P2Y2 receptor agonist with enhanced stability protects the heart from ischemic damage in vitro and in vivo

Extracellular nucleotides acting via P2 receptors play important roles in cardiovascular physiology/pathophysiology. Pyrimidine nucleotides activate four G protein-coupled P2Y receptors (P2YRs): P2Y₂ and P2Y₄ (UTP-activated), P2Y₆, and P2Y₁₄. Previously, we showed that uridine 5′-triphosphate (UTP) activating P2Y₂R reduced infarct size and improved mouse heart function after myocardial infarct (MI). Here, we examined the cardioprotective role of P2Y₂R in vitro and in vivo following MI using uridine-5′-tetraphosphate δ-phenyl ester tetrasodium salt (MRS2768), a selective and more stable P2Y₂R agonist. Cultured rat cardiomyocytes pretreated with MRS2768 displayed protection from hypoxia [as revealed by lactate dehydrogenase (LDH) release and propidium iodide (PI) binding], which was reduced by P2Y₂R antagonist, AR-C118925 (5-((5-(2,8-dimethyl-5H-dibenzo[a,d][7]annulen-5-yl)-2-oxo-4-thioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-(1H-tetrazol-5-yl)furan-2-carboxamide). In vivo, echocardiography and infarct size staining of triphenyltetrazolium chloride (TTC) in 3 groups of mice 24 h post-MI: sham, MI, and MI+MRS2768 indicated protection. Fractional shortening (FS) was higher in MRS2768-treated mice than in MI alone (40.0 ± 3.1 % vs. 33.4 ± 2.7 %, p < 0.001). Troponin T and tumor necrosis factor-α (TNF-α) measurements demonstrated that MRS2768 pretreatment reduced myocardial damage (p < 0.05) and c-Jun phosphorylation increased. Thus, P2Y₂R activation protects cardiomyocytes from hypoxia in vitro and reduces post-ischemic myocardial damage in vivo.     

The platelet P2Y12 receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [3H]PSB-0413

Various radioligands have been used to characterize and quantify the platelet P2Y₁₂ receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y₁ and P2Y₁₂. We used the [³H]PSB-0413 selective P2Y₁₂ receptor antagonist radioligand to reevaluate the number of P2Y₁₂ receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [³H]PSB-0413 bound to 425 ± 50 sites/platelet (K_D = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y₁₂, with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y₁ ligand MRS2179 and the P2X₁ ligand α,β-Met-ATP did not displace [³H]PSB-0413 binding. Patients with severe P2Y₁₂ deficiency displayed virtually no binding of [³H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y₁₂ receptor had normal binding. Studies in mice showed that: (1) [³H]PSB-0413 bound to 634 ± 87 sites/platelet (K_D = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [³H]PSB-0413 bound to 1 mg protein of platelet membranes (K_D = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [³H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y₁₂ receptors, to identify patients with P2Y₁₂ deficiencies or quantify the effect of P2Y₁₂ targeting drugs.     

UTP Controls Cell Surface Distribution and Vasomotor Activity of the Human P2Y2 Receptor through an Epidermal Growth Factor Receptor-transregulated Mechanism

Extracellular nucleotides transmit signals into the cells through the P2 family of cell surface receptors. These receptors are amply expressed in human blood vessels and participate in vascular tone control; however, their signaling mechanisms remain unknown. Here we show that in smooth muscle cells of isolated human chorionic arteries, the activation of the P2Y₂ receptor (P2Y₂R) induces not only its partition into membrane rafts but also its rapid internalization. Cholesterol depletion with methyl-β-cyclodextrin reduced the association of the agonist-activated receptor into membrane rafts but did not affect either the UTP-mediated vasoconstrictions or the vasomotor responses elicited by both serotonin and KCl. Ex vivo perfusion of human chorionic artery segments with 1–10 μm UTP, a selective P2Y₂R agonist, displaced the P2Y₂R localization into membrane rafts within 1 min, a process preceded by the activation of both RhoA and Rac1 GTPases. AG1478, a selective and potent inhibitor of the epidermal growth factor receptor tyrosine kinase activity, not only blocked the UTP-induced vasomotor activity but also abrogated both RhoA and Rac1 activation, the P2Y₂R association with membrane rafts, and its internalization. Altogether, these results show for the first time that the plasma membrane distribution of the P2Y₂R is transregulated by the epidermal growth factor receptor, revealing an unsuspected functional interplay that controls both the membrane distribution and the vasomotor activity of the P2Y₂R in intact human blood vessels.     

