Human Ntera-2/D1 neuronal progenitor cells endogenously express a functional P2Y1 receptor

We report here that human Ntera-2/D1 (NT-2) cells, an undifferentiated committed neuronal progenitor cell line, endogenously express a functional P2Y(1) receptor, while other P2Y subtypes, except perhaps P2Y(4), are not functionally expressed. Quantitative RT-PCR analysis showed that NT-2 cells abundantly express mRNA for P2Y(1) and P2Y(11) receptors, while P2Y(2) and P2Y(4) receptors were detected at considerably lower levels. Western blot analysis also demonstrated expression of P2Y(1) receptors and Galpha(q/11) subunits. Various nucleotides induced intracellular Ca(2+) mobilisation in NT-2 cells in a concentration-dependent manner with a rank order potency of 2-MeSADP > 2-MeSATP > ADP > ATP > UTP > ATPgammaS, a profile resembling that of human P2Y(1) receptors. Furthermore, P2Y(1) receptor-specific (A3P5P) and P2Y-selective (PPADS, suramin) antagonists inhibited adenine nucleotide-induced Ca(2+) responses in a concentration-dependent manner, consistent with expression of a P2Y(1) receptor. Moreover, of seven adenine nucleotides tested, only Bz-ATP and ATPgammaS elicited small increases in cAMP formation suggesting that few, if any, functional P2Y(11) receptors were expressed. P2Y(1) receptor-selective adenine nucleotides, including 2-MeSADP and ADP, also induced concentration-dependent phosphorylation and hence, activation of the extracellular-signal regulated protein kinases (ERK1/2). NT-2 cells, therefore, provide a useful neuronal-like cellular model for studying the precise signalling pathways and physiological responses mediated by a native P2Y(1) receptor.     

Evaluation of reactive blue 2 derivatives as selective antagonists for P2Y receptors

P2Y receptor pharmacology is hampered by a lack of subtype selective antagonists. However, a recent study evaluated series of compounds, structurally related to the dye reactive blue 2, for their antagonist selectivity at P2X vs. P2Y receptors. Acid blue 129, acid blue 80, acid blue 25 and acid violet 34 were found to be the most potent of the antagonists studied, at P2Y receptors [Naunyn Schmiedeberg's Arch. Pharmacol. 357 (1998) 111]. In this study, we have determined the ability of these four agents to selectively antagonize inositol phosphate turnover mediated by P2Y1 and P2Y2 receptors that are natively expressed in bovine aortic endothelial (BAE) cells. Acid blue 129, acid blue 80, and acid violet 34 shifted the dose-response curve of the P2Y1 agonist 2-methylthio adenosine trisphosphate (2MeSATP) to the right. Acid blue 129 and acid blue 80 were also very weak antagonists of the P2Y2 agonist uridine 5'-triphosphate (UTP). At 30 and 100 microM, acid violet 34 failed to have any significant effect on the dose-response to UTP. However, at 10 microM, acid violet 34 enhanced the UTP responses. Acid blue 80, acid blue 129 and acid violet 34 are P2Y vs. P2X selective, but show poor selectivity between P2Y1 and P2Y2 receptors and are therefore of limited use in the field of P2Y receptor pharmacology. Furthermore, contrary to previous reports, acid blue 25 is not a P2Y-selective antagonist.     

The influence of P2Y12 receptor deficiency on the platelet inhibitory activities of prasugrel in a mouse model: evidence for specific inhibition of P2Y12 receptors by prasugrel

