P2Y2 deficiency impacts adult neurogenesis and related forebrain functions.

Adult neurogenesis occurs particularly in the subgranular zone (SGZ) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricle. This continuous addition of neurons to pre-existing neuronal networks is essential for intact cognitive and olfactory functions, respectively. Purinergic signaling modulates adult neurogenesis, however, the role of individual purinergic receptor subtypes in this dynamic process and related cognitive performance is poorly understood. In this study, we analyzed the role of P2Y2 receptor in the neurogenic niches and in related forebrain functions such as spatial working memory and olfaction using mice with a targeted deletion of the P2Y2 receptor (P2Y2-/- ). Proliferation, migration, differentiation, and survival of neuronal precursor cells (NPCs) were analyzed by BrdU assay and immunohistochemistry; signal transduction pathway components were analyzed by immunoblot. In P2Y2-/- mice, proliferation of NPCs in the SGZ and the SVZ was reduced. However, migration, neuronal fate decision, and survival were not affected. Moreover, p-Akt expression was decreased in P2Y2-/- mice. P2Y2-/- mice showed an impaired performance in the Y-maze and a higher latency in the hidden food test. These data indicate that the P2Y2 receptor plays an important role in NPC proliferation as well as in hippocampus-dependent working memory and olfactory function.

Purinergic Signaling Mediates PTH and Fluid Flow-Induced Osteoblast Proliferation.

Both parathyroid hormone (PTH) and mechanical signals are able to regulate bone growth and regeneration. They also can work synergistically to regulate osteoblast proliferation, but little is known about the mechanisms how PTH and mechanical signals interact with each other during this process. In this study, we investigated responses of MC3T3-E1 osteoblasts to PTH and oscillatory fluid flow. We found that osteoblasts are more sensitive to mechanical signals in the presence of PTH according to ERK1/2 phosphorylation, ATP release, CREB phosphorylation, and cell proliferation. PTH may also reduce the osteoblast refractory period after desensitization due to mechanical signals. We further found that the synergistic responses of osteoblasts to fluid flow or ATP with PTH had similar patterns, suggesting that synergy between fluid flow and PTH may be through the ATP pathway. After we inhibited ATP effects using apyrase in osteoblasts, their synergistic responses to mechanical stimulation and PTH were also inhibited. Additionally, knocking down P2Y2 purinergic receptors can significantly attenuate osteoblast synergistic responses to mechanical stimulation and PTH in terms of ERK1/2 phosphorylation, CREB phosphorylation, and cell proliferation. Thus, our results suggest that PTH enhances mechanosensitivity of osteoblasts via a mechanism involving ATP and P2Y2 purinergic receptors.

Vascular control of the CO2/H+-dependent drive to breathe.

Respiratory chemoreceptors regulate breathing in response to changes in tissue CO2/H+. Blood flow is a fundamental determinant of tissue CO2/H+, yet little is known regarding how regulation of vascular tone in chemoreceptor regions contributes to respiratory behavior. Previously, we showed in rat that CO2/H+-vasoconstriction in the retrotrapezoid nucleus (RTN) supports chemoreception by a purinergic-dependent mechanism (Hawkins et al., 2017). Here, we show in mice that CO2/H+ dilates arterioles in other chemoreceptor regions, thus demonstrating CO2/H+ vascular reactivity in the RTN is unique. We also identify P2Y2 receptors in RTN smooth muscle cells as the substrate responsible for this response. Specifically, pharmacological blockade or genetic deletion of P2Y2 from smooth muscle cells blunted the ventilatory response to CO2, and re-expression of P2Y2 receptors only in RTN smooth muscle cells fully rescued the CO2/H+ chemoreflex. These results identify P2Y2 receptors in RTN smooth muscle cells as requisite determinants of respiratory chemoreception.

P2Y2 Receptor Promotes High-Fat Diet-Induced Obesity.