Association of P2Y2 receptor SNPs with bone mineral density and osteoporosis risk in a cohort of Dutch fracture patients

The P2Y₂ receptor is a G-protein-coupled receptor with adenosine 5′-triphosphate (and UTP) as natural ligands. It is thought to be involved in bone physiology in an anti-osteogenic manner. As several non-synonymous single nucleotide polymorphisms (SNPs) have been identified within the P2Y₂ receptor gene in humans, we examined associations between genetic variations in the P2Y₂ receptor gene and bone mineral density (BMD) (i.e., osteoporosis risk), in a cohort of fracture patients. Six hundred and ninety women and 231 men aged ≥50 years, visiting an osteoporosis outpatient clinic at Maastricht University Medical Centre for standard medical follow-up after a recent fracture, were genotyped for three non-synonymous P2Y₂ receptor gene SNPs. BMD was measured at three locations (total hip, lumbar spine, and femoral neck) using dual-energy X-ray absorptiometry. Differences in BMD between different genotypes were tested using analysis of covariance. In women, BMD values at all sites were significantly different between the genotypes for the Leu46Pro polymorphism, with women homozygous for the variant allele showing the highest BMD values (0.05 > p > 0.01). The Arg312Ser and Arg334Cys polymorphisms showed no differences in BMD values between the different genotypes. This is the first report that describes the association between the Leu46Pro polymorphism of the human P2Y₂ receptor and the risk of osteoporosis.     

P2Y2 Nucleotide Receptors Mediate Metalloprotease-dependent Phosphorylation of Epidermal Growth Factor Receptor and ErbB3 in Human Salivary Gland Cells

The G protein-coupled receptor P2Y₂ nucleotide receptor (P2Y₂R) has been shown to be up-regulated in a variety of tissues in response to stress or injury. Recent studies have suggested that P2Y₂Rs may play a role in immune responses, wound healing, and tissue regeneration via their ability to activate multiple signaling pathways, including activation of growth factor receptors. Here, we demonstrate that in human salivary gland (HSG) cells, activation of the P2Y₂R by its agonist induces phosphorylation of ERK1/2 via two distinct mechanisms, a rapid, protein kinase C-dependent pathway and a slower and prolonged, epidermal growth factor receptor (EGFR)-dependent pathway. The EGFR-dependent stimulation of UTP-induced ERK1/2 phosphorylation in HSG cells is inhibited by the adamalysin inhibitor tumor necrosis factor-α protease inhibitor or by small interfering RNA that selectively silences ADAM10 and ADAM17 expression, suggesting that ADAM metalloproteases are required for P2Y₂R-mediated activation of the EGFR. G protein-coupled receptors have been shown to promote proteolytic release of EGFR ligands; however, neutralizing antibodies to known ligands of the EGFR did not inhibit UTP-induced EGFR phosphorylation. Immunoprecipitation experiments indicated that UTP causes association of the EGFR with another member of the EGF receptor family, ErbB3. Furthermore, stimulation of HSG cells with UTP induced phosphorylation of ErbB3, and silencing of ErbB3 expression inhibited UTP-induced phosphorylation of both ErbB3 and EGFR. UTP-induced phosphorylation of ErbB3 and EGFR was also inhibited by silencing the expression of the ErbB3 ligand neuregulin 1 (NRG1). These results suggest that P2Y₂R activation in salivary gland cells promotes the formation of EGFR/ErbB3 heterodimers and metalloprotease-dependent neuregulin 1 release, resulting in the activation of both EGFR and ErbB3.     

From UTP to AR-C118925, the discovery of a potent non nucleotide antagonist of the P2Y2 receptor

The G protein-coupled P2Y₂ receptor, activated by ATP and UTP has been reported as a potential drug target for a wide range of important clinical conditions, such as tumor metastasis, kidney disorders, and in the treatment of inflammatory conditions. However, pharmacological studies on this receptor have been impeded by the limited reported availability of stable, potent and selective P2Y₂R antagonists. This article describes the design and synthesis of AR-C118925, a potent and selective non-nucleotide antagonist of the P2Y₂ receptor discovered using the endogenous P2Y₂R agonist UTP as the chemical starting point.     