Prasugrel is a novel orally active thienopyridine with faster, higher and more reliable inhibition of platelet aggregation than clopidogrel reflecting its metabolism in vivo to an active metabolite with selective P2Y(12) antagonistic activity. Several lines of evidence support the contention that prasugrel provides selective P2Y(12) receptor antagonistic activity. To date, however, direct evidence of P2Y(12) specific action by prasugrel in vivo is limited. In the present study, effects of prasugrel on ex vivo platelet aggregation were examined in wild type (WT) and P2Y(12)(-/-) mice. In WT mice, prasugrel showed platelet inhibition that was 8.2 times more potent than clopidogrel. In P2Y(12)(-/-) mice, ADP induced platelet aggregation was minimal, and its extent was similar to that in prasugrel-treated WT mice. In addition, no further inhibition of platelet aggregation was observed after administration of prasugrel to P2Y(12)(-/-) mice. Furthermore, prasugrel-treated WT mice showed similar aggregation patterns using collagen- and murine PAR-4 agonist peptide to those of P2Y(12)(-/-) mice treated with vehicle or prasugrel. Overall, these results clearly provide additional in vivo evidence that prasugrel has selective P2Y(12) antagonistic activity.     

Molecular cloning and characterization of rat P2Y4 nucleotide receptor

An intronless open reading frame encoding a protein (361aa in length) was isolated from a rat genomic library probed with a DNA fragment from rat heart. This protein showed 83% sequence identity with the human P2Y₄ (hP2Y₄) receptor and represents a homologue of the human pyrimidinoceptor. However, the rP2Y₄ receptor is not selective for uridine nucleotides and, instead, shows an agonist potency order of ITP=ATP=ADP(pure)=UTP=ATPγS=2-MeSATP=Ap₄A>UDP(pure). ADP, ATPγS, 2-MeSATP and UDP are partial agonists. Thus, in terms of agonist profile, rP2Y₄ is more like the P2U receptor subtype. The rP2Y₄ receptor was reversibly antagonized by Reactive blue 2 but not by suramin which, otherwise, inhibits the hP2Y₂ receptor (a known P2U receptor). Thus, rP2Y₄ and the P2Y₂ subtype appear to be structurally distinct forms of the P2U receptor (where ATP and UTP are equi-active) but can be distinguished as suramin-insensitive and suramin-sensitive P2U receptors, respectively.     

Effects of P2Y(1) receptor antagonism on the reactivity of platelets from patients with stable coronary artery disease using aspirin and clopidogrel

BACKGROUND AND PURPOSE

P2Y₁ is a purine receptor that triggers platelet aggregation. Its inhibition was studied in patients with stable coronary artery disease (CAD) receiving standard anti-platelet therapy.

EXPERIMENTAL APPROACH

Blood samples from 10 patients on aspirin therapy (ASA, 80 mg·day⁻¹) were withdrawn before and 24 h after the administration of 450 mg clopidogrel (ASA/C) and were anti-coagulated with citrate or hirudin/PPACK in the presence or absence of the P2Y₁ inhibitor MRS2179 (M, 100 µM). Platelet responses to ADP (2.5 µM) and TRAP (2.5 µM), and collagen-induced thrombosis under flow conditions were analysed.

KEY RESULTS

Compared with ASA, ASA + M strongly inhibited ADP-induced peak platelet aggregation (88%), late aggregation (84%), P-selectin expression (85%) and α_IIbβ₃ activation (62%) (28%, 65%, 70% and 51% inhibition, respectively, for ASA/C vs. ASA). ASA + M also inhibited platelet/monocyte and platelet/neutrophil conjugate formation by 69% and 71% (57% and 59% for ASA/C vs. ASA). In TRAP-activated blood, ASA + M unexpectedly inhibited α_IIbb₃ activation by 30%. In blood perfused in collagen-coated glass capillaries (shear rate of 1500 s⁻¹), ASA/C prevented thrombus growth beyond 5 min in relation to thrombus fragments embolization. ASA + M with or without clopidogrel completely prevented thrombus formation. Finally, ex vivo addition of MRS2179 and ASA to the blood of healthy donors markedly blocked thrombus formation on collagen in flow conditions, in contrast to ASA plus the P2Y₁₂ inhibitor 2-MeSAMP.

CONCLUSIONS AND IMPLICATIONS

Through particularly efficient complementarities with ASA to inhibit platelet activation and thrombus formation, the inhibition of P2Y₁ in the blood of patients with CAD appears to play a more important role than previously anticipated.     

