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Keywords:

  • antagonist;
  • ATP;
  • metabolism;
  • P2Y12 receptor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Preparation of washed mouse platelets and aggregation studies
  7. Cloning, sequencing and heterologous expression of the human P2Y12 receptor
  8. Cell culture
  9. cAMP assays
  10. Triphosphate nucleotide regeneration and purification
  11. Measurement of ectonucleotidase activity in 1321 N1 P2Y12 cells
  12. Results
  13. Effects of adenine diphosphate nucleotides on the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor
  14. The agonistic properties of adenine triphosphate nucleotides are lost in the presence of the regenerating system CP/CPK
  15. Adenine triphosphate nucleotides behave as antagonists at the mouse platelet P2Y12 receptor
  16. The poorly hydrolyzable ATP analogue ATPγS is an antagonist at both the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor expressed in 1321 N1 cells
  17. Discussion
  18. Acknowledgement
  19. References

Summary.   The aim of the present study was to characterize the pharmacological profile of the P2Y12 receptor for several adenine triphosphate nucleotides in view of their possible roles as partial agonists or true antagonists. Two distinct cellular systems were used: P2Y1 receptor deficient mouse platelets (inline image platelets) previously shown to express a native and functional P2Y12 receptor and 1321 N1 astrocytoma cells stably expressing the human P2Y12 receptor (1321 N1 P2Y12). ADP and its structural analogues inhibited cAMP accumulation in a dose-dependent manner in both inline image platelets and 1321 N1 P2Y12 cells with a similar rank order of potency, 2 methylthio-ADP (2MeSADP) >>ADP – Adenosine 5′-(βthio) diphosphate (ΑDPβS). Commercial ATP, 2 chloro; ATP (2ClATP) and 2 methylthio-ATP (2MeSATP) also inhibited cAMP accumulation in both cell systems. In contrast, after creatine phosphate (CP)/creatine phosphokinase (CPK) regeneration, adenine triphosphate nucleotides lost their agonistic effect on inline image platelets and behaved as antagonists of ADP (0.5 µm)-induced adenylyl cyclase inhibition with IC50 of 13.5 ± 4.8, 838 ± 610, 1280 ± 1246 µm for 2MeSATP, ATP and 2ClATP, respectively. In 1321 N1 P2Y12 cells, CP/CPK regenerated ATP and 2ClATP lost their agonistic effect only when CP/CPK was maintained during the cAMP assay. The stable ATP analogue ATPγS antagonized ADPβS-induced inhibition of cAMP accumulation in both inline image platelets and 1321 N1 P2Y12 cells. Thus, ATP and its triphosphate analogues are not agonists but rather antagonists at the P2Y12 receptor expressed in platelets or transfected cells, provided care is taken to remove diphosphate contaminants and to prevent the generation of diphosphate nucleotide derivatives by cell ectonucleotidases.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Preparation of washed mouse platelets and aggregation studies
  7. Cloning, sequencing and heterologous expression of the human P2Y12 receptor
  8. Cell culture
  9. cAMP assays
  10. Triphosphate nucleotide regeneration and purification
  11. Measurement of ectonucleotidase activity in 1321 N1 P2Y12 cells
  12. Results
  13. Effects of adenine diphosphate nucleotides on the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor
  14. The agonistic properties of adenine triphosphate nucleotides are lost in the presence of the regenerating system CP/CPK
  15. Adenine triphosphate nucleotides behave as antagonists at the mouse platelet P2Y12 receptor
  16. The poorly hydrolyzable ATP analogue ATPγS is an antagonist at both the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor expressed in 1321 N1 cells
  17. Discussion
  18. Acknowledgement
  19. References

Adenine nucleotides activate two different groups of receptors, the G protein-coupled receptors termed P2Y and the ligand-gated ion channels termed P2X. Molecular identification of P2 receptors on the surface of blood platelets has made it possible to separately assign the known effects of ADP and ATP to the three cloned platelet P2 receptors, namely P2X1, P2Y1 and P2Y12[1], all of which have been shown to play an important role in platelet activation and thrombosis.

The P2X1 receptor mediates a transient calcium entry leading to platelet shape change in response to ATP [2,3]. This receptor has been shown to be involved in collagen-induced platelet activation and to play a role in the thrombosis of small arteries [4–6]. The P2Y1 receptor is responsible for the initiation of platelet aggregation in response to ADP through the mobilization of intracellular calcium stores [7]. This receptor also appears to be of physiological and pathological importance, since its inhibition by molecular targeting or administration of a selective antagonist results in resistance to experimental thrombosis in mice [8–10]. Finally the P2Y12 receptor, coupled to the inhibition of adenylyl cyclase through the Gαi2 subunit of heterotrimeric G proteins [11–14], is responsible for completion and amplification of the platelet aggregation response to ADP [1]. This receptor plays a key role in hemostasis. Patients with P2Y12 deficiency exhibit a prolonged bleeding time linked to a selective impairment of ADP-induced platelet aggregation [15]. The cofactor role of ADP in platelet activation in response to strong agonists such as thrombin or collagen (i.e. stabilization of aggregates and enhancement of secretion) is largely mediated by activation of the P2Y12 receptor. As a consequence, this receptor constitutes a key target in antithrombotic therapy. Thus, P2Y12 is the molecular target which is covalently bound by the active metabolite of Clopidogrel (Plavix™), a thienopyridine currently in clinical use [16].