UTP is not a biased agonist at human P2Y11 receptors

Biased agonism describes a multistate model of G protein-coupled receptor activation in which each ligand induces a unique structural conformation of the receptor, such that the receptor couples differentially to G proteins and other intracellular proteins. P2Y receptors are G protein-coupled receptors that are activated by endogenous nucleotides, such as adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP). A previous report suggested that UTP may be a biased agonist at the human P2Y₁₁ receptor, as it increased cytosolic [Ca²⁺], but did not induce accumulation of inositol phosphates, whereas ATP did both. The mechanism of action of UTP was unclear, so the aim of this study was to characterise the interaction of UTP with the P2Y₁₁ receptor in greater detail. Intracellular Ca²⁺ was monitored in 1321N1 cells stably expressing human P2Y₁₁ receptors using the Ca²⁺-sensitive fluorescent indicator, fluo-4. ATP evoked a rapid, concentration-dependent rise in intracellular Ca²⁺, but surprisingly, even high concentrations of UTP were ineffective. In contrast, UTP was slightly, but significantly more potent than ATP in evoking a rise in intracellular Ca²⁺ in 1321N1 cells stably expressing the human P2Y₂ receptor, with no difference in the maximum response. Thus, the lack of response to UTP at hP2Y₁₁ receptors was not due to a problem with the UTP solution. Furthermore, coapplying a high concentration of UTP with ATP did not inhibit the response to ATP. Thus, contrary to a previous report, we find no evidence for an agonist action of UTP at the human P2Y₁₁ receptor, nor does UTP act as an antagonist.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.     

P2Y2 Nucleotide Receptor-Mediated Responses in Brain Cells

Acute inflammation is important for tissue repair; however, chronic inflammation contributes to neurodegeneration in Alzheimer's disease (AD) and occurs when glial cells undergo prolonged activation. In the brain, stress or damage causes the release of nucleotides and activation of the G_q protein-coupled P2Y₂ nucleotide receptor subtype (P2Y₂R) leading to pro-inflammatory responses that can protect neurons from injury, including the stimulation and recruitment of glial cells. P2Y₂R activation induces the phosphorylation of the epidermal growth factor receptor (EGFR), a response dependent upon the presence of a SH3 binding domain in the intracellular C terminus of the P2Y₂R that promotes Src binding and transactivation of EGFR, a pathway that regulates the proliferation of cortical astrocytes. Other studies indicate that P2Y₂R activation increases astrocyte migration. P2Y₂R activation by UTP increases the expression in astrocytes of α_Vβ_3/5 integrins that bind directly to the P2Y₂R via an Arg-Gly-Asp (RGD) motif in the first extracellular loop of the P2Y₂R, an interaction required for G_o and G₁₂ protein-dependent astrocyte migration. In rat primary cortical neurons (rPCNs) P2Y₂R expression is increased by stimulation with interleukin-1β (IL-1β), a pro-inflammatory cytokine whose levels are elevated in AD, in part due to nucleotide-stimulated release from glial cells. Other results indicate that oligomeric β-amyloid peptide (Aβ_1-42), a contributor to AD, increases nucleotide release from astrocytes, which would serve to activate upregulated P2Y₂Rs in neurons. Data with rPCNs suggest that P2Y₂R upregulation by IL-1β and subsequent activation by UTP are neuroprotective, since this increases the non-amyloidogenic cleavage of amyloid precursor protein. Furthermore, activation of IL-1β-upregulated P2Y₂Rs in rPCNs increases the phosphorylation of cofilin, a cytoskeletal protein that stabilizes neurite outgrowths. Thus, activation of pro-inflammatory P2Y₂Rs in glial cells can promote neuroprotective responses, suggesting that P2Y₂Rs represent a novel pharmacological target in neurodegenerative and other pro-inflammatory diseases.     