UDP induces intestinal epithelial migration via the P2Y6 receptor

BACKGROUND AND PURPOSE

Extracellular nucleotides are released at high concentrations from damaged cells and function through P2 receptor activation. Intestinal epithelial restitution, which is defined as cell migration independent of cell proliferation, is an important initial step in the process of wound healing. In this study, we investigated the role of extracellular nucleotides in intestinal epithelial migratory responses.

EXPERIMENTAL APPROACH

Wound-healing and trans-well migration assays were performed with a rat intestinal epithelial cell line (IEC-6). The concentrations of extracellular nucleotides released from injured IEC-6 cells were measured by HPLC. TGF-β expression was assessed by RT-PCR and elisa.

KEY RESULTS

Scratching the monolayer of IEC-6 cells induced cell migration. Pretreatment with apyrase or MRS2578, a selective P2Y₆ antagonist, inhibited the wound-induced cell migration. Among the cellular nucleotides, only ATP and uridine 5''-diphosphate (UDP) were detected in the culture medium after cell wounding. Exogenously applied UDP dose-dependently enhanced the migration more effectively than ATP but did not induce proliferation. In addition, cell wounding and UDP increased the expression of TGF-β, and both the wound-induced and UDP-enhanced migration were inhibited by MRS2578 or ALK5Inhibitor (ALK5i), a TGF-β receptor blocker. Furthermore, cell wounding and UDP stimulation up-regulated the expression of P2Y₆ receptor mRNA, and this effect was suppressed by MRS2578 or ALK5i.

CONCLUSION AND IMPLICATIONS

Wound-induced UDP evokes intestinal epithelial restitution by activation of P2Y₆ receptors, which mediates de novo synthesis of TGF-β. In addition, the expression of P2Y₆ receptors is increased by cell wounding and UDP, which constitutes a positive-feedback loop for mucosal repair.     

Novel consequences of voltage-dependence to G-protein-coupled P2Y1 receptors

BACKGROUND AND PURPOSE: Emerging evidence suggests that activation of G-protein-coupled receptors (GPCRs) can be directly regulated by membrane voltage. However, the physiological and pharmacological relevance of this effect remains unclear. We have further examined this phenomenon for P2Y1 receptors in the non-excitable megakaryocyte using a range of agonists and antagonists. EXPERIMENTAL APPROACH: Simultaneous whole-cell patch clamp and fura-2 fluorescence recordings of rat megakaryocytes, which lack voltage-gated Ca2+ influx, were used to examine the voltage-dependence of P2Y1 receptor-evoked IP3-dependent Ca2+ mobilization. RESULTS: Depolarization transiently and repeatedly enhanced P2Y1 receptor-evoked Ca2+ mobilization across a wide concentration range of both weak, partial and full, potent agonists. Moreover, the amplitude of the depolarization-evoked [Ca2+]i increase displayed an inverse relationship with agonist concentration, such that the greatest potentiating effect of voltage was observed at near-threshold levels of agonist. Unexpectedly, depolarization also stimulated an [Ca2+]i increase in the absence of agonist during exposure to the competitive antagonists A3P5PS and MRS2179, or the allosteric enhancer 2,2'-pyridylisatogen tosylate. A further effect of some antagonists, particularly suramin, was to enhance the depolarization-evoked Ca2+ responses during co-application of an agonist. Of several P2Y1 receptor inhibitors, only SCH202676, which has a proposed allosteric mechanism of action, could block ADP-induced voltage-dependent Ca2+ release. CONCLUSIONS AND IMPLICATIONS: The ability of depolarization to potentiate GPCRs at near-threshold agonist concentrations represents a novel mechanism for coincidence detection. Furthermore, the induction and enhancement of voltage-dependent GPCR responses by antagonists has implications for the design of therapeutic compounds.     