P2Y2, a G protein-coupled receptor (R), is expressed in all organs involved in the development of obesity and insulin resistance. To explore the role of it in diet-induced obesity, we fed male P2Y2-R whole body knockout (KO) and wild type (WT) mice (B6D2 genetic background) with regular diet (CNT; 10% calories as fat) or high-fat diet (HFD; 60% calories as fat) with free access to food and water for 16 weeks, and euthanized them. Adjusted for body weights (BW), KO mice consumed modestly, but significantly more HFD vs. WT mice, and excreted well-formed feces with no taint of fat or oil. Starting from the 2nd week, HFD-WT mice displayed significantly higher BW with terminal mean difference of 22% vs. HFD-KO mice. Terminal weights of white adipose tissue (WAT) were significantly lower in the HFD-KO vs. HFD-WT mice. The expression of P2Y2-R mRNA in WAT was increased by 2-fold in HFD-fed WT mice. Serum insulin, leptin and adiponectin levels were significantly elevated in the HFD-WT mice, but not in the HFD-KO mice. When induced in vitro, preadipocytes derived from KO mice fed regular diet did not differentiate and mature as robustly as those from the WT mice, as assessed by cellular expansion and accumulation of lipid droplets. Blockade of P2Y2-R by AR-C118925 in preadipocytes derived from WT mice prevented differentiation and maturation. Under basal conditions, KO mice had significantly higher serum triglycerides and showed slightly impaired lipid tolerance as compared to the WT mice. HFD-fed KO mice had significantly better glucose tolerance (GTT) as compared to HFD-fed WT mice. Whole body insulin sensitivity and mRNA expression of insulin receptor, IRS-1 and GLUT4 in WAT was significantly higher in HFD-fed KO mice vs. HFD-fed WT mice. On the contrary, the expression of pro-inflammatory molecules MCP-1, CCR2, CD68, and F4/80 were significantly higher in the WAT of HFD-fed WT vs. HFD-fed KO mice. These data suggest that P2Y2-R plays a significant role in the development of diet-induced obesity by promoting adipogenesis and inflammation, and altering the production of adipokines and lipids and their metabolism in adipose tissue, and thereby facilitates HFD-induced insulin resistance.

Ligand binding and activation of UTP-activated G protein-coupled P2Y2 and P2Y4 receptors elucidated by mutagenesis, pharmacological and computational studies.

The nucleotide receptors P2Y2 and P2Y4 are the most closely related G protein-coupled receptors (GPCRs) of the P2Y receptor (P2YR) family. Both subtypes couple to Gq proteins and are activated by the pyrimidine nucleotide UTP, but only P2Y2R is also activated by the purine nucleotide ATP. Agonists and antagonists of both receptor subtypes have potential as drugs e.g. for neurodegenerative and inflammatory diseases. So far, potent and selective, "drug-like" ligands for both receptors are scarce, but would be required for target validation and as lead structures for drug development. Structural information on the receptors is lacking since no X-ray structures or cryo-electron microscopy images are available. Thus, we performed receptor homology modeling and docking studies combined with mutagenesis experiments on both receptors to address the question how ligand binding selectivity for these closely related P2YR subtypes can be achieved. The orthosteric binding site of P2Y2R appeared to be more spacious than that of P2Y4R. Mutation of Y197 to alanine in P2Y4R resulted in a gain of ATP sensitivity. Anthraquinone-derived antagonists are likely to bind to the orthosteric or an allosteric site depending on their substitution pattern and the nature of the orthosteric binding site of the respective P2YR subtype. These insights into the architecture of P2Y2- and P2Y4Rs and their interactions with structurally diverse agonists and antagonist provide a solid basis for the future design of potent and selective ligands.

Nucleotides regulate the common molecular mechanisms that underlie neurodegenerative diseases; Therapeutic implications.

Neurodegenerative diseases (ND) are a heterogeneous group of neurological disorders characterized by a progressive loss of neuronal function which results in neuronal death. Although a specific toxic factor has been identified for each ND, all of them share common pathological molecular mechanisms favouring the disease development. In the final stages of ND, patients become unable to take care of themselves and decline to a total functional incapacitation that leads to their death. Some of the main factors which contribute to the disease progression include proteasomal dysfunction, neuroinflammation, synaptic alterations, protein aggregation, and oxidative stress. Over recent years, evidence has been accumulated to suggest that purinergic signaling plays a key role in the aforementioned molecular pathways. In this review, we revise the implications of the purinergic signaling in the common molecular mechanism underlying the ND. In particular, we focus on the role of the purinergic receptors P2X7, P2Y2 and the ectoenzyme tissue-nonspecific alkaline phosphatase (TNAP).

Mechanism underlying the contractile activity of UTP in the mammalian heart.