The recent cloning of the P2Y12 receptor has allowed its expression in many heterologous systems and several groups have characterized its pharmacological profile in either native P2Y12 expressing cells [17,18] or transfected cells [11,19]. In a number of these studies, ADP and its diphosphate analogues 2 methylthio-ADP (2MeSADP) and Adenosine 5′-(β-thio) diphosphate (ADPβS) and also ATP and its structural triphosphate analogues 2 methylthio-ATP (2MeSATP), 2 chloro-ATP (2ClATP), Adenosine 5′-(γ-thio) triphosphate (ATPγS) and ATPαS were found to be agonists at the P2Y12 receptor [11,17,19]. A Gi-coupled P2Y receptor long known to exist in rat C6 glioma cells [20] was recently identified as P2Y12[21]. Analogues of either ADP or ATP were likewise found to be agonists at this native P2Y12 receptor [20]. In contrast, at the native P2Y12 receptor expressed in a clonal population (B10) of rat brain capillary endothelial cells, Simon et al. reported that ADP, 2MeSADP and 2MeSATP were agonists, while ATP and 2ClATP were weak partial agonists [18]. Overall, these results do not fit the current knowledge of blood platelet pharmacology, whereby ATP and a wide range of its triphosphate analogues behave as antagonists of both ADP-induced adenylyl cyclase inhibition which depends on P2Y12 activation and the subsequent platelet aggregation [22–24].

Pharmacological characterization of P2 receptors is difficult due to the metabolism of nucleotides by a variety of ecto-kinases and hydrolases [25]. In addition, commercial sources of triphosphate nucleotides are frequently ‘contaminated’ with their di- and monophosphate analogues, which results in misleading apparent agonist selectivities [26–28]. Finally, apyrase (adenosine 5′-triphosphate diphosphohydrolase, EC 3.6.1.5) is required in most experiments involving P2 receptors to prevent their loss of function through desensitization [29]. All these factors make it difficult to assess the true effect of triphosphate nucleotides and suggest that their agonistic activity at the P2Y12 receptor might be only apparent.

Similar conflicting results have been reported concerning the pharmacology of the P2Y1 receptor. ATP and its analogues were in fact shown to be true antagonists at the P2Y1 receptor in some studies [27,28], while in others they behaved as partial agonists in several cellular systems [30].

Therefore, the aim of the present study was to carefully characterize the pharmacological profile of the P2Y12 receptor for several adenine triphosphate nucleotides in view of their possible roles as partial agonists or true antagonists. Two distinct cellular systems were used: P2Y1 receptor deficient mouse platelets (inline image platelets) previously shown to express a native and functional P2Y12 receptor [9,31] and 1321 N1 astrocytoma cells stably expressing the human P2Y12 receptor (1321 N1 P2Y12). Pharmacological characterization of this receptor is a prerequisite for the establishment of structure-activity relationships which represent the starting point for the design of new drugs.

Materials

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Preparation of washed mouse platelets and aggregation studies
  7. Cloning, sequencing and heterologous expression of the human P2Y12 receptor
  8. Cell culture
  9. cAMP assays
  10. Triphosphate nucleotide regeneration and purification
  11. Measurement of ectonucleotidase activity in 1321 N1 P2Y12 cells
  12. Results
  13. Effects of adenine diphosphate nucleotides on the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor
  14. The agonistic properties of adenine triphosphate nucleotides are lost in the presence of the regenerating system CP/CPK
  15. Adenine triphosphate nucleotides behave as antagonists at the mouse platelet P2Y12 receptor
  16. The poorly hydrolyzable ATP analogue ATPγS is an antagonist at both the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor expressed in 1321 N1 cells
  17. Discussion
  18. Acknowledgement
  19. References

ATP, 2MeSADP, ADPβS, 2MeSATP, 2ClATP, ATPγS, prostaglandin E1 (PGE1), CGS 15943, forskolin, creatine phosphate (CP), creatine phosphokinase (CPK), 3-isobutyl-1-methylxanthine (IBMX), adrenaline and human serum albumin were from Sigma-Aldrich (Saint Quentin-Fallavier, France). ADP was from Boehringer (Mannheim, Germany), the cyclic adenosine 3′-5′-monophosphate (cAMP) assay kit and [3H]ATP (40 Ci mmole-1) were from Amersham Pharmacia Biotech (Les Ulis, France) and human fibrinogen from Kabi (Stockholm, Sweden). DMEM, G418 (geneticin) and PBS were from Gibco BRL (Paris, France). Apyrase (adenosine 5′-triphosphate diphosphohydrolase, EC 3.6.1.5) was purified from potatoes as previously described [32].

Preparation of washed mouse platelets and aggregation studies

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Preparation of washed mouse platelets and aggregation studies
  7. Cloning, sequencing and heterologous expression of the human P2Y12 receptor
  8. Cell culture
  9. cAMP assays
  10. Triphosphate nucleotide regeneration and purification
  11. Measurement of ectonucleotidase activity in 1321 N1 P2Y12 cells
  12. Results
  13. Effects of adenine diphosphate nucleotides on the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor
  14. The agonistic properties of adenine triphosphate nucleotides are lost in the presence of the regenerating system CP/CPK
  15. Adenine triphosphate nucleotides behave as antagonists at the mouse platelet P2Y12 receptor
  16. The poorly hydrolyzable ATP analogue ATPγS is an antagonist at both the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor expressed in 1321 N1 cells
  17. Discussion
  18. Acknowledgement
  19. References

Washed mouse platelets were prepared from blood (6 vol) drawn from the abdominal aorta of mice into a plastic syringe containing ACD (acid citrate dextrose) anticoagulant (1 vol). Pooled blood (6 mL) was centrifuged at 2300 g for 90 s at 37 °C and platelet rich plasma (PRP) was removed and centrifuged at 2200 g for 5 min at 37 °C. The platelet pellet was washed twice in Tyrode's buffer (137 mm NaCl, 2 mm KCl, 12 mm NaHCO3, 0.3 mm NaH2PO4, 1 mm MgCl2, 2 mm CaCl2) containing 5.5 mm glucose, 5 mm Hepes and 0.35% human serum albumin (pH 7.3) and finally resuspended at a density of 105 platelets μL-1 in the same buffer. Apyrase (0.02 U mL-1) was added to the final suspension of washed platelets for experiments with non hydrolysable nucleotide analogues (ATPγS and ADPβS) but otherwise not.

