at 900×g for 10 min, then resuspended in Tyrode's buffer with 20 μg ml−1 apyrase.
96-well plate method
Suspensions of washed platelets were prepared by differential centrifugation of human blood obtained from either healthy male or female volunteers by venepuncture using 1/10 volume 3.2% trisodium citrate as anti-coagulant or from Isoflurane anaesthetized rats from the dorsal aorta using 1/10 volume 3.2% trisodium citrate as anti-coagulant. A two-stage washing procedure was used with prostacyclin (PGI2, 300 ng ml−1) included at each stage to prevent platelet activation (Humphries et al., 1994). The final suspension in calcium-free Tyrode's buffer was adjusted to a platelet count of 2×105 μl−1 and stored in a capped syringe at 8°C for at least 2 h prior to use in aggregation experiments.
Platelet aggregation (0.5 ml sample volume) was measured in a Chronolog lumi-aggregometer with stirring at 37°C.
96-well plate method
Aliquots (150 μl) of platelet suspensions containing CaCl2 (1 mM) and human fibrinogen (0.2 mg ml−1) were added to individual wells of 96-well plates. The plate was read (R1) at 650 nm to establish baseline. Saline or the appropriate solution of test compound was added to each well and the plate was then shaken for 5 min on an orbital shaker on setting 10 and read (R2) at 650 nm. Saline or ADP (3 or 30 μM) was then added to each well and the plate shaken for a further 5 min before reading (R3) again at 650 nm.
Responses were measured as extent of aggregation calculated as follows after subtraction of baseline values:-
Effects on ADP-induced platelet aggregation were calculated as % inhibition. The concentration of compound producing 50% inhibition (pIC50) was derived by graphical interpolation.
Measurement of cyclic AMP
Cells were incubated with 2 μCi ml−1 [3H]-adenine (25 Ci mmol−1) for 2 h at 37°C. The cells were washed once with DMEM. The medium was replaced by medium containing 100 μM forskolin. Various concentrations of agonist and antagonist were added after 1 min 30 s. The reactions were terminated by addition of 1 M HCl containing 2000 c.p.m. of [14C]-cyclic AMP (2 GBq mmol−1) as a recovery standard. Cyclic AMP levels were determined as described earlier (Daniel et al., 1998) and expressed as percentage of total [3H]-adenine nucleotides.
Reverse transcription-coupled polymerase chain reaction (RT – PCR)
The total RNA was isolated from C6-2B cells by RNAzol procedure (Tel-Test Inc., Friendswood, TX, U.S.A.) and the cDNA was prepared from 5 μg of total RNA using the first strand synthesis kit (Gibco-BRL, Gaithersburg, MD, U.S.A.). As a control, total RNA without reverse transcription was used to eliminate the possible contamination of genomic DNA in the RNA preparation. The PCR was carried out using forward and reverse primers specific for rat P2Y12 receptor cDNA [GenBank Accession No. AF313450] (Hollopeter et al., 2001). The forward primer was 5′-CAGGTTCTCTTCCCATTGCT-3′ (corresponds to 214 – 233 nt) and the reverse primer was 5′-CAGCAATGATGATGAAAACC-3′ (852 – 871 nt). After initial denaturation for 5 min at 94°C the amplifications were carried out for 35 cycles using 5.0 units of pfu DNA polymerase as follows: denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min. The final cycle was followed by an additional extension for 7 min at 72°C.
Effects of three P2T receptor antagonists on ADP-induced platelet aggregation
Subsequent to the characterization of AR-C66096 as an antagonist at the human platelet P2TAC receptor (Humphries et al., 1994; Daniel et al., 1998; Jin et al., 1998), a number of other selective P2T receptor antagonists have been synthesized. The structures of the compounds used in the present study are shown in Figure 1. The compounds were chosen because of the non-overlapping potency for inhibition of ADP-induced platelet aggregation. All these compounds inhibited ADP (30 μM)-induced aggregation of human washed platelets in a concentration-dependent manner (Figure 2A). The rank order of antagonist potency was AR-C78511>AR-C69581>AR-C70300 (Table 1). Since the C6-2B cells are of rat origin, we investigated the effect of these compounds on ADP-induced aggregation of rat washed platelets. As shown in Figure 2B and Table 1, all these compounds inhibited ADP (3 μM) -induced aggregation in a concentration-dependent manner, with an identical rank order of antagonist potency (AR-C78511>AR-C69581>AR-C70300) to that seen in human platelets. Since platelet aggregation can be blocked either byantagonizing the P2Y1 receptor or the P2TAC receptor (Jin & Kunapuli, 1998), we investigated the effect of these compounds on ADP-induced platelet shape change. When platelets are stimulated with agonists they first undergo change in their shape from smooth discs to spiculated spheres, resulting in a decrease in light transmission in an aggregometer. Upon aggregation, when platelets are cross-linked through fibrinogen bound to activated integrin αIIbβ3, and settle to the bottom of the cuvette, the light transmission increases. As shown in Figure 3, all three compounds inhibited ADP (3 μM)-induced rat platelet aggregation but not shape change, suggesting that they inhibit only the P2TAC receptor but not the P2Y1 receptor.