We previously reported that uridine 5'-triphosphate (UTP), a pyrimidine nucleoside triphosphate produced a concentration- and time-dependent increase in the contraction force in isolated right atrial preparations from patients undergoing cardiac bypass surgery due to angina pectoris. The stimulation of the force of contraction was sustained rather than transient. In the present study, we tried to elucidate the underlying receptor and signal transduction for this effect of UTP. Therefore, we measured the effect of UTP on force of contraction, phosphorylation of p38 and ERK1/2, in human atrial preparations, atrial preparations from genetically modified mice, cardiomyocytes from adult mice and cardiomyocytes from neonatal rats. UTP exerted a positive inotropic effect in isolated electrically driven left atrial preparations from wild-type (WT) mice and P2Y2-, P2Y4- and P2Y6-receptor knockout mice. Therefore, we concluded that these P2Y receptors did not mediate the inotropic effects of UTP in atrial preparations from mice. However, UTP (like ATP) increased the phosphorylation states of p38 and ERK1/2 in neonatal rat cardiomyocytes, adult mouse cardiomyocytes and human atrial tissue in vitro. U0126, a MEK 1/2- signal cascade inhibitor, attenuated this phosphorylation and the positive inotropic effects of UTP in murine and human atrial preparations. We suggest that presently unknown receptors mediate the positive inotropic effect of UTP in murine and human atria. We hypothesize that UTP stimulates inotropy via p38 or ERK1/2 phosphorylation. We speculate that UTP may be a valuable target in the development of new drugs aimed at treating human systolic heart failure.

Adenosine triphosphate induces P2Y2 activation and interleukin-8 release in human esophageal epithelial cells.

BACKGROUND AND AIM: Immune-mediated mucosal inflammation characterized by the release of interleukin (IL)-8 is associated with gastroesophageal reflux disease. ATP released by human esophageal epithelial cells (HEECs) mediates the release of cytokines through P2 nucleotide receptors that are present on various cells, including HEECs. This study characterized and identified human esophageal epithelial P2 receptors that are responsible for ATP-mediated release of IL-8 by using a human esophageal stratified squamous epithelial model. METHODS: Primary HEECs were cultured with the use of an air-liquid interface (ALI) system. The ATP analogue adenosine 5'-O-3-thiotriphosphate (ATP-γ-S) was added to the basolateral compartment, and IL-8 release was measured. Involvement of the P2Y2 receptor was assessed with the use of selective and non-selective receptor antagonists and a P2Y2 receptor agonist. Expression of the P2Y2 receptor was assessed using western blotting and immunohistochemistry. RESULTS: Adenosine triphosphate-γ-S induced IL-8 release through the P2Y2 receptor. A P2Y2 receptor antagonist but not a P2X3 receptor antagonist or a P2Y1 receptor antagonist blocked ATP-γ-S-mediated IL-8 release. Conversely, a P2Y2 receptor agonist induced IL-8 release. Western blotting and immunohistochemistry of the P2Y2 receptor showed strong expression of the P2Y2 receptor on ALI-cultured HEECs and in human esophagus. Inhibition of extracellular signal-regulated kinase but not of protein kinase C blocked the ATP-mediated release of IL-8. ATP-γ-S induced phosphorylation of extracellular signal-regulated kinase, and a P2Y2 receptor antagonist blocked this phosphorylation. CONCLUSIONS: Interleukin-8 release after purinergic stimulation in ALI-cultured HEECs is mediated through P2Y2 receptor activation. ATP-induced IL-8 release maybe involved in the pathogenesis of refractory gastroesophageal reflux disease.

Functional and molecular evidence for heteromeric association of P2Y1 receptor with P2Y2 and P2Y4 receptors in mouse granulocytes.

BACKGROUND: All hematopoietic cells express P2 receptors, however pharmacological characteristics such as expression and affinity in granulocytes are unknown. METHODS: Pharmacological characteristics of P2 receptors were evaluated by Ca(2+) measurements using Fura-2 fluorophore. P2 receptors expression were analyzed by flow cytometry and RT-PCR. P2 interaction were shown by coimmunoprecipitation, western blotting and FRET. RESULTS: Granulocytes were responsive to P2Y agonists, whereas P2X agonists were ineffective. Ca(2+) increase, elicited by ADP and UTP was dependent on intracellular stocks and sensitive to G-coupled receptor inhibition. Moreover, MRS2179, a specific antagonist of the P2Y1 receptor, abolished ADP response. Interestingly, ADP and UTP exhibited full heterologous desensitization, suggesting that these agonists interact with the same receptor. The heteromeric association between P2Y1 receptor and the P2Y2 and P2Y4 receptors was shown by immunoprecipitation and FRET analysis. CONCLUSION: Clear evidence of heteromeric association of P2Y receptors was found during the evaluation of P2 receptors present in mice granulocytes, which could impact in the classical pharmacology of P2Y receptors in granulocytes.