Aggregation was measured at 37 °C by a turbidimetric method in a dual-channel Payton aggregometer (Payton Associates, Scarborough, ON, Canada). A 450 μL aliquot of platelet suspension was stirred at 1100 r.p.m. and activated in the presence of human fibrinogen (0.25 mg mL-1) by addition of different agonists to a final volume of 500 μL.

Cloning, sequencing and heterologous expression of the human P2Y12 receptor

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Preparation of washed mouse platelets and aggregation studies
  7. Cloning, sequencing and heterologous expression of the human P2Y12 receptor
  8. Cell culture
  9. cAMP assays
  10. Triphosphate nucleotide regeneration and purification
  11. Measurement of ectonucleotidase activity in 1321 N1 P2Y12 cells
  12. Results
  13. Effects of adenine diphosphate nucleotides on the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor
  14. The agonistic properties of adenine triphosphate nucleotides are lost in the presence of the regenerating system CP/CPK
  15. Adenine triphosphate nucleotides behave as antagonists at the mouse platelet P2Y12 receptor
  16. The poorly hydrolyzable ATP analogue ATPγS is an antagonist at both the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor expressed in 1321 N1 cells
  17. Discussion
  18. Acknowledgement
  19. References

A 1100-bp fragment corresponding to the human P2Y12 coding sequence was amplified by PCR from human platelet cDNA using primers (5′-AACGAGTTCTGAACACAAAGAGATTGAA-3′ and 5′-ATCAACAGTTATCAGGTA ACCAACAAGA-3′) derived from the human cDNA sequence [11] and subcloned into the pCDNA3 vector (Invitrogen, Groningen, the Netherlands). The Hind III (Boehringer, Mannheim, Germany) linearized vector was transfected into 1321 N1 human astrocytoma cells using FuGENETM6 transfection reagent (Roche Molecular Biochemicals, Meylan, France). Transfected cells were selected with G418 (0.8 mg mL-1) and cloned. The functional expression of the P2Y12 receptor was determined by cAMP assays and positive clones were selected for their ability to suppress forskolin (10 µm)-stimulated cAMP levels in response to 2MeSADP (1 µm).

cAMP assays

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Preparation of washed mouse platelets and aggregation studies
  7. Cloning, sequencing and heterologous expression of the human P2Y12 receptor
  8. Cell culture
  9. cAMP assays
  10. Triphosphate nucleotide regeneration and purification
  11. Measurement of ectonucleotidase activity in 1321 N1 P2Y12 cells
  12. Results
  13. Effects of adenine diphosphate nucleotides on the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor
  14. The agonistic properties of adenine triphosphate nucleotides are lost in the presence of the regenerating system CP/CPK
  15. Adenine triphosphate nucleotides behave as antagonists at the mouse platelet P2Y12 receptor
  16. The poorly hydrolyzable ATP analogue ATPγS is an antagonist at both the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor expressed in 1321 N1 cells
  17. Discussion
  18. Acknowledgement
  19. References

A washed platelet suspension (450 μL) was stirred at 1100 r.p.m. in an aggregometer cuvette and nucleotides were added 30 s before PGE1. One minute after addition of PGE1 (10 µm), the reaction (500 µL) was stopped by adding 50 µL of ice-cold 6 N perchloric acid (PCA). cAMP was isolated from the supernatant by extraction with a freon/trioctylamine (v/v) mixture. The upper aqueous phase was lyophilized and the dry residue dissolved in the buffer provided with the commercial cAMP radioimmunoassay kit. cAMP was then quantified according to the manufacturer's instructions [27].

1321 N1 P2Y12 cells were grown in 24-well tissue culture dishes to 80–90% confluence. After two washes in PBS, the cells were preincubated in DMEM for 30 min at 37 °C in the presence of the phosphodiesterase inhibitor IBMX (100 µm) and CGS 15943 (10 µm), a mixed adenosine receptor antagonist, to block the effects of adenosine generated by degradation of nucleotides [17]. Nucleotides were then added to a final volume of 500 µL, 30 s before forskolin (10 μM). The reaction was stopped 4 min later by adding 50 µL of ice-cold 6 N PCA and cAMP extraction was performed as described above.

Triphosphate nucleotide regeneration and purification

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Preparation of washed mouse platelets and aggregation studies
  7. Cloning, sequencing and heterologous expression of the human P2Y12 receptor
  8. Cell culture
  9. cAMP assays
  10. Triphosphate nucleotide regeneration and purification
  11. Measurement of ectonucleotidase activity in 1321 N1 P2Y12 cells
  12. Results
  13. Effects of adenine diphosphate nucleotides on the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor
  14. The agonistic properties of adenine triphosphate nucleotides are lost in the presence of the regenerating system CP/CPK
  15. Adenine triphosphate nucleotides behave as antagonists at the mouse platelet P2Y12 receptor
  16. The poorly hydrolyzable ATP analogue ATPγS is an antagonist at both the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor expressed in 1321 N1 cells
  17. Discussion
  18. Acknowledgement
  19. References