P2X7 receptor activation mediates organic cation uptake into human myeloid leukaemic KG-1 cells

The P2X7 purinergic receptor is an ATP-gated cation channel with an emerging role in neoplasia. In this study we demonstrate that the human KG-1 cell line, a model of acute myelogenous leukaemia, expresses functional P2X7. RT-PCR and immunochemical techniques demonstrated the presence of P2X7 mRNA and protein respectively in KG-l cells, as well as in positive control multiple myeloma RPMI 8226 cells. Flow cytometric measurements demonstrated that ATP induced ethidium⁺ uptake into KG-l cells suspended in sucrose medium (EC₅₀ of ∼3 μM), but not into cells in NaCl medium. In contrast, ATP induced ethidium⁺ uptake into RPMI 8226 cells suspended in either sucrose or NaCl medium (EC₅₀ of ∼3 or ∼99 μM, respectively), as well as into RPMI 8226 cells in KCl medium (EC₅₀ of ∼18 μM). BzATP and to a lesser extent ATPγS and αβ-methylene ATP, but not ADP or UTP, also induced ethidium⁺ uptake into KG-1 cells. ATP-induced ethidium⁺ uptake was completely impaired by the P2X7 antagonists, AZ10606120 and A-438079. ATP-induced ethidium⁺ uptake was also impaired by probenecid but not by carbenoxolone, both pannexin-1 antagonists. ATP induced YO-PRO-1²⁺ and propidium²⁺ uptake into KG-1 cells. Finally, sequencing of full-length P2X7 cDNA identified several single nucleotide polymorphisms (SNPs) in KG-1 cells including H155Y, A348T, T357S and Q460R. RPMI 8226 cells contained A348T, A433V and H521Q SNPs. In conclusion, the KG-1 cell line expresses functional P2X7. This cell line may help elucidate the signalling pathways involved in P2X7-induced survival and invasiveness of myeloid leukaemic cells.     

Up‐regulation and activation of the P2Y2 nucleotide receptor mediate neurite extension in IL‐1β‐treated mouse primary cortical neurons

The pro‐inflammatory cytokine interleukin‐1β (IL‐1β), whose levels are elevated in the brain in Alzheimer's and other neurodegenerative diseases, has been shown to have both detrimental and beneficial effects on disease progression. In this article, we demonstrate that incubation of mouse primary cortical neurons (mPCNs) with IL‐1β increases the expression of the P2Y₂ nucleotide receptor (P2Y₂R) and that activation of the up‐regulated receptor with UTP, a relatively selective agonist of the P2Y₂R, increases neurite outgrowth. Consistent with the accepted role of cofilin in the regulation of neurite extension, results indicate that incubation of IL‐1β‐treated mPCNs with UTP increases the phosphorylation of cofilin, a response absent in PCNs isolated from P2Y₂R^?/? mice. Other findings indicate that function‐blocking anti‐α_vβ_3/5 integrin antibodies prevent UTP‐induced cofilin activation in IL‐1β‐treated mPCNs, suggesting that established P2Y₂R/α_vβ_3/5 interactions that promote G₁₂‐dependent Rho activation lead to cofilin phosphorylation involved in neurite extension. Cofilin phosphorylation induced by UTP in IL‐1β‐treated mPCNs is also decreased by inhibitors of Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII), suggesting a role for P2Y₂R‐mediated and G_q‐dependent calcium mobilization in neurite outgrowth. Taken together, these studies indicate that up‐regulation of P2Y₂Rs in mPCNs under pro‐inflammatory conditions can promote cofilin‐dependent neurite outgrowth, a neuroprotective response that may be a novel pharmacological target in the treatment of neurodegenerative diseases.     

The P2Y12 Antagonists, 2MeSAMP and Cangrelor,Inhibit Platelet Activation through P2Y12/Gi-Dependent Mechanism

Background

ADP is an important physiological agonist that induces integrin activation and platelet aggregation through its receptors P2Y₁ (Gα_q-coupled) and P2Y₁₂ (Gα_i-coupled). P2Y₁₂ plays a critical role in platelet activation and thrombosis. Adenosine-based P2Y₁₂ antagonists, 2-methylthioadenosine 5′-monophosphate triethylammonium salt hydrate (2MeSAMP) and Cangrelor (AR-C69931MX) have been widely used to demonstrate the role of P2Y₁₂ in platelet function. Cangrelor is being evaluated in clinical trials of thrombotic diseases. However, a recent study reported that both 2MeSAMP and Cangrelor raise intra-platelet cAMP levels and inhibit platelet aggregation through a P2Y₁₂-independent mechanism.