P2Y2 receptors regulate osteoblast mechanosensitivity during fluid flow.

Mechanical stimulation of osteoblasts activates many cellular mechanisms including the release of ATP. Binding of ATP to purinergic receptors is key to load-induced osteogenesis. Osteoblasts also respond to fluid shear stress (FSS) with increased actin stress fiber formation (ASFF) that we postulate is in response to activation of the P2Y2 receptor (P2Y2R). Furthermore, we predict that ASFF increases cell stiffness and reduces the sensitivity to further mechanical stimulation. We found that small interfering RNA (siRNA) suppression of P2Y2R attenuated ASFF in response to FSS and ATP treatment. In addition, RhoA GTPase was activated within 15 min after the onset of FSS or ATP treatment and mediated ASFF following P2Y2R activation via the Rho kinase (ROCK)1/LIM kinase 2/cofilin pathway. We also observed that ASFF in response to FSS or ATP treatment increased the cell stiffness and was prevented by knocking down P2Y2R. Finally, we confirmed that the enhanced cell stiffness and ASFF in response to RhoA GTPase activation during FSS drastically reduced the mechanosensitivity of the osteoblasts based on the intracellular Ca(2+) concentration ([Ca(2+)]i) response to consecutive bouts of FSS. These data suggest that osteoblasts can regulate their mechanosensitivity to continued load through P2Y2R activation of the RhoA GTPase signaling cascade, leading to ASFF and increased cell stiffness.

Involvement of P2Y(2,4) receptors in the regulation of myocardial contractility in growing rats.

Experiments with R2Y receptor blockers allowed identification of R2Y subtypes mediating the inhibitory effects of uridine triphosphate on myocardial contractility. In 100-day-old animals, the myocardial inotropic response to the administration of uridine triphosphate was mediated by R2Y2 receptors. R2Y4 receptors took part in the realization of negative inotropic response to uridine triphosphate in all age groups, but the most pronounced effects of this substance on myocardial contractility were found in 100-day-old rats. It was found that R2Y receptor blockers PPADS and reagent blue-2 affect amplitude-time parameters of myocardial contractility in rats of various ages.

[Nucleotide receptors and actin cytoskeleton dynamics]. / Receptory nukleotydowe a dynamika cytoszkieletu aktynowego.

Signaling cascades evoked by P2Y2 receptor plays an important role in the phenomena dependent on the actin cytoskeleton dynamics endocy-tosis, cell division, adhesion, intracellular transport and migration. P2Y2R coupled with G proteins, in response to ATP or UTP activates Rac1 and RhoA proteins important factors in actin cytoskeletal reorganization and regulates the level of phosphatidylinositol-4,5-bisphosphate (PIP2) that binds directly to a variety of actin regulatory proteins and modulates their function. The P2Y2 nucleotide receptor contains the integrin-binding domain enables it to interact selectively with α(v)ß3 and α(v)ß5 integrins and is required for G0-mediated Rac1 activation. Interaction with α(v)ß5 is necessary for coupling the P2Y2 receptor to G12 and subsequent activation of RhoA.

Platelet-derived nucleotides promote tumor-cell transendothelial migration and metastasis via P2Y2 receptor.

Tumor cells can activate platelets, which in turn facilitate tumor cell survival and dissemination. The exact mechanisms by which platelets promote metastasis have remained unclear. Here, we show that adenine nucleotides released from tumor cell-activated platelets induce opening of the endothelial barrier to allow transendothelial migration of tumor cells and thereby promote cancer cell extravasation. We identified the endothelial P2Y2 receptor, which is activated by ATP, as the primary mediator of this effect. Mice deficient in P2Y2 or lacking ATP secretion from platelets show strongly reduced tumor cell metastasis. These findings demonstrate a mechanism by which platelets promote cancer cell metastasis and suggest the P2Y2 receptor and its endothelial downstream signaling mechanisms as a target for antimetastatic therapies.

