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.     

P2X7 nucleotide receptors mediate caspase-8/9/3-dependent apoptosis in rat primary cortical neurons

Apoptosis is a major cause of cell death in the nervous system. It plays a role in embryonic and early postnatal brain development and contributes to the pathology of neurodegenerative diseases. Here, we report that activation of the P2X₇ nucleotide receptor (P2X₇R) in rat primary cortical neurons (rPCNs) causes biochemical (i.e., caspase activation) and morphological (i.e., nuclear condensation and DNA fragmentation) changes characteristic of apoptotic cell death. Caspase-3 activation and DNA fragmentation in rPCNs induced by the P2X₇R agonist BzATP were inhibited by the P2X₇R antagonist oxidized ATP (oATP) or by pre-treatment of cells with P2X₇R antisense oligonucleotide indicating a direct involvement of the P2X₇R in nucleotide-induced neuronal cell death. Moreover, Z-DEVD-FMK, a specific and irreversible cell permeable inhibitor of caspase-3, prevented BzATP-induced apoptosis in rPCNs. In addition, a specific caspase-8 inhibitor, Ac-IETD-CHO, significantly attenuated BzATP-induced caspase-9 and caspase-3 activation, suggesting that P2X₇R-mediated apoptosis in rPCNs occurs primarily through an intrinsic caspase-8/9/3 activation pathway. BzATP also induced the activation of C-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated kinases (ERK1/2) in rPCNs, and pharmacological inhibition of either JNK1 or ERK1/2 significantly reduced caspase activation by BzATP. Taken together, these data indicate that extracellular nucleotides mediate neuronal apoptosis through activation of P2X₇Rs and their downstream signaling pathways involving JNK1, ERK and caspases 8/9/3.     

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.     

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 G_q 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.     

Differential deregulation of astrocytic calcium signalling by amyloid-β, TNFα, IL-1β and LPS

In Alzheimer's disease (AD), astrocytes undergo complex morphological and functional changes that include early atrophy, reactive activation and Ca²⁺ deregulation. Recently, we proposed a mechanism by which nanomolar Aβ₄₂ deregulates mGluR5 and InsP₃ receptors, the key elements of astrocytic Ca²⁺ signalling toolkit. To evaluate the specificity of these changes, we have now investigated whether the effects of Aβ₄₂ on Ca²⁺ signalling machinery can be reproduced by pro-inflammatory agents (TNFα, IL-1β, LPS). Here we report that Aβ₄₂ (100 nM, 72 h) significantly increased mRNA levels of mGluR5, InsP₃R1 and InsP₃R2, whereas pro-inflammatory agents reduced expression of these specific mRNAs. Furthermore, DHPG-induced Ca²⁺ signals and store operated Ca²⁺ entry (SOCE) were augmented in Aβ₄₂-treated cells due to up-regulation of a set of Ca²⁺ signalling-related genes including TRPC1 and TRPC4. Opposite changes were observed when astrocytes were treated with TNFα, IL-1β and LPS. Last, the effects observed on SOCE by treating wild-type astrocytes with Aβ₄₂ were also identified in untreated astrocytes from 3×Tg-AD animals, suggesting a link to the AD pathology. Our results demonstrate that effects of Aβ₄₂ on astrocytic Ca²⁺ signalling differ from and may contrast to the effects of pro-inflammatory agents.     

P2Y nucleotide receptor interaction with alpha integrin mediates astrocyte migration

Astrocytes become activated in response to brain injury, as characterized by increased expression of glial fibrillary acidic protein (GFAP) and increased rates of cell migration and proliferation. Damage to brain cells causes the release of cytoplasmic nucleotides, such as ATP and uridine 5'-triphosphate (UTP), ligands for P2 nucleotide receptors. Results in this study with primary rat astrocytes indicate that activation of a G protein-coupled P2Y(2) receptor for ATP and UTP increases GFAP expression and both chemotactic and chemokinetic cell migration. UTP-induced astrocyte migration was inhibited by silencing of P2Y(2) nucleotide receptor (P2Y(2)R) expression with siRNA of P2Y(2)R (P2Y(2)R siRNA). UTP also increased the expression in astrocytes of alpha(V)beta(3/5) integrins that are known to interact directly with the P2Y(2)R to modulate its function. Anti-alpha(V) integrin antibodies prevented UTP-stimulated astrocyte migration, suggesting that P2Y(2)R/alpha(V) interactions mediate the activation of astrocytes by UTP. P2Y(2)R-mediated astrocyte migration required the activation of the phosphatidylinositol-3-kinase (PI3-K)/protein kinase B (Akt) and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways, responses that also were inhibited by anti-alpha(V) integrin antibody. These results suggest that P2Y(2)Rs and their associated signaling pathways may be important factors regulating astrogliosis in brain disorders.     

