P2Y<Subscript>2</Subscript> and P2Y<Subscript>6</Subscript> receptor activation elicits intracellular calcium responses in human adipose-derived mesenchymal stromal cells

Adipose tissue contains self-renewing multipotent cells termed mesenchymal stromal cells. In situ, these cells serve to expand adipose tissue by adipogenesis, but their multipotency has gained interest for use in tissue regeneration. Little is known regarding the repertoire of receptors expressed by adipose-derived mesenchymal stromal cells (AD-MSCs). The purpose of this study was to undertake a comprehensive analysis of purinergic receptor expression. Mesenchymal stromal cells were isolated from human subcutaneous adipose tissue and confirmed by flow cytometry. The expression profile of purinergic receptors was determined by quantitative real-time PCR and immunocytochemistry. The molecular basis for adenine and uracil nucleotide-evoked intracellular calcium responses was determined using Fura-2 measurements. All the known subtypes of P2X and P2Y receptors, excluding P2X2, P2X3 and P2Y₁₂ receptors, were detected at the mRNA and protein level. ATP, ADP and UTP elicited concentration-dependent calcium responses in mesenchymal cells (N?=?7–9 donors), with a potency ranking ADP (EC₅₀ 1.3 ± 1.0 μM)?>?ATP (EC₅₀ 2.2 ± 1.1 μM)?=?UTP (3.2 ± 2.8 μM). Cells were unresponsive to UDP (<?30 μM) and UDP-glucose (<?30 μM). ATP responses were attenuated by selective P2Y₂ receptor antagonism (AR-C118925XX; IC₅₀ 1.1 ± 0.8 μM, 73.0?±?8.5% max inhibition; N?=?7 donors), and UTP responses were abolished. ADP responses were attenuated by the selective P2Y₆ receptor antagonist, MRS2587 (IC₅₀ 437 ± 133nM, 81.0?±?8.4% max inhibition; N?=?6 donors). These data demonstrate that adenine and uracil nucleotides elicit intracellular calcium responses in human AD-MSCs with a predominant role for P2Y₂ and P2Y₆ receptor activation. This study furthers understanding about how human adipose-derived mesenchymal stromal cells can respond to external signalling cues.     

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.     

Cerebellar astrocytes co-express several ADP receptors. Presence of functional P2Y<Subscript>13</Subscript>-like receptors

Astrocytes exhibit a form of excitability based on variations of intracellular Ca²⁺ concentration in response to various stimuli, including ADP, ATP, UTP and dinucleotides. Here, we investigate the presence of the recently cloned ADP-sensitive receptors, P2Y₁₂ and P2Y₁₃ subtypes, which are negatively coupled to adenylate cyclase, in cerebellar astrocytes. We checked the effect of specific agonists, 2-methylthioadenosine diphosphate (2MeSADP) and ADP, on adenylate cyclase stimulation induced by isoproterenol. Both agonists significantly reduced the cAMP accumulation induced by isoproterenol. The inhibitory effect was concentration-dependent with IC₅₀ values of 46 ± 13 and 23 ± 14 nM for 2MeSADP and ADP, respectively. The experiments were carried out in the presence of MRS-2179, a specific antagonist of P2Y₁ receptor, to avoid any contribution of this receptor. Using fura-2 microfluorimetry we also proved that astrocytes responded to 2MeSADP stimulations with calcium responses in the absence and also in the presence of MRS-2179. Both effects, inhibition of adenylate cyclase and intracellular calcium mobilization, were not modified by 2MeSAMP, an antagonist of P2Y₁₂ receptor, suggesting that were mediated by P2Y₁₃-like receptors.¹These authors equally contributed to this work.     

Distribution of P2Y<Subscript>2</Subscript> receptors in the guinea pig enteric nervous system and its coexistence with P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> receptors,neuropeptide Y,nitric oxide synthase and calretinin