Nucleotide solutions were regenerated with creatine phosphokinase (CPK, 20 U mL-1) and creatine phosphate (CP, 10 mm) at 37 °C for 120 min, before adding the entire mixture to the platelet suspension or to plated cells. Where indicated, CP/CPK (5 mm, 20 U mL-1) was also added to the platelet suspension or to plated cells. In this case, in order to determine whether ADP was still able to inhibit cAMP accumulation, ADP was mixed with cold CP/CPK immediately before a cAMP assay. The purity of nucleotide solutions was checked by high performance liquid chromatography (HPLC) analysis on a Partisil 10 µm SAX column (Interchrom, Interchim, Montluçon, France) eluted with a linear gradient from 0 to 100% of 1 m ammonium phosphate buffer (pH 3.8), as previously described [27]. In competition experiments, adenine triphosphate nucleotides were treated with CP/CPK (10 mm, 20 U mL-1) at 37 °C for 120 min to regenerate triphosphate nucleotides, after which CP/CPK was precipitated with 6 N PCA and the nucleotides were extracted with a freon/trioctylamine (v/v) mixture. ATPγS stock solutions were contaminated with up to about 20% ADP. Since CP/CPK treatment of ATPγS would generate ATP, this nucleotide was purified by HPLC on a Partisil 10 µm SAX column eluted with a linear gradient of 0–500 mm triethylammonium bicarbonate buffer (pH 8.4). The triphosphate nucleotide fraction was collected on ice and added immediately to platelets or cells and the ATPγS concentration was determined from its absorbance at 259 nm.

Effects of adenine diphosphate nucleotides on the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Preparation of washed mouse platelets and aggregation studies
  7. Cloning, sequencing and heterologous expression of the human P2Y12 receptor
  8. Cell culture
  9. cAMP assays
  10. Triphosphate nucleotide regeneration and purification
  11. Measurement of ectonucleotidase activity in 1321 N1 P2Y12 cells
  12. Results
  13. Effects of adenine diphosphate nucleotides on the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor
  14. The agonistic properties of adenine triphosphate nucleotides are lost in the presence of the regenerating system CP/CPK
  15. Adenine triphosphate nucleotides behave as antagonists at the mouse platelet P2Y12 receptor
  16. The poorly hydrolyzable ATP analogue ATPγS is an antagonist at both the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor expressed in 1321 N1 cells
  17. Discussion
  18. Acknowledgement
  19. References

The pharmacology of the P2Y12 receptor was examined with respect to different adenine diphosphate nucleotides: ADP, its stronger analogue 2MeSADP and its non-hydrolyzable analogue ADPβS. All three nucleotides were tested on inline image mouse platelets which express a native P2Y12 receptor and on 1321 N1 P2Y12 astrocytoma cells stably expressing the human P2Y12 receptor. The ability of ADP, 2MeSADP or ADPβS to activate the P2Y12 receptor was evaluated by quantifying the effect of the agonist on intracellular cAMP accumulation. None of these nucleotides affected cAMP accumulation in non-transfected 1321 N1 cells (data not shown). Conversely, all three nucleotides reduced the production of cAMP induced by PGE1 (10 µm) in inline image mouse platelets in a dose-dependent manner, with a maximal effect reaching 70–80% inhibition (Fig. 1A). Basal cAMP levels in resting platelets were 10.1 ± 1.5 pmoles cAMP/108 platelets. After addition of PGE1 (10 µm), cAMP levels increased to 179 ± 37 pmoles/108 platelets and were reduced to 64 ± 9 pmoles/108 platelets in the presence of each diphosphate nucleotide. The rank order of potency was 2MeSADP >> ADP ≃ ADPβS with respective EC50 values of 0.8 ± 0.3, 219 ± 52 and 122 ± 24 nm (mean ± SEM in three independent experiments).

image

Figure 1. Inhibition of adenylyl cyclase activity by adenine diphosphate nucleotides in inline image mouse platelets and 1321 N1 P2Y12 cells. A. 2MeSADP, ADP or ADPβS reduced the production of cAMP induced by PGE1 (10 µm) in inline image mouse platelets in a dose-dependent manner, with respective EC50 values of 0.8 ± 0.3, 219 ± 52 and 122 ± 24 nm (mean ± SEM in three independent experiments). B. In 1321 N1 P2Y12 cells, cAMP production was stimulated with forskolin (10 µm) in the presence of IBMX (100 µm) and CGS 15943 (10 µm) (see Methods). 2MeSADP, ADP or ADPβS induced a dose-dependent inhibition of forskolin-stimulated intracellular cAMP accumulation, reducing stimulated cAMP levels by up to 70–80%. EC50 values were, respectively, 0.07 ± 0.01, 6.8 ± 0.8 and 74 ± 34 nm (mean ± SEM in 3–6 independent experiments). Data represent the percentage cell content of cAMP relative to the maximum level induced by either PGE1 or forskolin.

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In 1321 N1 P2Y12 cells, 2MeSADP, ADP or ADPβS likewise induced a dose-dependent inhibition of forskolin-stimulated intracellular cAMP accumulation, reducing stimulated cAMP levels by up to 70–80% (Fig. 1B). Basal levels in resting cells were about 20 pmoles cAMP/108 cells. After stimulation with forskolin (10 µm), cAMP levels increased to 500–700 pmoles/108 cells and were reduced to about 50–100 pmoles/108 cells in the presence of each diphosphate nucleotide. EC50 values were, respectively, 0.07 ± 0.01, 6.8 ± 0.8 and 74 ± 34 nm (mean ± SEM in 3–6 independent experiments). The rank order of potency was similar for mouse platelets and transfected cells: 2MeSADP>> ADP ≃ ADPβS and in good agreement with previous reports that 2MeSADP is a 100-fold more potent inhibitor of adenylyl cyclase than ADP in human platelets [24,33].