Human adenosine A1 receptor and P2Y2-purinoceptor-mediated activation of the mitogen-activated protein kinase cascade in transfected CHO cells

1. The mitogen-activated protein (MAP) kinase signalling pathway can be activated by a variety of heterotrimeric G_i/G_o protein-coupled and G_q/G₁₁ protein-coupled receptors. The aims of the current study were: (i) to investigate whether the G_i/G_o protein-coupled adenosine A₁ receptor activates the MAP kinase pathway in transfected Chinese hamster ovary cells (CHO-A1) and (ii) to determine whether adenosine A1 receptor activation would modulate the MAP kinase response elicited by the endogenous P2Y2 purinoceptor.
2. The selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC50 7.1±0.4 nM). CPA-mediated increases in MAP kinase activity were blocked by PD 98059 (50 μM; 89±4% inhibition), an inhibitor of MAP kinase kinase 1 (MEKI) activation, and by pre-treating cells with pertussis toxin (to block G_i/G_o-dependent pathways).
3. Adenosine A₁ receptor-mediated activation of MAP kinase was abolished by pre-treatment with the protein tyrosine inhibitor, genistein (100 μM; 6±10% of control). In contrast, daidzein (100 μM), the inactive analogue of genistein had no significant effect (96±12 of control). MAP kinase responses to CPA (1 μM) were also sensitive to the phosphatidylinositol 3-kinase inhibitors wortmannin (100 nM; 55±8% inhibition) and LY 294002 (30 μM; 40±5% inhibition) but not to the protein kinase C (PKC) inhibitor Ro 31-8220 (10 μM).
4. Activation of the endogenous P2Y₂ purinoceptor with UTP also stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC₅₀=1.6±0.3 μM). The MAP kinase response to UTP was partially blocked by pertussis toxin (67±3% inhibition) and by the PKC inhibitor Ro 31-8220 (10 μM; 45±5% inhibition), indicating the possible involvement of both G_i/G_o protein and G_q protein-dependent pathways in the overall response to UTP.
5. CPA and UTP stimulated concentration-dependent increases in the phosphorylation state of the 42 kDa and 44 kDa forms of MAP kinase as demonstrated by Western blotting.
6. Co-activation of CHO-A1 cells with CPA (10 nM) and UTP (1 μM) produced synergistic increases in MAP kinase activity which were not blocked by the PKC inhibitor Ro 31-8220 (10 μM).
7. Adenosine A1 and P2Y2 purinoceptor activation increased the expression of luciferase in CHO cells transfected with a luciferase reporter gene containing the c-fos promoter. However, co-activating these two receptors produced only additive increases in luciferase expression.
8. In conclusion, our studies have shown that the transfected adenosine A₁ receptor and the endogenous P2Y₂ purinoceptor couple to the MAP kinase signalling pathway in CHO-A1 cells. Furthermore, co-stimulation of the adenosine A1 receptor and the P2Y₂ purinoceptor produced synergistic increases in MAP kinase activity but not c-fos mediated luciferase expression.

     

Diastereoselectivity of the P2Y11 nucleotide receptor: mutational analysis

Background and purpose:

The P2Y₁₁ receptor, a member of the group of metabotropic nucleotide receptors, shows a stereospecific ligand recognition of P_α-substituted ATP derivatives (ATP-α-S isomers). These compounds are suitable candidates for the development of selective P2Y₁₁ receptor agonists that might be used as immune modulators. We have analysed the binding mode of ATP at the P2Y₁₁ receptor by molecular modeling and site-directed mutagenesis. Based on our recent findings, we decided to decipher the molecular determinants of stereoselective recognition at the P2Y₁₁ receptor.

Experimental approach:

Two amino acid residues [Glu186 in the extracellular loop 2 and Arg268 in the transmembrane domain 6 (TM6)], which are part of the nucleotide-binding pocket, were selected and studied by mutational analyses. We expected these residues to be involved in determining the stereospecificity of the P2Y₁₁ receptor.

Key results:

After mutation of Arg268 to alanine or glutamine, the stereospecific recognition of the ATP-α-S isomers at the P2Y₁₁ receptor was lost. In contrast, at the Glu186Ala receptor mutant, the stereoselective differentiation between these isomers was increased. On the Arg268Gln/Glu186Ala double mutant we observed no further effect, except for additivity in the decrease in potency of both isomers, as compared with the single-point mutants.