The distribution of P2Y₂ receptor-immunoreactive (ir) neurons and fibers and coexistence of P2Y₂ with P2X₂ and P2X₃ receptors, neuropeptide Y (NPY), calretinin (CR), calbindin (CB) and nitric oxide synthase (NOS) was investigated with immunostaining methods. The results showed that P2Y₂-ir neurons and fibers were distributed widely in myenteric and submucous plexuses of the guinea pig stomach corpus, jejunum, ileum and colon. The typical morphology of P2Y₂-ir neurons was a long process with strong positive staining on the same side of the cell body. The P2Y₂-ir neurons could be Dogiel type 1. About 40–60% P2X₃-ir neurons were immunoreactive for P2Y₂ in the myenteric plexus and all the P2X₃-ir neurons expressed the P2Y₂ receptor in the submucosal plexus; almost all the NPY-ir neurons and the majority of CR-ir neurons were also immunoreactive for P2Y₂, especially in the myenteric plexus of the small intestine; no P2Y₂-ir neurons were immunoreactive for P2X₂ receptors, CB and NOS. It is shown for the first time that S type/Dogiel type 1 neurons with fast P2X and slow P2Y receptor-mediated depolarizations could be those neurons expressing both P2Y₂-ir and P2X₃-ir and that they are widely distributed in myenteric and submucosal plexuses of guinea pig gut.     

2-Aminothiazole based P2Y1 antagonists as novel antiplatelet agents

ADP receptors, P2Y₁ and P2Y₁₂ have been recognized as potential targets for antithrombotic drugs. A series of P2Y₁ antagonists that contain 2-aminothiazoles as urea surrogates were discovered. Extensive SAR of the thiazole ring is described. The most potent compound 7j showed good P2Y₁ binding (K_i = 12 nM), moderate antagonism of platelet aggregation (PA IC₅₀ = 5.2 μM) and acceptable PK in rats.     

Evidence for the possible involvement of the P2Y<Subscript>6</Subscript> receptor in Ca<Superscript>2+</Superscript> mobilization and insulin secretion in mouse pancreatic islets

Subtypes of purinergic receptors involved in modulation of cytoplasmic calcium ion concentration ([Ca²⁺]_i) and insulin release in mouse pancreatic β-cells were examined in two systems, pancreatic islets in primary culture and beta-TC6 insulinoma cells. Both systems exhibited some physiological responses such as acetylcholine-stimulated [Ca²⁺]_i rise via cytoplasmic Ca²⁺ mobilization. Addition of ATP, ADP, and 2-MeSADP (each 100 μM) transiently increased [Ca²⁺]_i in single islets cultured in the presence of 5.5 mM (normal) glucose. The potent P2Y₁ receptor agonist 2-MeSADP reduced insulin secretion significantly in islets cultured in the presence of high glucose (16.7 mM), whereas a slight stimulation occurred at 5.5 mM glucose. The selective P2Y₆ receptor agonist UDP (200 μM) transiently increased [Ca²⁺]_i and reduced insulin secretion at high glucose, whereas the P2Y_2/4 receptor agonist UTP and adenosine receptor agonist NECA were inactive. [Ca²⁺]_i transients induced by 2-MeSADP and UDP were antagonized by suramin (100 μM), U73122 (2 μM, PLC inhibitor), and 2-APB (10 or 30 μM, IP₃ receptor antagonist), but neither by staurosporine (1 μM, PKC inhibitor) nor depletion of extracellular Ca²⁺. The effect of 2-MeSADP on [Ca²⁺]_i was also significantly inhibited by MRS2500, a P2Y₁ receptor antagonist. These results suggested that P2Y₁ and P2Y₆ receptor subtypes are involved in Ca²⁺ mobilization from intracellular stores and insulin release in mouse islets. In beta-TC6 cells, ATP, ADP, 2-MeSADP, and UDP transiently elevated [Ca²⁺]_i and slightly decreased insulin secretion at normal glucose, while UTP and NECA were inactive. RT-PCR analysis detected mRNAs of P2Y₁ and P2Y₆, but not P2Y₂ and P2Y₄ receptors.     