The agonistic properties of adenine triphosphate nucleotides are lost in the presence of the regenerating system CP/CPK

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Preparation of washed mouse platelets and aggregation studies
  7. Cloning, sequencing and heterologous expression of the human P2Y12 receptor
  8. Cell culture
  9. cAMP assays
  10. Triphosphate nucleotide regeneration and purification
  11. Measurement of ectonucleotidase activity in 1321 N1 P2Y12 cells
  12. Results
  13. Effects of adenine diphosphate nucleotides on the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor
  14. The agonistic properties of adenine triphosphate nucleotides are lost in the presence of the regenerating system CP/CPK
  15. Adenine triphosphate nucleotides behave as antagonists at the mouse platelet P2Y12 receptor
  16. The poorly hydrolyzable ATP analogue ATPγS is an antagonist at both the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor expressed in 1321 N1 cells
  17. Discussion
  18. Acknowledgement
  19. References

In a first set of experiments, the adenine triphosphate nucleotide analogues ATP, 2ClATP and 2MeSATP were found to inhibit the accumulation of cAMP in both inline image mouse platelets and 1321 N1 P2Y12 cells (Fig. 2). However, as previously reported [27,28], commercial nucleotide powders are often contaminated with degradation products which can be responsible for misleading results. HPLC analyses in fact indicated that each of the three commercial adenine triphosphate nucleotides was contaminated with its diphosphate derivative: 1–2% ADP in ATP, 8–10% 2MeSADP in 2MeSATP and 10–12% 2 chloro-ADP (2ClADP) in 2ClATP preparations. Hence, to determine whether the agonistic activity might be attributable to the contaminating diphosphate analogue, ATP, 2ClATP and 2MeSATP solutions were incubated with the triphosphate nucleotide regenerating system CP/CPK, as described in Materials and Methods. In the presence of CP, CPK phosphorylates diphosphate nucleotides into their triphosphate analogues. HPLC control analyses showed that CP/CPK treatment reduced the diphosphate nucleotide contamination to less than 0.5% (data not shown).

image

Figure 2. Apparent inhibition of adenylyl cyclase activity by adenine triphosphate nucleotides is abolished in the presence of CP/CPK. Commercial ATP, 2ClATP or 2MeSATP (10 μM) strongly inhibited the accumulation of cAMP induced by PGE1 (10 μM) in inline image mouse platelets. Similarly, commercial ATP, 2ClATP or 2MeSATP (100 μM) strongly inhibited the accumulation of cAMP induced by forskolin (10 μM) in 1321 N1 P2Y12 cells. When contaminating diphosphate nucleotides were enzymatically removed using CP/CPK (10 mm, 20 U mL-1, 2 h at 37 °C) and the enzymatic treatment was maintained during incubation of cells with the nucleotides, ATP, 2ClATP or 2MeSATP (10 μM) no longer inhibited cAMP production in inline image mouse platelets. In 1321 N1 P2Y12 cells, while ATP and 2ClATP (100 μM) lost their agonistic activity, 2MeSATP (100 μM) still induced a slight inhibition of cAMP accumulation. Data represent the percentage of the stimulated cAMP production induced by PGE1 or forskolin and are the mean ± SEM of three independent experiments performed in duplicate.

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ATP and 2ClATP no longer inhibited cAMP accumulation in either inline image mouse platelets or 1321 N1 P2Y12 cells under these conditions. 2MeSATP lost its agonistic effect on inline image mouse platelets, but still slightly inhibited cAMP accumulation in 1321 N1 P2Y12 cells (Fig. 2). Results were normalized as percentage of stimulated cAMP values. However, it is noteworthy that CP/CPK treatment led to a 1.5-fold greater cAMP production in response to forskolin in 1321 N1 P2Y12 cells. This effect was dependent on the presence of the P2Y12 receptor, since it was not observed in non-transfected cells (data not shown). It has been reported previously that relatively large amounts of endogeneous nucleotides are released from 1321 N1 cells upon mechanical stimulation [34]. Hence, regeneration of diphosphate nucleotides into their triphosphate analogues using CP/CPK, prevents activation of the P2Y12 receptor, explaining the increased cAMP generation in response to forskolin. In the presence of CP/CPK, inhibition of adenylyl cyclase induced by adrenaline (10 µm) in inline image mouse platelets, which depends on activation of the Gi-coupled α2A adrenergic receptor, remained unchanged (data not shown), while in 1321 N1 P2Y12 cells, ADP (100 μM) was still able to inhibit forskolin-induced cAMP accumulation by up to 80%. Thus the molecular processes involved in adenylyl cyclase inhibition were not affected by CP/CPK. Furthermore, as previously reported [27], this effect was dependent on the presence of both the enzyme CPK and the phosphate provider CP, since when added separately they did not prevent the adenylyl cyclase inhibition produced by non regenerated adenine triphosphate nucleotides (data not shown).

Adenine triphosphate nucleotides behave as antagonists at the mouse platelet P2Y12 receptor

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Preparation of washed mouse platelets and aggregation studies
  7. Cloning, sequencing and heterologous expression of the human P2Y12 receptor
  8. Cell culture
  9. cAMP assays
  10. Triphosphate nucleotide regeneration and purification
  11. Measurement of ectonucleotidase activity in 1321 N1 P2Y12 cells
  12. Results
  13. Effects of adenine diphosphate nucleotides on the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor
  14. The agonistic properties of adenine triphosphate nucleotides are lost in the presence of the regenerating system CP/CPK
  15. Adenine triphosphate nucleotides behave as antagonists at the mouse platelet P2Y12 receptor
  16. The poorly hydrolyzable ATP analogue ATPγS is an antagonist at both the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor expressed in 1321 N1 cells
  17. Discussion
  18. Acknowledgement
  19. References