Conclusions and implications:

Our results show that the stereospecificity of the P2Y₁₁ receptor for P_α-substituted ATP derivatives is largely determined by the basic residue Arg268 in TM6. This will allow the design of receptor-subtype selective ligands.     

Cloning, pharmacological characterisation and distribution of the rat G-protein-coupled P2Y(13) receptor

The human P2Y(13) receptor is a new receptor characterized by coupling to Gi, responsiveness to adenine di-phospho-nucleotides and blockade by the P2Y antagonist AR-C69931MX. The mouse P2Y(13) ortholog has also been reported. Here we report, for the first time, the cloning of rat P2Y(13) receptor, its pharmacological analysis and tissue distribution. Rat P2Y(13) is 79% and 87% identical to human and mouse P2Y(13) receptors, respectively. Expression of rP2Y(13) receptor in 1321N1 cells induced the appearance of responses to the typical P2Y(13) receptor agonists ADP and 2MeSADP, as detected by stimulation of [(35)S]GTPgammaS binding. Agonist activities were higher in cells transfected with rP2Y(13) receptor in the presence of the Galpha(16) subunit; in all cases agonist effects were abolished by pertussis toxin pre-treatment. At variance from both human and mouse receptors, ADP was more potent than 2MeSADP. Other nucleotides and sugar-nucleotides were ineffective. Both in the absence and presence of Galpha(16), activation of rP2Y(13) receptor by ADP and 2MeSADP was completely inhibited by nM concentrations of AR-C69931MX. In contrast, no inhibition of rP2Y(13) receptor was induced by the selective P2Y(1) receptor antagonist MRS2179. rP2Y(13) receptor showed highest expression levels in spleen, followed by liver and brain (with particularly high levels in cortex and striatum as reported in man), suggesting important roles in the nervous and immune systems. Expression levels comparable to those of the other cloned P2Y receptors were found in primary rat astrocytes, indicating a possible role in reactive astrogliosis. Hence, rat P2Y(13) receptor displays several similarities but also interesting differences with its human and mouse orthologs, that will have to be taken into account when characterizing the pathophysiological roles of this receptor in the rat animal models.     

The novel suramin analogue NF864 selectively blocks P2X1 receptors in human platelets with potency in the low nanomolar range

The role of ATP-stimulated P2X₁ receptors in human platelets is still unclear. They may act alone or in synergy with other pathways, such as P2Y₁ or P2Y₁₂ receptors, to accelerate and enhance calcium mobilisation, shape change and aggregation. To date very few pharmacological means of selectively inhibiting platelet P2X₁ receptors have been described, although recent work has shown that suramin is a useful lead compound for the development of high-affinity P2X₁ antagonists. We therefore investigated the effects of a series of bivalent and tetravalent suramin analogues on meATP (P2X₁ receptors)-induced or ADP (P2Y₁ receptors)-induced intracellular calcium increases and shape change, as well as on ADP-induced aggregation (P2Y₁ & P2Y₁₂ receptors) in human platelets. Changes in intracellular calcium were measured using standard fluorescence techniques, while shape change and aggregation were determined by turbidimetry. The novel tetravalent compound NF864 (8,8,8,8-(carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakis-naphthalene-1,3,5-trisulfonic acid-dodecasodium salt) proved to be the most potent platelet P2X₁ antagonist reported to date, blocking meATP-induced Ca²⁺ increases and shape change in a concentration-dependent manner, with a pA₂ of 8.17 and 8.49, respectively. The ability to inhibit the platelet P2X₁ receptor displayed the following order : NF864>NF449NF110>NF023=MK-HU1=suramin. A different antagonistic profile was observed for ADP-induced Ca²⁺ increases, shape change and aggregation; however, overall four compounds showed sufficient ability to selectively inhibit P2X₁ responses, with the order NF110>NF449NF864MK-HU1. Therefore, these compounds should prove useful tools for investigating the functional significance of platelet P2X₁ receptors in thrombosis and haemostasis, NF864 being the most promising compound.     

Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2′-deoxyadenosine 3′,5′-bisphosphate

The antagonist activity of N⁶-methyl 2′-deoxyadenosine 3′,5′-bisphosphate (N6MABP) has been examined at the phospholipase C-coupled P2Y₁ receptor of turkey erythrocyte membranes. N6MABP antagonized 2MeSATP-stimulated inositol phosphate hydrolysis with a potency approximately 20 fold greater than the previously studied parent molecule, adenosine 3′,5′-bisphosphate. The P2Y₁ receptor antagonism observed with N6MABP was competitive as revealed by Schild analysis (pK_B=6.99±0.13). Whereas N6MABP was an antagonist at the human P2Y₁ receptor, no antagonist effect of N6MABP was observed at the human P2Y₂, human P2Y₄ or rat P2Y₆ receptors.     

P2Y12 receptor inhibitors for secondary prevention of ischemic stroke

Introduction: Ischemic stroke (IS) is a major cause of death and disability worldwide. The P2Y₁₂ receptor plays a critical role in the formation of a stable thrombus leading to ischemic complications. Therefore, P2Y₁₂ receptor inhibitors constitute a major antiplatelet strategy in the secondary prevention of IS.

Areas covered: We searched articles about P2Y₁₂ receptor inhibitors and stroke in PubMed published until December 2014. This is a comprehensive review of the role of P2Y₁₂ receptor inhibitors alone and in combination with aspirin in the secondary prevention of noncardioembolic stroke.

Expert opinion: The potential benefit of more potent antiplatelet therapy for secondary stroke prevention must be weighed against the risk of bleeding in patients with IS. Short-term (≤ 3 months) dual antiplatelet therapy with clopidogrel and aspirin that is initiated early after IS or transient ischemic attack due to large artery atherosclerosis appears most efficient.     

UTP reduces infarct size and improves mice heart function after myocardial infarct via P2Y2 receptor

Pyrimidine nucleotides are signaling molecules, which activate G protein-coupled membrane receptors of the P2Y family. P2Y₂ and P2Y₄ receptors are part of the P2Y family, which is composed of 8 subtypes that have been cloned and functionally defined. We have previously found that uridine-5′-triphosphate (UTP) reduces infarct size and improves cardiac function following myocardial infarct (MI). The aim of the present study was to determine the role of P2Y₂ receptor in cardiac protection following MI using knockout (KO) mice, in vivo and wild type (WT) for controls. In both experimental groups used (WT and P2Y₂^−/− receptor KO mice) there were 3 subgroups: sham, MI, and MI + UTP. 24 h post MI we performed echocardiography and measured infarct size using triphenyl tetrazolium chloride (TTC) staining on all mice. Fractional shortening (FS) was higher in WT UTP-treated mice than the MI group (44.7 ± 4.08% vs. 33.5 ± 2.7% respectively, p < 0.001). However, the FS of P2Y₂^−/− receptor KO mice were not affected by UTP treatment (34.7 ± 5.3% vs. 35.9 ± 2.9%). Similar results were obtained with TTC and hematoxylin and eosin stainings. Moreover, troponin T measurements demonstrated reduced myocardial damage in WT mice pretreated with UTP vs. untreated mice (8.8 ± 4.6 vs. 12 ± 3.1 p < 0.05). In contrast, P2Y₂^−/− receptor KO mice pretreated with UTP did not demonstrate reduced myocardial damage. These results indicate that the P2Y₂ receptor mediates UTP cardioprotection, in vivo.     

Pharmacology of neuropeptide Y receptor antagonists. Focus on cardiovascular functions

Neuropeptide Y is one of the most abundant mammalian neuropeptides identified to date. The possible actions of neuropeptide Y, that is co-localized and released with noradrenaline, as a sympathetic co-transmitter has attracted much attention during the last decade. In recent years, several non-peptide antagonists with high subtype selectivity for neuropeptide Y receptors have been introduced. With them, the status of neuropeptide Y as a sympathetic transmitter has been established, and so have profound cardiovascular effects mediated by neuropeptide Y Y(1) and Y(2) receptors. Significant release of neuropeptide Y occurs especially upon stronger sympathetic activation, and recent data suggest that the importance of neuropeptide Y seems enhanced in stress-related cardiovascular disorders. The true significance of neuropeptide Y has thus started to unfold, owing to the presence of the first generation of selective neuropeptide Y receptor antagonists. This review concerns the pharmacology of these agents, what we have learnt from them, and might find out in the future.     