Methodology/Principal Findings

The present work, using P2Y₁₂ deficient mice, sought to clarify previous conflicting reports and to elucidate the mechanisms by which 2MeSAMP and Cangrelor inhibit platelet activation and thrombosis. 2MeSAMP and Cangrelor inhibited aggregation and ATP release of wild-type but not P2Y₁₂ deficient platelets. 2MeSAMP and Cangrelor neither raised intracellular cAMP concentrations nor induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in washed human or mouse platelets. Furthermore, unlike the activators (PGI₂ and forskolin) of the cAMP pathway, 2MeSAMP and Cangrelor failed to inhibit Ca²⁺ mobilization, Akt phosphorylation, and Rap1b activation in P2Y₁₂ deficient platelets. Importantly, while injection of Cangrelor inhibited thrombus formation in a FeCl₃-induced thrombosis model in wild-type mice, it failed to affect thrombus formation in P2Y₁₂ deficient mice.

Conclusions

These data together demonstrate that 2MeSAMP and Cangrelor inhibit platelet function through the P2Y₁₂-dependent mechanism both in vitro and in vivo.     

Agonist-induced phosphorylation and desensitization of the P2Y2 nucleotide receptor

Purification of HA-tagged P2Y₂ receptors from transfected human 1321N1 astrocytoma cells yielded a protein with a molecular size determined by SDS-PAGE to be in the range of 57–76 kDa, which is typical of membrane glycoproteins with heterogeneous complex glycosylation. The protein phosphatase inhibitor, okadaic acid, attenuated the recovery of receptor activity from the agonist-induced desensitized state, suggesting a role for P2Y₂ receptor phosphorylation in desensitization. Isolation of HA-tagged P2Y₂ nucleotide receptors from metabolically [³²P]-labelled cells indicated a (3.8 ± 0.2)-fold increase in the [³²P]-content of the receptor after 15 min of treatment with 100 μM UTP, as compared to immunoprecipitated receptors from untreated control cells. Receptor sequestration studies indicated that ∼40% of the surface receptors were internalized after a 15-min stimulation with 100 μM UTP. Point mutation of three potential GRK and PKC phosphorylation sites in the third intracellular loop and C-terminal tail of the P2Y₂ receptor (namely, S243A, T344A, and S356A) extinguished agonist-induced receptor phosphorylation, caused a marked reduction in the efficacy of UTP to desensitize P2Y₂ receptor signalling to intracellular calcium mobilization, and impaired agonist-induced receptor internalization. Activation of PKC isoforms with phorbol 12-myristate 13-acetate that caused heterologous receptor desensitization did not increase the level of P2Y₂ receptor phosphorylation. Our results indicate a role for receptor phosphorylation by phorbol-insensitive protein kinases in agonist-induced desensitization of the P2Y₂ nucleotide receptor. (Mol Cell Biochem xxx: 35–45, 2005)     