Since purified adenine triphosphate nucleotides did not display agonistic activity, we next tested whether they could act as antagonists of the P2Y12 receptor. In these experiments, contaminating diphosphate nucleotide derivatives were removed from the triphosphate nucleotide solutions by treatment with CP/CPK followed by PCA precipitation to eliminate the enzymatic system, after which the nucleotides were extracted with a mixture of freon/trioctylamine and used immediately. It has been shown previously that ADP is able to induce aggregation of inline image mouse platelets through activation of the P2Y12 receptor [9,31]. Therefore, we investigated whether the various adenine triphosphate nucleotides could inhibit ADP-induced platelet aggregation. Whereas unpurified ATP, 2ClATP or 2MeSATP (100 μM) were able to induce aggregation of inline image mouse platelets (data not shown), after CP/CPK regeneration and extraction, neither ATP (1 mm), 2ClATP (100 μM) nor 2MeSATP (100 μM) induced aggregation (Fig. 3A). Moreover, at the same concentrations all three adenine triphosphate nucleotides inhibited ADP (10 μM)-induced platelet aggregation (Fig. 3A). ATP was a less potent antagonist than 2ClATP or 2MeSATP, since 1 mm ATP was required to abolish ADP (10 μM)-induced aggregation, while 100 μM 2ClATP or 2MeSATP was sufficient. This inhibitory effect of adenine triphosphate nucleotides on ADP-induced platelet aggregation could be overcome at high concentrations of ADP (data not shown). Concerning adenylyl cyclase inhibition in inline image platelets, neither ATP (1 mm), 2ClATP (100 µm) nor 2MeSATP (100 µm) significantly inhibited PGE1 (10 µm)-induced cAMP accumulation. On the other hand, all three nucleotides were able to reverse the ADP (0.5 μM)-induced inhibition of PGE1 (10 μM)-stimulated cAMP levels with IC50 of 13.5 ± 4.8, 838 ± 610, 1280 ± 1246 µm for 2MeSATP, ATP and 2ClATP, respectively (Fig. 3B). Overall, these results indicate that ATP, 2ClATP and 2MeSATP are true antagonists of the platelet P2Y12 receptor.

image

Figure 3. Adenine triphosphate nucleotides are antagonists at the mouse platelet P2Y12 receptor. Adenine triphosphate nucleotides were enzymatically regenerated with CP/CPK and tested for their capacity to antagonize the effects of ADP on inline image platelets. (A). ADP (10 µm)-induced inline image platelet aggregation, which depends only on activation of the P2Y12 receptor, was inhibited in the presence of CP/CPK regenerated ATP, 2ClATP or 2MeSATP. (B). ADP (0.5 µm)-induced inhibition of PGE1 (10 µm)-stimulated cAMP levels was inhibited in a dose-dependent manner by increasing concentrations of CP/CPK regenerated adenine triphosphate nucleotides. IC50 were 13.5 ± 4.8, 838 ± 610, 1280 ± 1246 µm for 2MeSATP (▪), ATP (♦) and 2ClATP (•), respectively. Data are the mean ± SEM of three independent experiments performed in duplicate.

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In 1321 N1 P2Y12 cells, CP/CPK regenerated and extracted triphosphate nucleotides still inhibited forskolin-induced accumulation of cAMP (Fig. 4A). However, when CP/CPK was maintained during incubation of the nucleotides with 1321 N1 P2Y12 cells, ATP, 2ClATP and 2MeSATP lost their agonistic effect (Fig. 2), suggesting that enzymatic metabolism of the purified nucleotides by cell ectonucleotidases might be responsible for their apparent agonistic activity. In order to test this hypothesis, the amount of ADP generated through hydrolysis of ATP on the surface of 1321 N1 P2Y12 cells was determined. Using various concentrations of ATP (1, 10, 100 µm) in the presence of [3H]ATP (0.5 µCi) as a tracer, the amount of [3H]ADP generated after 4 min incubation with 1321 N1 P2Y12 cells was determined by HPLC analysis. As shown in Fig. 4(B), the concentrations of ADP generated were 0.044 ± 0.001, 0.203 ± 0.019 and 1.55 ± 0.20 µm in the presence of 1, 10 or 100 µm ATP, respectively. It was thus not possible to test the P2Y12 antagonistic activity of these triphosphate nucleotides in the transfected cell system owing to their rapid metabolism.

image

Figure 4. Apparent adenine triphosphate nucleotides agonism at the cellular P2Y12 receptor. (A). In 1321 N1 P2Y12 cells, CP/CPK regenerated nucleotides (10 µm) still inhibited forskolin (10 µm)-induced accumulation of cAMP. (B). The amount of ADP generated through hydrolysis of ATP on 1321 N1 P2Y12 cells was quantified.[3H]ATP (0.5 µCi) was added as a tracer with 1321 N1 P2Y12 cells and the amount of [3H]ADP generated after 4 min was determined by HPLC analysis. In the presence of ATP 1, 10 or 100 µm, the concentrations of ADP generated were 0.044 ± 0.001, 0.203 ± 0.019 and 1.55 ± 0.2 µm, respectively. Data are the mean ± SEM of three independent experiments performed in duplicate.

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The poorly hydrolyzable ATP analogue ATPγS is an antagonist at both the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor expressed in 1321 N1 cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Preparation of washed mouse platelets and aggregation studies
  7. Cloning, sequencing and heterologous expression of the human P2Y12 receptor
  8. Cell culture
  9. cAMP assays
  10. Triphosphate nucleotide regeneration and purification
  11. Measurement of ectonucleotidase activity in 1321 N1 P2Y12 cells
  12. Results
  13. Effects of adenine diphosphate nucleotides on the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor
  14. The agonistic properties of adenine triphosphate nucleotides are lost in the presence of the regenerating system CP/CPK
  15. Adenine triphosphate nucleotides behave as antagonists at the mouse platelet P2Y12 receptor
  16. The poorly hydrolyzable ATP analogue ATPγS is an antagonist at both the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor expressed in 1321 N1 cells
  17. Discussion
  18. Acknowledgement
  19. References