Development of selective agonists and antagonists of P2Y receptors

Although elucidation of the medicinal chemistry of agonists and antagonists of the P2Y receptors has lagged behind that of many other members of group A G protein-coupled receptors, detailed qualitative and quantitative structure–activity relationships (SARs) were recently constructed for several of the subtypes. Agonists selective for P2Y₁, P2Y₂, and P2Y₆ receptors and nucleotide antagonists selective for P2Y₁ and P2Y₁₂ receptors are now known. Selective nonnucleotide antagonists were reported for P2Y₁, P2Y₂, P2Y₆, P2Y₁₁, P2Y₁₂, and P2Y₁₃ receptors. At the P2Y₁ and P2Y₁₂ receptors, nucleotide agonists (5′-diphosphate derivatives) were converted into antagonists of nanomolar affinity by altering the phosphate moieties, with a focus particularly on the ribose conformation and substitution pattern. Nucleotide analogues with conformationally constrained ribose-like rings were introduced as selective receptor probes for P2Y₁ and P2Y₆ receptors. Screening chemically diverse compound libraries has begun to yield new lead compounds for the development of P2Y receptor antagonists, such as competitive P2Y₁₂ receptor antagonists with antithrombotic activity. Selective agonists for the P2Y₄, P2Y₁₁, and P2Y₁₃ receptors and selective antagonists for P2Y₄ and P2Y₁₄ receptors have not yet been identified. The P2Y₁₄ receptor appears to be the most restrictive of the class with respect to modification of the nucleobase, ribose, and phosphate moieties. The continuing process of ligand design for the P2Y receptors will aid in the identification of new clinical targets.     

P2Y1 and P2Y12 receptor cross-talk in calcium signalling: Evidence from nonstarved and long-term serum-deprived glioma C6 cells

The current work presents results of experiments on the calcium response evoked by the stimulation by extracellular nucleotides occurring in control, nonstarved glioma C6 cells and in cells after long-term (96 h) serum starvation. Three nucleotide receptors were studied: P2Y₁, P2Y₂ and P2Y₁₂. Two of them, P2Y₁ and P2Y₂, directly stimulate calcium response. The protein level of the P2Y₂ receptor did not change during the serum starvation, while P2Y₁ protein level fell dramatically. Observed changes in the calcium response generated by P2Y₁ are directly correlated with the receptor protein level as well as with the amount of calcium present in the intracellular calcium stores, partially depleted during starvation process. The third receptor, P2Y₁₂, did not directly evoke calcium response, however it is activated by the same ligand as P2Y₁. The experiments with AR-C69941MX, the P2Y₁₂-specific antagonist, indicated that in control and serum-starved cells, calcium response evoked by P2Y₁ receptor is potentiated by the activity of P2Y₁₂-dependent signaling pathways. This potentiation may be mediated by P2Y₁₂ inhibitory effect on the plasma membrane calcium pump. The calcium influx enhanced by the cooperation of P2Y₁ and P2Y₁₂ receptor activity directly depends on the capacitative calcium entrance mechanism.     

P2Y13 Receptor-Mediated Rapid Increase in Intracellular Calcium Induced by ADP in Cultured Dorsal Spinal Cord Microglia

P2Y receptors have been implicated in the calcium mobilization by the response to neuroexcitatory substances in neurons and astrocytes, but little is known about P2Y receptors in microglia cells. In the present study, the effects of ADP on the intracellular calcium concentration ([Ca²⁺]i) in cultured dorsal spinal cord microglia were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescence indicator that could monitor real-time alterations of [Ca²⁺]i. Here we show that ADP (0.01–100 μM) causes a rapid increase in [Ca²⁺]i with a dose-dependent manner in cultured microglia. The action of ADP on [Ca²⁺]i was significantly blocked by MRS2211 (a selective P2Y₁₃ receptor antagonist), but was unaffected by MRS2179 (a selective P2Y₁ receptor antagonist) or MRS2395 (a selective P2Y₁₂ receptor antagonist), which suggest that P2Y₁₃ receptor may be responsible for ADP-evoked Ca²⁺ mobilization in cultured microglia. P2Y₁₃-evoked Ca²⁺ response can be obviously inhibited by BAPTA-AM and U-73122, respectively. Moreover, removal of extracellular Ca²⁺ (by EGTA) also can obvious suppress the Ca²⁺ mobilization. These results means both intracellular calcium and extracellular calcium are potentially important mechanisms in P2Y₁₃ receptor-evoked Ca²⁺ mobilization. However, P2Y₁₃ receptor-evoked Ca²⁺ response was not impaired after CdCl₂ and verapamil administration, which suggest that voltage-operated Ca²⁺ channels may be not related with P2Y₁₃-evoked Ca²⁺ response. In addition, Ca²⁺ mobilization induced by ADP was abolished by different store-operated Ca²⁺ channels (SOCs) blocker, 2-APB (50 μM) and SKF-96365 (1 mM), respectively. These observations suggest that the activation of P2Y₁₃ receptor might be involved in the effect of ADP on [Ca²⁺]i in cultured dorsal spinal cord microglia. Furthermore, our results raise a possibility that P2Y₁₃ receptor activation causes Ca²⁺ release from Ca²⁺ store, which leads to the opening of SOCs.     