Platelets and tumor cells: a new form of border control.

Tumor metastasis is the primary cause of death due to cancer, but the mechanisms by which tumor cells metastasize remain incompletely understood. In this issue of Cancer Cell, Schumacher and colleagues suggest that ATP released from tumor-associated platelets in the blood facilitates tumor metastasis by relaxing endothelial barrier function.

Lifelong physical activity preserves functional sympatholysis and purinergic signalling in the ageing human leg.

Ageing is associated with an impaired ability to modulate sympathetic vasoconstrictor activity (functional sympatholysis) and a reduced exercise hyperaemia. The purpose of this study was to investigate whether a physically active lifestyle can offset the impaired functional sympatholysis and exercise hyperaemia in the leg and whether ATP signalling is altered by ageing and physical activity. Leg haemodynamics, interstitial [ATP] and P2Y(2) receptor content was determined in eight young (23 ± 1 years), eight lifelong sedentary elderly (66 ± 2 years) and eight lifelong active elderly (62 ± 2 years) men at rest and during one-legged knee extensions (12 W and 45% maximal workload (WL(max))) and arterial infusion of ACh and ATP with and without tyramine. The vasodilatory response to ACh was lowest in the sedentary elderly, higher in active elderly (P < 0.05) and highest in the young men (P < 0.05), whereas ATP-induced vasodilatation was lower in the sedentary elderly (P < 0.05). During exercise (12 W), leg blood flow, vascular conductance and VO2 was lower and leg lactate release higher in the sedentary elderly compared to the young (P < 0.05), whereas there was no difference between the active elderly and young. Interstitial [ATP] during exercise and P2Y(2) receptor content were higher in the active elderly compared to the sedentary elderly (P < 0.05). Tyramine infusion lowered resting vascular conductance in all groups, but only in the sedentary elderly during exercise (P < 0.05). Tyramine did not alter the vasodilator response to ATP infusion in any of the three groups. Plasma [noradrenaline] increased more during tyramine infusion in both elderly groups compared to young (P < 0.05). A lifelong physically active lifestyle can maintain an intact functional sympatholysis during exercise and vasodilator response to ATP despite a reduction in endothelial nitric oxide function. A physically active lifestyle increases interstitial ATP levels and skeletal muscle P2Y(2) receptor content.

Purinergic P2Y2 receptors promote neutrophil infiltration and hepatocyte death in mice with acute liver injury.

BACKGROUND & AIMS: During progression of liver disease, inflammation affects survival of hepatocytes. Endogenous release of adenosine triphosphate (ATP) in the liver activates purinergic P2 receptors (P2R), which regulate inflammatory responses, but little is known about the roles of these processes in the development of acute hepatitis. METHODS: We induced acute hepatitis in C57BL/6 mice by intravenous injection of concanavalin A and then analyzed liver concentrations of ATP and expression of P2R. We assessed P2Y(2)R(-/-) mice and C57BL/6 wild-type mice injected with suramin, a pharmacologic inhibitor of P2YR. Toxic liver failure was induced in mice by intraperitoneal injection of acetaminophen. Hepatocyte-specific functions of P2R signaling were analyzed in primary mouse hepatocytes. RESULTS: Induction of acute hepatitis in wild-type C57BL/6 mice released large amounts of ATP from livers and induced expression of P2Y(2)R. Liver damage and necrosis were greatly reduced in P2Y(2)R(-/-) mice and C57BL/6 mice given injections of suramin. Acetaminophen-induced liver damage was reduced in P2Y(2)R(-/-) mice. Analysis of liver-infiltrating immune cells during acute hepatitis revealed that expression of P2Y(2)R in bone marrow-derived cells was required for liver infiltration by neutrophils and subsequent liver damage. Hepatic expression of P2Y(2)R interfered with expression of genes that regulate cell survival, and promoted tumor necrosis factor-α-mediated cell death, in a cell-autonomous manner. CONCLUSIONS: Extracellular ATP and P2Y(2)R have cell-type specific, but synergistic functions during liver damage that regulate cellular immune responses and promote hepatocyte death. Reagents designed to target P2Y(2)R might be developed to treat inflammatory liver disease.