To circumvent the problem of the rapid metabolism of adenine triphosphate nucleotides, we used the poorly hydrolyzable ATP analogue ATPγS, of which the rate of ATPase hydrolysis is only 1/200th of that of ATP [35]. Its potential antagonism at the P2Y12 receptor was assessed by performing competition experiments between ATPγS and the non-hydrolyzable ADP analogue ADPβS. This use of hydrolysis-resistant nucleotides (ADPβS and ATPγS) made it possible to work in the presence of apyrase (0.02 U mL-1), to remove any traces of released nucleotides. HPLC-purified ATPγS (60 µm) had no significant effect on stimulated cAMP levels, but caused a parallel right shift of the dose–response curve of cAMP inhibition induced by ADPβS in both inline image platelets and 1321 N1 P2Y12 cells (Fig. 5). The EC50 value of ADPβS-induced cAMP inhibition was 207 ± 65 nm in the presence of 60 µm ATPγS and 56 ± 7 nm in its absence in 1321 N1 P2Y12 cells and similarly 221 ± 110 nm in the presence of 60 µm ATPγS and 60 ± 19 nm in its absence in inline image platelets. ATPγS (60 µm) was also able to inhibit ADP (10 μM)-induced inline image platelet aggregation (data not shown), while its inhibitory effects could be overcome at a high concentration of ADP (100 μM). Hence ATPγS appeared to be a competitive antagonist of the P2Y12 receptor in both P2Y1-/- platelets and 1321 N1 P2Y12 cells.

image

Figure 5. The poorly hydrolyzable ATP analogue ATPγS behaves as an antagonist at the mouse platelet and recombinant human P2Y12 receptors. ADPβS inhibited the cAMP production induced by PGE1 in inline image mouse platelets or by forskolin in 1321 N1 P2Y12 cells in a dose-dependent manner. Experiments were performed in the presence of apyrase (0.02 U mL-1) to scavenge any traces of released nucleotides. In both inline image platelets and 1321 N1 P2Y12 cells, HPLC purified ATPγS (60 µm) caused a right shift of the dose–response curve. Data represent the percentage cell content of cAMP relative to the maximum level induced by either PGE1 or forskolin. Data are the mean ± SEM of three independent experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Preparation of washed mouse platelets and aggregation studies
  7. Cloning, sequencing and heterologous expression of the human P2Y12 receptor
  8. Cell culture
  9. cAMP assays
  10. Triphosphate nucleotide regeneration and purification
  11. Measurement of ectonucleotidase activity in 1321 N1 P2Y12 cells
  12. Results
  13. Effects of adenine diphosphate nucleotides on the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor
  14. The agonistic properties of adenine triphosphate nucleotides are lost in the presence of the regenerating system CP/CPK
  15. Adenine triphosphate nucleotides behave as antagonists at the mouse platelet P2Y12 receptor
  16. The poorly hydrolyzable ATP analogue ATPγS is an antagonist at both the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor expressed in 1321 N1 cells
  17. Discussion
  18. Acknowledgement
  19. References

The pharmacological profile of the P2Y12 receptor was investigated here both under native conditions using inline image mouse platelets and in a heterologous expression system, 1321 N1 cells transfected with the human P2Y12 receptor. The main objective of our work was to re-examine the effects of adenine triphosphate nucleotides at this receptor, since in several recent studies ATP and its triphosphate analogues were found to be full agonists at native or heterologously expressed P2Y12[11,17–19], which does not fit the data previously obtained in blood platelets. Thus, ATP and its adenine triphosphate analogues 2MeSATP and 2ClATP have been shown to be antagonists of ADP-induced adenylyl cyclase inhibition in platelets, a response mediated solely by activation of the P2Y12 receptor [22–24].

ADP and its diphosphate nucleotide analogues were found to be highly potent agonists at the P2Y12 receptor with a similar rank order of potency for inline image mouse platelets and 1321 N1 P2Y12 cells (Fig. 1). However, EC50 values were lower at the human P2Y12 receptor expressed in 1321 N1 cells than at the endogenous mouse platelet P2Y12 receptor. This might have been due to the high level of P2Y12 expression on 1321 N1 cells (∼ 1 × 106 receptors per cell as determined in binding studies using [33P]2MeSADP), but probably not to interspecies variations as the potency of diphosphate nucleotide analogues for mouse platelets was close to that reported for the P2Y12 receptor of human platelets [36].

When contaminating diphosphates were enzymatically removed using CP/CPK, ATP, 2ClATP and 2MeSATP lost all agonistic activity towards inline image platelets (Fig. 2), indicating that the diphosphate contaminants of commercial stock solutions were responsible for their apparent agonistic properties. In 1321 N1 P2Y12 cells, even after CP/CPK treatment, ATP, 2ClATP and 2MeSATP still inhibited cAMP accumulation (Fig. 4A). However, when the regenerated nucleotides were incubated with the cells in the continuous presence of CP/CPK, ATP and 2ClATP again lost their agonistic activity, confirming that these triphosphate nucleotides are not agonists at the P2Y12 receptor. These results also indicate that hydrolysis of adenine triphosphate nucleotides was more important on 1321 N1 cells than on platelets, which might be attributable to a higher ectonucleotidase activity on 1321 N1 P2Y12 cells as compared to platelets or to the longer incubation time before cAMP assay in experiments with cells (4 min vs. 1 min for platelets). Indeed, as shown in Fig. 4(B), considerable amounts of ADP were generated when ATP was incubated with 1321 N1 P2Y12 cells. Furthermore, there exists in fact previous reports of a high ectonucleotidase activity on 1321 N1 cells [26,30,37].

2MeSATP still slightly inhibited cAMP accumulation in 1321 N1 P2Y12 cells, despite the presence of CP/CPK during incubation with the cells. One possible explanation is that some metabolism of the triphosphate nucleotide still occurred, generating the highly potent P2Y12 receptor agonist 2MeSADP.