Extracellular NAD induces a rise in [Ca]i in activated human monocytes via engagement of P2Y1 and P2Y11 receptors

Extracellular nicotinamide adenine dinucleotide (NAD⁺) is known to increase the intracellular calcium concentration [Ca²⁺]_i in different cell types and by various mechanisms. Here we show that NAD⁺ triggers a transient rise in [Ca²⁺]_i in human monocytes activated with lipopolysaccharide (LPS), which is caused by a release of Ca²⁺ from IP₃-responsive intracellular stores and an influx of extracellular Ca²⁺. By the use of P2 receptor-selective agonists and antagonists we demonstrate that P2 receptors play a role in the NAD⁺-induced calcium response in activated monocytes. Of the two subclasses of P2 receptors (P2X and P2Y) the P2Y receptors were considered the most likely candidates, since they share calcium signaling properties with NAD⁺. The identification of P2Y₁ and P2Y₁₁ as receptor subtypes responsible for the NAD⁺-triggered increase in [Ca²⁺]_i was supported by several lines of evidence. First, specific P2Y₁ and P2Y₁₁ receptor antagonists inhibited the NAD⁺-induced increase in [Ca²⁺]_i. Second, NAD⁺ was shown to potently induce calcium signals in cells transfected with either subtype, whereas untransfected cells were unresponsive. Third, NAD⁺ caused an increase in [cAMP]_i, prevented by the P2Y₁₁ receptor-specific antagonist NF157.     

Allosteric Inhibition of Serotonin 5-HT<Subscript>7</Subscript> Receptors by Zinc Ions

The allosteric regulation of G protein-coupled receptors (GPCRs) is a well-known phenomenon, but there are only a few examples of allosteric modulation within the metabotropic serotonergic receptor family. Recently, we described zinc non-competitive interactions toward agonist binding at serotonin 5-HT_1A receptors, in which biphasic effects, involving potentiation at sub-micromolar concentrations (10 μM) and inhibition at sub-millimolar concentrations (500 μM) of Zn²⁺ in radioligand binding assays, were consistent with both the agonist and antagonist-like effects of zinc ions observed in in vivo studies. Here, we showed new data demonstrating zinc allosteric inhibition of both agonist and antagonist binding at human recombinant 5-HT₇ receptors stably expressed in HEK293 cells as observed by radioligand binding studies as well as zinc neutral antagonism displayed by the concentration of 10 μM in the functional LANCE assay. The allosteric nature of the effect of Zn on 5-HT₇ receptors was confirmed (1) in saturation studies in which zinc inhibited the binding of potent orthosteric 5-HT₇ receptor radioligands, the agonist [³H]5-CT, and the two antagonists [³H]SB-269970 and [³H]mesulergine, showing ceiling effect and differences in the magnitude of negative cooperativity (α = 0.15, 0.06, and 0.25, respectively); (2) in competition experiments in which 500 μM of zinc inhibited all radioligand displacements by non-labeled orthosteric ligands (5-CT, SB-269970, and clozapine), and the most significant reduction in affinity was observed for the 5-CT agonist (4.9–16.7-fold) compared with both antagonists (1.4–3.9-fold); and (3) in kinetic experiments in which 500 μM zinc increased the dissociation rate constants for [³H]5-CT and [³H]mesulergine but not for [³H]SB-269970. Additionally, in the functional LANCE test using the constitutively active HEK293 cell line expressing the 5-HT₇ receptor, 10 μM zinc had features of neutral antagonism and increased the EC₅₀ value of the 5-CT agonist by a factor of 3.2. Overall, these results showed that zinc can act as a negative allosteric inhibitor of 5-HT₇ receptors. Given that the inhibiting effects of low concentrations of zinc in the functional assay represent the most likely direction of zinc activity under physiological conditions, among numerous zinc-regulated proteins, the 5-HT₇ receptor can be considered a serotonergic target for zinc modulation in the CNS.     