UDP Facilitates Microglial Phagocytosis Through P2Y6 Receptors

Microglia engage in the clearance of dead cells or dangerous debris. When neighboring cells are injured, the cells release or leak ATP into extracellular space and microglia rapidly move toward or extend a process to the nucleotides as chemotaxis through P2Y12 receptors. In the meanwhile, microglia express the metabotropic P2Y6 receptors, the activation of which by uridine 5′-diphosphate (UDP) triggers microglial phagocytosis in a concentration-dependent fashion. UDP/UTP was leaked when hippocampal neurons were damaged by kainic acid in vivo and in vitro. Systemic administration of kainic acid in rats resulted in neuronal cell death in the hippocampal CA1 and CA3 regions, where increases in mRNA for P2Y6 receptors in activated microglia. Thus, the P2Y6 receptor is upregulated when neurons are damaged, and would function as a sensor for phagocytosis by sensing diffusible UDP signals.Key Words: microglia, phagocytosis, P2Y6 receptors, UDPAccumulating findings indicate that nucleotides play an important role in neuron to glia communication through P2 purinoceptors, even though ATP is recognized primarily to be a source of free energy and nucleotides are key molecules in cells. P2 purinoceptors are divided into two families, ionotropic receptors (P2X) and metabotropic receptors (P2Y) (Fig. 1). P2X receptors (seven types; P2X₁-P2X₇) contain intrinsic pores that open by binding with ATP. P2Y (eight types; P2Y_{1,2,4,6 and 11–14}) are activated by nucleotides and couple to intracellular second-messenger systems through heteromeric G-proteins.¹ Microglia express P2X₄, P2X₇, P2Y₂, P2Y₆ and P2Y₁₂¹ and are known as resident macrophages in CNS, accounting for 5–10% of the total population of glia.^2,3 When neurons are injured or dead, microglia are activated, resulting in their interaction with immune cells, active migration to the site of injury, release of pro-inflammatory substances and the phagocytosis of damaged cells or debris. For such activation of microglial motilities, extracellular nucleotides have a central role. Extracellular ATP functions as a chemoattractant. Microglial chemotaxis by ATP via P2Y₁₂ receptors was originally found by Honda et al.,⁴ and has recently been confirmed in vivo in P2Y₁₂ receptor knockout animals.⁵ Neuronal injury results in the release or leakage of ATP that appears to be a “find-me” signal from damaged neurons to microglia to cause chemotaxis. In addition to microglial migration by ATP, another nucleotide, UDP, an endogenous agonist of the P2Y₆ receptor, greatly activates the motility of microglia and orders microglia to engulf damaged neurons.⁶Open in a separate windowFigure 1P2 purinergic receptors (ATP receptors).Phagocytosis is a specialized form of endocytosis taking relatively large particles (> 1.0 µm) into vacuoles and has a central role in tissue remodeling, inflammation and the defense against infectious agents.⁷ Phagocytosis is initiated by the activation of cell-surface phagocytosis receptors, including Fc receptors, complement receptors, integrins, endotoxin receptors (CD18, CD14), mannose receptors and scavenger receptors⁸ which are activated by corresponding extracellular ligands called as “eat-me” signals. Since recognition is the most important step for phagocytosis, extensive studies on phagocytosis receptors have been reported. With regard to apoptotic cells, it is well known that dying cells express so called “eat-me” signals such as phosphatidylserine (PS) on their surface membrane,⁸ by which microglia recognize the apoptotic cells in order to catch and remove them.⁸ As for amyloid β protein (Aβ), a key molecule that mediates Alzheimer''s disease, microglia remove Aβ presumably via Fc receptor-dependent phagocytosis.^9,10 It, however, is unclear how phagocytotic cells come to the target cells or debris. Our findings suggest that nucleotides might be the molecules to guide phagocytotic cells to the targets.We found that exogenously applied UDP caused microglial phagocytosis through P2Y₆ in a concentration-dependent manner, and that neuronal injury caused by kainic acid (KA) upregulated P2Y₆ receptors in microglia, the KA evoked neuronal injury resulted in an increase in extracellular UTP, which was immediately metabolized into UDP in vivo and in vitro. We also found that UDP leaked from injured neurons caused P2Y₆ receptor-dependent phagocytosis in vivo and in vitro. Thus, UDP could be a diffusible molecule that signals the crisis of damaged neurons to microglia, triggering phagocytosis. Nucleotides seem to have the ability to act as “eat-us” signals for necrotic cells suffering traumatic or ischemic injury because such necrotic cells cause swelling, followed by shrinkage, leading to the leakage of cytoplasmic molecules including a large amount of ATP and UTP and extracellular nucleotides are immediately degraded by ecto-nucleotideases, suggesting that leaked nucleotides could be transient and localized signals that alert to the crisis created by the presence of the necrotic cells. These findings suggest that microglia might be attracted by ATP/ADP^4,5,11,12 and subsequently recognize UDP, starting to recognize “eat-me” signals attached to the targets and engulf them (Fig. 2). It is interesting that ATP/ADP is not able to efficiently activate P2Y₆ receptors, nor can UDP act on P2Y₁₂ receptors. Thus, adenine and uridine nucleotides would regulate microglial motilities, i.e. chemotaxis and phagocytosis, in a coordinated fashion.Open in a separate windowFigure 2Illustration of nucleotide-activated microglial chemotaxix and phagocytosis. Activated microglia might be attracted by ATP/ADP is not able to efficiently activate P2Y6 receptors, nor ca UDP act on P2Y12 receptors.     

Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor

UTP activates P2Y₂ receptors in both 1321N1 cell transfectants expressing the P2Y₂ receptor and human HT-29 epithelial cells expressing endogenous P2Y₂ receptors with an EC₅₀ of 0.2- 1.0 M. Pretreatment of these cells with UTP diminished the effectiveness of a second dose of UTP (the IC₅₀ for UTP-induced receptor desensitization was 0.3 - 1.0 M for both systems). Desensitization and down-regulation of the P2Y₂ nucleotide receptor may limit the effectiveness of UTP as a therapeutic agent. The present studies investigated the phenomenon of P2Y₂ receptor desensitization in human 1321N1 astrocytoma cells expressing recombinant wild type and C-terminal truncation mutants of the P2Y,₂ receptor. In these cells, potent P2Y₂ receptor desensitization was observed after a 5 min exposure to UTP. Full receptor responsiveness returned 5-10 min after removal of UTP. Thapsigargin, an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, induced an increase in the intracellular free calcium concentration, [Ca²⁺]_i, after addition of desensitizing concentrations of UTP, indicating that P2Y₂ receptor desensitization is not due to depletion of calcium from intracellular stores. Single cell measurements of increases in [Ca²⁺]_i induced by UTP in 1321N1 cell transfectants expressing the P2Y₂ receptor indicate that time- and UTP concentration-dependent desensitization occurred uniformly across a cell population. Other results suggest that P2Y₂ receptor phosphorylation/dephosphorylation regulate receptor desensitization/resensitization. A 5 min preincubation of 1321N1 cell transfectants with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), reduced the subsequent response to UTP by about 50% whereas co-incubation of PMA with UTP caused a greater inhibition in the response. The protein phosphatases - 1 and -2A inhibitor, okadaic acid, partially blocked resensitization of the receptor. Furthermore, C-terminal truncation mutants of the P2Y₂ receptor that eliminated several potential phosphorylation sites including two for PKC were resistant to UTP-, but not phorbol ester-induced desensitization. Down regulation of protein kinase C isoforms prevented phorbol ester-induced desensitization but had no effect on agonist-induced desensitization of wild type or truncation mutant receptors. These results suggest that phosphorylation of the C-terminus of the P2Y₂ receptor by protein kinases other than protein kinase C mediates agonist-induced receptor desensitization. A better understanding of the molecular mechanisms of P2Y₂ nucleotide receptor desensitization may help optimize a promising cystic fibrosis pharmacotherapy based on the activation of anion secretion in airway epithelial cells by P2Y₂ receptor agonists.     

P2Y2R activation by nucleotides released from oxLDL-treated endothelial cells (ECs) mediates the interaction between ECs and immune cells through RAGE expression and reactive oxygen species production

Lipoprotein oxidation, inflammation, and immune responses involving the vascular endothelium and immune cells contribute to the pathogenesis of atherosclerosis. In an atherosclerotic animal model, P2Y₂ receptor (P2Y₂R) upregulation and stimulation were previously shown to induce intimal hyperplasia and increased intimal monocyte infiltration. Thus, we investigated the role of P2Y₂R in oxidized low-density lipoprotein (oxLDL)-mediated oxidative stress and the subsequent interaction between endothelial cells (ECs) and immune cells. The treatment of human ECs with oxLDL caused the rapid release of ATP (maximum after 5 min). ECs treated with oxLDL or the P2Y₂R agonists ATP/UTP for 1 h exhibited significant reactive oxygen species (ROS) production, but this effect was not observed in P2Y₂R siRNA-transfected ECs. In addition, oxLDL and ATP/UTP both induced RAGE expression, which was P2Y₂R dependent. Oxidized LDL- and ATP/UTP-mediated ROS production was diminished in RAGE siRNA-transfected ECs, suggesting that RAGE is an important mediator in P2Y₂R-mediated ROS production. Treatment with oxLDL for 24 h induced P2Y₂R expression in the human monocyte cell line THP-1 and increased THP-1 cell migration toward ECs. The addition of apyrase, an enzyme that hydrolyzes nucleotides, or diphenyleneiodonium (DPI), a well-known inhibitor of NADPH oxidase, significantly inhibited the increase in cell migration caused by oxLDL. P2Y₂R siRNA-transfected THP-1 cells did not migrate in response to oxLDL or ATP/UTP treatment, indicating a critical role for P2Y₂R and nucleotide release in oxLDL-induced monocyte migration. Last, oxLDL and ATP/UTP effectively increased ICAM-1 and VCAM-1 expression and the subsequent binding of THP-1 cells to ECs, which was inhibited by pretreatment with DPI or by siRNA against P2Y₂R or RAGE, suggesting that P2Y₂R is an important mediator in oxLDL-mediated monocyte adhesion to ECs through the regulation of ROS-dependent adhesion molecule expression in ECs. Taken together, our findings suggest that P2Y₂R could be a therapeutic target for the prevention of vascular disorders, including atherosclerosis.     