Purified ATP, 2ClATP or 2MeSATP inhibited ADP-induced adenylyl cyclase inhibition and aggregation in inline image platelets (Fig. 3), showing that these triphosphate nucleotides are antagonists of the mouse platelet P2Y12 receptor. Owing to their rapid metabolism by 1321 N1 ectonucleotidases, it was not possible to test the putative antagonistic activity of the triphosphate nucleotides in the 1321 N1 P2Y12 cell system. Hence the poorly hydrolyzable ATP analogue ATPγS was employed. ATPγS caused a parallel right shift of the dose–response curve of cAMP inhibition induced by ADPβS in both inline image mouse platelets and 1321 N1 P2Y12 cells, which could be overcome by using a high concentration of ADPβS (100 μM) (Fig. 5). ATPγS therefore appeared to be a competitive antagonist of the P2Y12 receptor in inline image platelets and 1321 N1 P2Y12 cells. Overall, these results indicate that adenine triphosphate nucleotides are not agonists of the P2Y12 receptor but rather antagonists, provided care is taken to remove diphosphate contaminants and to prevent the generation of diphosphate nucleotide derivatives by cell ectonucleotidases.

The discrepancies between our results and those of some previous reports might be due to the facts that firstly unpurified adenine triphosphate nucleotide analogues were used in these studies [11,19,20] and secondly the triphosphate nucleotides were incubated with cells for a long period of time (10–30 min), which could have enabled ectonucleotidases to generate diphosphate nucleotide derivatives. Furthermore, since diphosphate nucleotides are highly potent P2Y12 receptor agonists, trace amount of these diphosphate nucleotides might be sufficient to overcome the antagonism of the triphosphate nucleotides. Simon et al. found that ATP and 2ClATP behaved as weak partial agonists at the P2Y12 receptor of rat B10 cells. Here the discrepancy with our results is difficult to explain, since these authors used CP/CPK regenerated nucleotides and CP/CPK was maintained during the adenylyl cyclase assay. It is not likely that interspecies variation may be responsible for the difference observed in the agonistic effects of the triphosphate nucleotides between the rat P2Y12 receptor from B10 cells and the human platelet P2Y12 receptor. Indeed it has been shown previously that ATP is an antagonist of ADP-induced adenylyl cyclase inhibition in rat platelets [38].

It has also been reported that the extent of the receptor reserve can lead to an important increase in agonist potency and even convert a ligand of lower intrinsic efficacy from an apparent antagonist to an agonist [30]. However, this would appear unlikely in the case of 1321 N1 P2Y12 cells, since these cells display a high level of P2Y12 expression (∼ 1 × 106 receptors per cell) as evaluated in binding studies using [33P]2MeSADP (data not shown).

At least concerning the human P2Y12 receptor, ATP was shown recently to behave as a weak antagonist in a proteoliposomes reconstituted system, in which no enzymatic breakdown or interconversion of ATP to other adenine nucleotides occurs [39].

Overall findings indicate that the P2Y12 receptor is very sensitive to alterations of the phosphate side chain of adenine nucleotides. Thus, the diphosphate group is required for the agonistic activity of the nucleotide and substituents on C2 (2MeSADP or 2ClATP) greatly enhance its potency. The 5′-diphosphate chain is required for full agonist potency, while addition (ATP and its analogues 2ClATP and 2MeSATP) or removal of a phosphate group (AMP and its analogue 2MeSAMP) generates antagonists. Substituents on C2 of adenine triphosphate nucleotides confer gains in antagonistic activity, ATP being a less potent inhibitor of ADP-induced inhibition of adenylyl cyclase activity than 2ClATP or 2MeSATP (Fig. 3). Moreover, it is noteworthy that the most potent known P2Y12 antagonist, an AR-C compound, is an analogue of ATP substituted in position C2 with a 3,3,3-trifluoropropylthio group [40].

In vivo, ATP can be released from numerous cell types like neurones or glial cells [41], or coreleased with ADP from the dense granules of activated platelets. Although we show here that ATP is an antagonist of ADP at the P2Y12 receptor, it seems to have weak potency. ATP was likewise found to be a weak antagonist of the ADP-induced calcium response mediated by activation of the platelet P2Y1 receptor [27]. Therefore, at least for platelets and under in vivo conditions, where the local ADP and ATP concentrations are estimated to be 7–10 and 15–20 µm, respectively, after platelet secretion [42], ATP might not interfere with ADP signaling through the P2Y1 or P2Y12 receptor, while it most probably could activate the platelet P2X1 receptor [2]. Furthermore, in view of the active extracellular adenine nucleotide metabolism present on the vessel wall and on circulating cells, released ATP might serve as an extracellular reservoir for ADP generation. As a consequence, ectonucleotidases could play an important role in determining the nucleotide content of the extracellular environment and act as regulators of responsiveness to tri- and diphosphate nucleotides.

In summary, our results show that the P2Y12 receptor expressed on platelets or cells shares a common pharmacology with the P2Y1 receptor, at which adenine diphosphate nucleotides are highly potent agonists while ATP and other adenine triphosphate nucleotides are antagonists, which is of importance in term of structure-activity relationship and development of new drug-designed antagonists.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Materials
  6. Preparation of washed mouse platelets and aggregation studies
  7. Cloning, sequencing and heterologous expression of the human P2Y12 receptor
  8. Cell culture
  9. cAMP assays
  10. Triphosphate nucleotide regeneration and purification
  11. Measurement of ectonucleotidase activity in 1321 N1 P2Y12 cells
  12. Results
  13. Effects of adenine diphosphate nucleotides on the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor
  14. The agonistic properties of adenine triphosphate nucleotides are lost in the presence of the regenerating system CP/CPK
  15. Adenine triphosphate nucleotides behave as antagonists at the mouse platelet P2Y12 receptor
  16. The poorly hydrolyzable ATP analogue ATPγS is an antagonist at both the mouse platelet P2Y12 receptor and the recombinant human P2Y12 receptor expressed in 1321 N1 cells
  17. Discussion
  18. Acknowledgement
  19. References
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