Localisation of P2Y<Subscript>1</Subscript> and P2Y<Subscript>4</Subscript> receptors in dorsal root,nodose and trigeminal ganglia of the rat

The presence and distribution of P2Y (nucleotide) receptor subtypes in rat sensory neurons has been investigated. RT-PCR showed that P2Y₁, P2Y₂, P2Y₄ and P2Y₆ receptor mRNA is expressed in sensory ganglia [dorsal root ganglion (DRG), nodose ganglion (NG) and trigeminal ganglion (TG)]. The regional and cellular distribution of P2Y₁ and P2Y₄ receptor proteins in these ganglia was investigated using immunohistochemistry. P2Y₁ polyclonal antibodies stained over 80% of the sensory neurons, particularly the small-diameter (neurofilament-negative) neurons. The P2Y₄ receptor antibody stained more medium- and large- (neurofilament-positive) diameter neurons than small-diameter neurons. P2Y₁ and P2Y₄ receptor immunoreactivity (P2Y₁-IR and P2Y₄-IR) was often coexpressed with P2X₃ receptor immunoreactivity (P2X₃-IR) in subpopulations of neurons. Double immunohistochemistry showed that 73–84% of P2X₃ receptor-positive neurons also stained for the P2Y₁ receptor in DRG, TG and NG while only 25–35% also stained for the P2Y₄ receptor. Subpopulations of P2Y₁-IR neurons were coexpressed with NF200, CGRP and IB₄; most P2Y₄-IR neurons were coexpressed with NF200, while only a few neurons were coexpressed with CGRP (10–20%) or with IB₄ (1–2%). The results suggest that P2Y as well as P2X receptor subtypes contribute to purinergic signalling in sensory ganglia.     

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.     

Signaling of the human P2Y<Subscript>1</Subscript> receptor measured by a yeast growth assay with comparisons to assays of phospholipase C and calcium mobilization in 1321N1 human astrocytoma cells

The human P2Y₁ receptor was expressed in the yeast Saccharomyces cerevisiae strain MPY578q5, which is engineered to couple to mammalian G protein-coupled receptors (GPCRs) and requires agonist-induced activation for growth. A range of known P2Y₁ receptor agonists were examined with the yeast growth assay system, and the results were validated by comparing with potencies in the transfected 1321N1 astrocytoma cell line, in which calcium mobilization was measured with a FLIPR (fluorometric-imaging plate reader). The data were also compared with those from phospholipase C activation and radioligand binding with the use of a newly available radioligand [³H]MRS2279 (2-chloro-N ⁶-methyl-(N)-methanocarba-2’-deoxyadenosine-3’,5’bisphosphate). In the yeast growth assay, the rank order of potency of 2-MeSADP (2-methylthioadenosine 5’-diphosphate), ADP (adenosine 5’-diphosphate), and ATP (adenosine 5’-triphosphate) is the same as those in other assay systems, i.e., 2-MeSADP>ADP>ATP. The P2Y₁-selective antagonist MRS2179 (N ⁶-methyl-2-deoxyadenosine-3’,5’-bisphosphate) was shown to act as an antagonist with similar potency in all systems. The results suggest that the yeast expression system is suitable for screening P2Y₁ receptor ligands, both agonists and antagonists. The yeast system should be useful for random mutagenesis of GPCRs to identify mutants with certain properties, such as selective potency enhancement for small synthetic molecules and constitutive activity.     