Table 1. Comparison of pIC50 values for inhibition of ADP-induced platelet aggregation
Effects of three P2T receptor antagonists at the P2Y1 receptor
In order to investigate the effect of these compounds at the P2Y1 receptor, we developed a 1321N1 astrocytoma cell line expressing the human P2Y1 receptor. ADP caused inositol phosphate formation in these cells and the P2Y1 selective antagonists, A3P5PS, A3P5P and A2P5P, significantly blocked ADP-induced inositol phosphate formation (not shown). The P2Y1 receptor selective antagonist, A3P5PS, caused a rightward shift of the ADP concentration – dependence curve, consistent with the competitive nature of A3P5PS at the P2Y1 receptor (Figure 4). As shown in Figure 5, the P2T receptor antagonists, AR-C78511, AR-C69581 and AR-C70300, had no effect on ADP-stimulated inositol phosphate formation in P2Y1 receptor-expressing astrocytoma cells. These data clearly demonstrate that these three compounds do not antagonize the stimulation of the P2Y1 receptor by ADP in the concentration range (AR-C78511 and AR-C69581 up to 1 μM, and AR-C 70300 up to 300 μM) tested.
Effects of three P2T receptor antagonists on ADP-induced reduction in [cyclic AMP] in rat platelets and C6-2B glioma cells
To determine if the platelet P2TAC receptor is the same as the P2Y receptor in C6-2B cells coupled to inhibition of adenylyl cyclase, we used the three AR-C compounds to inhibit ADP-induced responses in rat platelets and C6-2B cells. We used rat platelets, instead of human platelets, since C6-2B cells are derived from rat brain. These antagonists blocked ADP-induced inhibition of forskolin-stimulated adenylyl cyclase in rat platelets (Figure 6A) and C6-2B cells (Figure 6B) with similar antagonist profiles. The rank order of antagonist potency to block ADP-induced adenylyl cyclase in rat platelets is identical to that in C6-2B cells (AR-C78511>AR-C69581>AR-C70300; Table 2).
Table 2. Comparison of pIC50 values for reversal of ADP-induced inhibition of adenylyl cyclase in rat platelets and C6 cells
Detection of the P2Y12 receptor mRNA in C6-2B cells
Since pharmacological evidence indicated that the C6-2B cell P2YAC receptor is similar to the P2Y12 receptor, and the rat P2Y12 receptor is cloned from platelets, we have investigated the expression of the P2Y12 receptors in C6-2B cells. The RNA (5 μg) from C6-2B cells was analysed for the P2Y12 receptor by RT – PCR analysis. As shown in Figure 7, ethidium bromide staining of the 657 bp PCR product reveals that the C6-2B cells express the P2Y12 receptor mRNA. Nucleotide sequence analysis of the PCR product revealed its identity with the rat P2Y12 receptor (GenBank Accession No. AF313450) (Hollopeter et al., 2001). The PCR product could have originated from the contaminating genomic DNA. To rule out this possibility we carried out PCR using RNA without reverse transcription which did not amplify any PCR product (Figure 7).
Platelet responses to ADP are mediated by at least three P2 receptor subtypes: P2X1; P2Y1; P2TAC. The P2TAC receptor has been demonstrated pharmacologically (Kunapuli, 1998) and a recent report indicated that this receptor has been cloned and designated P2Y12 (Hollopeter et al., 2001). Most of the G protein-coupled P2 receptors activate phospholipase C whereas stimulation of the P2Y receptors in C6 glioma cells and B10 microvascular endothelial cells causes inhibition of adenylyl cyclase (Feolde et al., 1995; Planet et al., 1989). The purpose of this study was: (a) to further characterize the P2YAC receptor in the C6-2B cells; (b) to compare the pharmacological profiles of the C6 cell P2YAC receptor and the platelet P2Y12 receptor.