Differential agonist-induced desensitization of P2Y2 nucleotide receptors by ATP and UTP

The equal potency and efficacy of the agonists, ATP and UTP, pharmacologically distinguish the P2Y₂ receptor from other nucleotide receptors. Investigation of the desensitization of the P2Y₂ receptors is complicated by the simultaneous expression of different P2 nucleotide receptor subtypes. The co-expression of multiple P2 receptor subtypes in mammalian cells may have led to contradictory reports on the efficacy of the natural agonists of the P2Y₂ receptor to induce desensitization. We decided to investigate the desensitization of human and murine isoforms of the P2Y₂ receptor, and to rigorously examine their signaling and desensitization properties. For these purposes, we used 1321N1 astrocytoma cells stably transfected with the human or murine P2Y₂ receptor cDNA, as well as human A431 cells that endogenously express the receptor. The mobilization of intracellular calcium by extracellular nucleotides was used as a functional assay for the P2Y₂ receptors. While ATP and UTP activated the murine and human P2Y₂ receptors with similar potencies (EC₅₀ values were 1.5-5.8 M), ATP was ~ 10-fold less potent (IC₅₀ = 9.1-21.2 M) than UTP (IC₅₀ = 0.7-2.9 M) inducing homologous receptor desensitization in the cell systems examined. Individual cell analyses of the rate and dose dependency of agonist-induced desensitization demonstrated that the murine receptor was slightly more resistant to desensitization than its human counterpart. To our knowledge, this is the first individual cell study that has compared the cellular heterogeneity of the desensitized states of recombinant and endogenously expressed receptors. This comparison demonstrated that the recombinant system conserved the cellular regulatory elements needed to attenuate receptor signaling by desensitization.     

Characterisation of P2Y12 Receptor Responsiveness to Cysteinyl Leukotrienes

Leukotriene E₄ (LTE₄), the most stable of the cysteinyl leukotrienes (cysLTs), binds poorly to classical type 1 and 2 cysLT receptors although in asthmatic individuals it may potently induce bronchial constriction, airway hyperresponsiveness and inflammatory cell influx to the lung. A recent study has suggested that the purinergic receptor P2Y₁₂ is required for LTE₄ mediated pulmonary inflammation in a mouse model of asthma and signals in response to cysLTs. The aim of the study was to characterise the responsiveness of human P2Y₁₂ to cysteinyl leukotrienes. Models of human CysLT₁, CysLT₂ and P2Y₁₂ overexpressed in HEK293, CHO cells and human platelets were used and responsiveness to different agonists was measured using intracellular calcium, cAMP and β-arrestin recruitment assays. CysLTs induced concentration dependent calcium mobilisation in cells overexpressing CysLT₁ and CysLT₂ but failed to induce any calcium response in cells expressing P2Y₁₂ or P2Y₁₂+ Gα₁₆. In contrast, selective P2Y₁₂ agonists ADP and 2-MeS-ADP induced specific calcium flux in cells expressing P2Y₁₂+ Gα₁₆. Similarly, specific response to 2-MeS-ADP, but not to cysLTs was also observed in cells expressing P2Y₁₂ when intracellular cAMP and β-arrestin signalling were analysed. Platelets were used as a model of human primary cells expressing P2Y₁₂ to analyse potential signalling and cell activation through P2Y₁₂ receptor or receptor heterodimers but no specific LTE₄ responses were observed. These results show that LTE₄ as well as other cysLTs do not activate intracellular signalling acting through P2Y₁₂ and suggest that another LTE₄ specific receptor has yet to be identified.