Pharmacochemistry of the platelet purinergic receptors

Platelets contain at least five purinergic G protein-coupled receptors, e.g., the pro-aggregatory P2Y₁ and P2Y₁₂ receptors, a P2Y₁₄ receptor (GPR105) of unknown function, and anti-aggregatory A_2A and A_2B adenosine receptor (ARs), in addition to the ligand-gated P2X1 ion channel. Probing the structure–activity relationships (SARs) of the P2X and P2Y receptors for extracellular nucleotides has resulted in numerous new agonist and antagonist ligands. Selective agents derived from known ligands and novel chemotypes can be used to help define the subtypes pharmacologically. Some of these agents have entered into clinical trials in spite of the challenges of drug development for these classes of receptors. The functional architecture of P2 receptors was extensively explored using mutagenesis and molecular modeling, which are useful tools in drug discovery. In general, novel drug delivery methods, prodrug approaches, allosteric modulation, and biased agonism would be desirable to overcome side effects that tend to occur even with receptor subtype-selective ligands. Detailed SAR analyses have been constructed for nucleotide and non-nucleotide ligands at the P2Y₁, P2Y₁₂, and P2Y₁₄ receptors. The thienopyridine antithrombotic drugs Clopidogrel and Prasugrel require enzymatic pre-activation in vivo and react irreversibly with the P2Y₁₂ receptor. There is much pharmaceutical development activity aimed at identifying reversible P2Y₁₂ receptor antagonists. The screening of chemically diverse compound libraries has identified novel chemotypes that act as competitive, non-nucleotide antagonists of the P2Y₁ receptor or the P2Y₁₂ receptor, and antithrombotic properties of the structurally optimized analogues were demonstrated. In silico screening at the A_2A AR has identified antagonist molecules having novel chemotypes. Fluorescent and other reporter groups incorporated into ligands can enable new technology for receptor assays and imaging. The A_2A agonist CGS21680 and the P2Y₁ receptor antagonist MRS2500 were derivatized for covalent attachment to polyamidoamine dendrimeric carriers of MW 20,000, and the resulting multivalent conjugates inhibited ADP-promoted platelet aggregation. In conclusion, a wide range of new pharmacological tools is available to control platelet function by interacting with cell surface purine receptors.     

The P2Y<Subscript>1</Subscript> receptor-mediated leukocyte adhesion to endothelial cells is inhibited by melatonin

Extracellular ATP (released by endothelial and immune cells) and its metabolite ADP are important pro-inflammatory mediators via the activation of purinergic P2 receptors (P2Y and P2X), which represent potential new targets for anti-inflammatory therapy. Endothelial P2Y₁ receptor (P2Y₁R) induces endothelial cell activation triggering leukocyte adhesion. A number of data have implicated melatonin as a modulator of immunity, inflammation, and endothelial cell function, but to date no studies have investigated whether melatonin modulates endothelial P2YR signaling. Here, we evaluated the putative effect of melatonin on P2Y₁R-mediated leukocyte adhesion to endothelial cells and TNF-α production, using mesenteric endothelial cells and fresh peripheral blood mononuclear cells isolated from rats. Endothelial cells were treated with the P2Y₁R agonist 2MeSATP, alone or in combination with melatonin, and then exposed to mononuclear cells. 2MeSATP increased leukocyte adhesion to endothelial cells and TNF-α production in vitro, and melatonin inhibited both effects without altering P2Y₁R protein expression. In addition, assays with the Ca²⁺ chelator BAPTA-AM indicate that the effect of melatonin on 2MeSATP-stimulated leukocyte adhesion depends on intracellular Ca²⁺ modulation. P2Y₁R is considered a potential target to control chronic inflammation. Therefore, our data unveiled a new endothelial cell modulator of purinergic P2Y₁ receptor signaling.     

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.     

Lung injury during LPS-induced inflammation occurs independently of the receptor P2Y<Subscript>1</Subscript>

Disruption of the lung endothelial and epithelial barriers during acute inflammation leads to excessive neutrophil migration. It is likely that activated platelets promote pulmonary recruitment of neutrophils during inflammation, and previous studies have found that anti-platelet therapy and depletion of circulating platelets have lung-protective effects in different models of inflammation. Because ADP signaling is important for platelet activation, I investigated the role of the ADP-receptor P2Y₁, a G protein-coupled receptor expressed on the surface of circulating platelets, during lipopolysaccharide (LPS)-induced inflammation and lung injury in P2Y₁-null and wild-type mice. Systemic inflammation was induced by a single intraperitoneal dose of LPS (3 mg/kg), and the mice were analyzed 24 h posttreatment. The data show that the LPS-induced inflammation levels were comparable in the P2Y₁-null and wild-type mice. Specifically, splenomegaly, counts of circulating platelets and white blood cells (lymphocytes and neutrophils), and assessments of lung injury (tissue architecture and cell infiltration) were similar in the P2Y₁-null and wild-type mice. Based on my results, I conclude that lung injury during LPS-induced inflammation in mice is independent of P2Y₁ signaling. I propose that if a blockade of purinergic signaling in platelets is a potential lung-protective strategy in the treatment of acute inflammation, then it is more likely to be a result of the disruption of the signaling pathway mediated by P2Y₁₂, another G protein-coupled receptor that mediates platelet responses to ADP.