We propose that the P2YAC receptor in C6-2B cells, is the same receptor subtype as the P2Y12 receptor in platelets based on the following observations. The agonist profiles of these two receptors are very similar (Boyer et al., 1993; Mills, 1996) and ATP has been shown to be an antagonist at the P2YAC receptor on C6-2B cells (Planet et al., 1989) and the P2Y12 (P2TAC) receptor (Daniel et al., 1998). Several selective P2T antagonists, tested in this study, inhibited ADP-induced rat platelet aggregation (P2TAC-mediated), but not shape change (P2Y1-mediated). Furthermore, these P2T receptor antagonists blocked ADP-induced inhibition of stimulated adenylyl cyclase in rat platelets and in C6-2B cells, with identical potencies and rank order (Table 2). The possibility of confounding influence of effects at the P2X1 receptors can be discounted since, under the assay conditions used the P2X1 receptors would not be activated (Jin & Kunapuli, 1998). These observations indicate that the C6-2B cell P2YAC receptor is pharmacologically similar to the P2Y12 receptor.
Webb et al. (1996) identified abundant P2Y1 receptor mRNA in both C6-2B glioma cells and B10 microvascular endothelial cells, and proposed that the P2Y receptor in these cells is the P2Y1 receptor. It was suggested that the P2Y1 receptor could couple to different G proteins in different cells (Webb et al., 1996). Since the pharmacological agents used in the study of Webb et al. (1996) are not specific for the P2Y1 receptor, we believe that C6-2B cells and B10 cells express mRNAs for at least two P2Y receptor subtypes: the P2Y1 receptor, that was detected by RT – PCR and at least one other P2Y receptor coupled to inhibition of adenylyl cyclase. Boyer et al. (1994) demonstrated differential effects of a non specific P2 receptor antagonist, PPADS, on the turkey erythrocyte P2Y receptor, coupled to phospholipase C, and the C6-2B P2YAC receptor. PPADS inhibited nucleotide-induced phospholipase C activation in turkey erythrocytes, while even at 100 μM it had no effect on agonist-induced adenylyl cyclase inhibition in C6-2B cells (Boyer et al., 1994). It was argued that the substitution of 41-lysine by arginine in the rat P2Y1 receptor might prevent a Schiff's base formation with PPADS, an essential step in antagonism by PPADS. However, Schachter et al. (1997) demonstrated that PPADS antagonizes the rat P2Y1 receptor.
Based on the studies of Webb et al. (1996), Gachet et al. (1997) suggested that the P2Y1 receptor in platelets couples to inhibition of adenylyl cyclase. However, we have shown that in human platelets AR-C66096 (previously FPL or ARL 66096) failed to inhibit ADP-induced mobilization of calcium from intracellular stores, while blocking the ADP-induced inhibition of adenylyl cyclase (Daniel et al., 1998; Jin et al., 1998). Even at high concentrations, A3P5PS, A3P5P, and A2P5P did not block ADP-induced inhibition of adenylyl cyclase in platelets (Jin et al., 1998). Furthermore, in platelets of mice deficient in the P2Y1 receptor, ADP causes inhibition of adenylyl cyclase (Leon et al., 1999, Fabre et al., 1999) confirming that the receptor coupled to Gi signalling in platelets is distinct from the P2Y1 receptor. The recent cloning of the Gi coupled brain and platelet P2Y receptors (Zhang et al., 2001; Hollopeter et al., 2001), further demonstrate that the Gi coupled receptors are molecularly distinct from the P2Y1 receptor.
It was recently shown that stem cells could be differentiated into glial cells (the origin of C6 cells) when treated with hematopoietic growth factors and cytokines (Reyes & Verfaillie, 1999). In addition, brain cells have been shown to differentiate into hematopoietic cells (Bjornson et al., 1999). Hence, it is conceivable that C6 cells, derived from glial cells, and platelets, a hematopoietic cell, have at least some common signal transduction mechanisms, including the P2 receptor subtype coupled to Gi. Although pharmacological evidence suggests that the P2Y12 and the P2YAC receptors are similar, only molecular cloning of these receptors could provide conclusive evidence. To achieve this goal, we used RT – PCR of the C6-2B cell mRNA using primers specific for the rat P2Y12 receptor and confirmed the identity of the RT – PCR product by nucleotide sequence analysis. Our data demonstrate that the P2Y12 mRNA is expressed in C6-2B cells. This molecular evidence, coupled with the pharmacological data, indicates that the C6-2B cell P2YAC receptor is identical to the platelet P2Y12 receptor.
We are grateful to Dr T. Kendall Harden, Department of Pharmacology, University of North Carolina, Chapel Hill, NC for providing us with the astrocytoma cells and C6-2B glioma cells. This work was supported by research grants HL60683 and HL64943 grant HL64943 from the National Heart Lung and Blood Institute, National Institutes of Health. This work was performed during the tenure of an Established Investigator award in Thrombosis from American Heart Association and Genentech to S.P. Kunapuli.