2-Alkylthio-substituted platelet P2Y12 receptor antagonists reveal pharmacological identity between the rat brain Gi-linked ADP receptors and P2Y12

The Gi-linked platelet ADP receptor, now designated as P2Y12, accounts for ADP-induced inhibition of adenylyl cyclase in platelets and certain clonal rat cell lines. The pharmacology of this receptor is well characterized. Based on the functional approach of [35S]GTPgammaS autoradiography, we recently disclosed the widespread presence of Gi-linked ADP receptors in the rat nervous system. Based on initial pharmacological analysis, these receptors were strikingly similar with P2Y12. Here, we extend this analysis by comparing the potencies of six 2-alkylthio-substituted ATP analogues, including the adenosine-aspartate conjugate 2-hexylthio-AdoOC(O)Asp2 and five AR-C compounds (AR-C67085, AR-C69931, AR-C78511, AR-C69581, AR-C70300) with wide range of affinities towards P2Y12, in reversing 2-methylthio-ADP stimulated G protein activity in rat brain sections and human platelet membranes. Closely matching pIC50 values (r2=0.99) revealed pharmacological similarity between the two receptors with one exception: AR-C67085 more avidly recognized the platelet P2Y12. Further analysis of the rat brain pIC50 data against those available for three of the AR-C compounds in reversing P2Y12-mediated adenylyl cyclase inhibition in rat platelets (r2=0.96) and rat C6 glioma cells (r2=1.00) demonstrated that the three P2Y receptors are pharmacologically indistinguishable. We conclude that the rat brain Gi-linked ADP receptors, as revealed using [35S]GTPgammaS autoradiography, correspond to P2Y12.     

P2Y12 receptor inhibitors: an evolution in drug design to prevent arterial thrombosis

Introduction: P2Y12 inhibitors are a critical component of dual antiplatelet therapy (DAPT), which is the superior strategy to prevent arterialthrombosis in patients with acute coronary syndromes (ACS) and undergoing stent implantation..

Areas covered: Basic science articles, clinical studies, and reviews from 1992–2017 were searched using Pubmed library to collet impactful literature. After an introduction to the purinergic receptor biology, this review summarizes current knowledge on P2Y12 receptor inhibitors. Furthermore, we describe the subsequent improvements of next-generation P2Y12 receptor inhibitors facing the ambivalent problem of bleeding events versus prevention of arterial thrombosis in a variety of clinical settings. Therefore, we summarize data from relevant preclinical and clinical trials of currently approved P2Y12 receptor inhibitors (clopidogrel, prasugrel, ticagrelor, cangrelor) and provide strategies of drug switching and management of bleeding events.

Expert opinion: An enormous amount of pharmacologic and clinical data is available for the application of P2Y12 receptor inhibitors. Today prasugrel, ticagrelor and clopidogrel are the standard of care drugs during dual antiplatelet therapy for ACS patients, but have considerable rates of bleeding. Recent and future clinical trials will provide evidence for subsequent escalation and de-escalation strategies of P2Y12 receptor inhibition. These data may pave the way for an evidence-based, individualized P2Y12 receptor inhibitor therapy.     

Cangrelor: a novel P2Y12 receptor antagonist

Antiplatelet therapy is critical in the prevention of thrombotic complications of acute coronary syndrome and percutaneous coronary interventions. Current antiplatelet agents (aspirin, clopidogrel and glycoprotein IIb/IIIa antagonists) have demonstrated the capacity to reduce major adverse cardiac events. However, these agents have limitations that compromise their clinical utility. The platelet P2Y₁₂ receptor plays a central role in platelet function and is a focus in the development of antiplatelet therapies. Cangrelor is a potent, competitive inhibitor of the P2Y₁₂ receptor that is administered by intravenous infusion and rapidly achieves near complete inhibition of ADP-induced platelet aggregation. This investigational drug has been studied for use during coronary procedures and the management of patients experiencing acute coronary syndrome and is undergoing evaluation for use in the prevention of perioperative stent thrombosis.     

P2Y receptors and atherosclerosis in apolipoprotein E-deficient mice

Background and purpose:

P2Y nucleotide receptors are involved in the regulation of vascular tone, smooth muscle cell (SMC) proliferation and inflammatory responses. The present study investigated whether they are involved in atherosclerosis.

Experimental approach:

mRNA of P2Y receptors was quantified (RT-PCR) in atherosclerotic and plaque-free aorta segments of apolipoprotein E-deficient (apoE^–/–) mice. Macrophage activation was assessed in J774 macrophages, and effects of non-selective purinoceptor antagonists on atherosclerosis were evaluated in cholesterol-fed apoE^–/– mice.

Key results:

P2Y₆ receptor mRNA was consistently elevated in segments with atherosclerosis, whereas P2Y₂ receptor expression remained unchanged. Expression of P2Y₁ or P2Y₄ receptor mRNA was low or undetectable, and not influenced by atherosclerosis. P2Y₆ mRNA expression was higher in cultured J774 macrophages than in cultured aortic SMCs. Furthermore, immunohistochemical staining of plaques demonstrated P2Y₆-positive macrophages, but few SMCs, suggesting that macrophage recruitment accounted for the increase in P2Y₆ receptor mRNA during atherosclerosis. In contrast to ATP, the P2Y₆-selective agonist UDP increased mRNA expression and activity of inducible nitric oxide synthase and interleukin-6 in J774 macrophages; this effect was blocked by suramin (100–300 µM) or pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS, 10–30 µM). Finally, 4-week treatment of cholesterol-fed apoE^–/– mice with suramin or PPADS (50 and 25 mg·kg⁻¹·day⁻¹ respectively) reduced plaque size, without changing plaque composition (relative SMC and macrophage content) or cell replication.

Conclusions and implications:

These results suggest involvement of nucleotide receptors, particularly P2Y₆ receptors, during atherosclerosis, and warrant further research with selective purinoceptor antagonists or P2Y₆ receptor-deficient mice.     

ERK1/2 activation is involved in the neuroprotective action of P2Y13 and P2X7 receptors against glutamate excitotoxicity in cerebellar granule neurons

Cerebellar granule neurons express several types of nucleotide receptors, with the metabotropic P2Y₁₃ and the ionotropic P2X7 being the most relevant in this model. In the present study we investigated the role of P2Y₁₃ and P2X7 nucleotide receptors in ERK1/2 signalling. The nucleotidic agonists 2MeSADP (2-methylthioadenosine-5′-diphosphate) for P2Y₁₃ and BzATP (2′(3′)-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate) for P2X7 receptors were coupled to ERK1/2 activation in granule neurons, being able to increase around two-fold the levels of ERK1/2 phosphorylation. These effects were sensitive to the inhibitory action of the antagonists MRS-2211 and A-438079, specific for P2Y₁₃ and P2X7 receptors, respectively. Although both receptor subtypes shared the same pattern of transient ERK1/2 phosphorylation, they differed in the intracellular cascades they triggered, being PI3K-dependent for P2Y₁₃ and calcium/calmodulin kinase II (CaMKII)-dependent for P2X7. These two different ERK-mediated pathways were involved in the neuroprotective effects displayed by both P2Y₁₃ and P2X7 receptors against apoptosis induced by an excitotoxic concentration of glutamate, in a similar manner to the neurotrophin, BDNF. In addition, P2Y₁₃ and P2X7 receptor agonists were also able to phosphorylate and activate the ERK-dependent target CREB, which could be involved in their neuroprotective effect. These results indicate that nucleotide receptors share with trophic factors the same survival routes in neurons, such as the ERK signalling route, and therefore, can contribute to the maintenance of granule neurons in conditions in which survival is being compromised.