P2Y6 receptor-dependent microglial phagocytosis of synapses mediates synaptic and memory loss in aging

Aging causes loss of brain synapses and memory, and microglial phagocytosis of synapses may contribute to this loss. Stressed neurons can release the nucleotide UTP, which is rapidly converted into UDP, that in turn activates the P2Y₆ receptor (P2Y₆R) on the surface of microglia, inducing microglial phagocytosis of neurons. However, whether the activation of P2Y₆R affects microglial phagocytosis of synapses is unknown. We show here that inactivation of P2Y₆R decreases microglial phagocytosis of isolated synapses (synaptosomes) and synaptic loss in neuronal–glial co-cultures. In vivo, wild-type mice aged from 4 to 17 months exhibited reduced synaptic density in cortical and hippocampal regions, which correlated with increased internalization of synaptic material within microglia. However, this aging-induced synaptic loss and internalization were absent in P2Y₆R knockout mice, and these mice also lacked any aging-induced memory loss. Thus, P2Y₆R appears to mediate aging-induced loss of synapses and memory by increasing microglial phagocytosis of synapses. Consequently, blocking P2Y₆R has the potential to prevent age-associated memory impairment.     

Astrocytes Protect Neurons against Methylmercury via ATP/P2Y1 Receptor-Mediated Pathways in Astrocytes

Methylmercury (MeHg) is a well known environmental pollutant that induces serious neuronal damage. Although MeHg readily crosses the blood-brain barrier, and should affect both neurons and glial cells, how it affects glia or neuron-to-glia interactions has received only limited attention. Here, we report that MeHg triggers ATP/P2Y₁ receptor signals in astrocytes, thereby protecting neurons against MeHg via interleukin-6 (IL-6)-mediated pathways. MeHg increased several mRNAs in astrocytes, among which IL-6 was the highest. For this, ATP/P2Y₁ receptor-mediated mechanisms were required because the IL-6 production was (i) inhibited by a P2Y₁ receptor antagonist, MRS2179, (ii) abolished in astrocytes obtained from P2Y₁ receptor-knockout mice, and (iii) mimicked by exogenously applied ATP. In addition, (iv) MeHg released ATP by exocytosis from astrocytes. As for the intracellular mechanisms responsible for IL-6 production, p38 MAP kinase was involved. MeHg-treated astrocyte-conditioned medium (ACM) showed neuro-protective effects against MeHg, which was blocked by anti-IL-6 antibody and was mimicked by the application of recombinant IL-6. As for the mechanism of neuro-protection by IL-6, an adenosine A₁ receptor-mediated pathway in neurons seems to be involved. Taken together, when astrocytes sense MeHg, they release ATP that autostimulates P2Y₁ receptors to upregulate IL-6, thereby leading to A₁ receptor-mediated neuro-protection against MeHg.     

Coupling switch of P2Y-IP<Subscript>3</Subscript> receptors mediates differential Ca<Superscript>2+</Superscript> signaling in human embryonic stem cells and derived cardiovascular progenitor cells

Purinergic signaling mediated by P2 receptors (P2Rs) plays important roles in embryonic and stem cell development. However, how it mediates Ca²⁺ signals in human embryonic stem cells (hESCs) and derived cardiovascular progenitor cells (CVPCs) remains unclear. Here, we aimed to determine the role of P2Rs in mediating Ca²⁺ mobilizations of these cells. hESCs were induced to differentiate into CVPCs by our recently established methods. Gene expression of P2Rs and inositol 1,4,5-trisphosphate receptors (IP₃Rs) was analyzed by quantitative/RT-PCR. IP₃R3 knockdown (KD) or IP₃R2 knockout (KO) hESCs were established by shRNA- or TALEN-mediated gene manipulations, respectively. Confocal imaging revealed that Ca²⁺ responses in CVPCs to ATP and UTP were more sensitive and stronger than those in hESCs. Consistently, the gene expression levels of most P2YRs except P2Y₁ were increased in CVPCs. Suramin or PPADS blocked ATP-induced Ca²⁺ transients in hESCs but only partially inhibited those in CVPCs. Moreover, the P2Y₁ receptor-specific antagonist MRS2279 abolished most ATP-induced Ca²⁺ signals in hESCs but not in CVPCs. P2Y₁ receptor-specific agonist MRS2365 induced Ca²⁺ transients only in hESCs but not in CVPCs. Furthermore, IP₃R2KO but not IP₃R3KD decreased the proportion of hESCs responding to MRS2365. In contrast, both IP₃R2 and IP₃R3 contributed to UTP-induced Ca²⁺ responses while ATP-induced Ca²⁺ responses were more dependent on IP₃R2 in the CVPCs. In conclusion, a predominant role of P2Y₁ receptors in hESCs and a transition of P2Y-IP₃R coupling in derived CVPCs are responsible for the differential Ca²⁺ mobilization between these cells.     

The role of P2Y14 and other P2Y receptors in degranulation of human LAD2 mast cells

Mast cell degranulation affects many conditions, e.g., asthma and urticaria. We explored the potential role of the P2Y₁₄ receptor (P2Y₁₄R) and other P2Y subtypes in degranulation of human LAD2 mast cells. All eight P2YRs were expressed at variable levels in LAD2 cells (quantitative real-time RT-PCR). Gene expression levels of ADP receptors, P2Y₁R, P2Y₁₂R, and P2Y₁₃R, were similar, and P2Y₁₁R and P2Y₄R were highly expressed at 5.8- and 3.8-fold of P2Y₁R, respectively. Least expressed P2Y₂R was 40-fold lower than P2Y₁R, and P2Y₆R and P2Y₁₄R were ≤50 % of P2Y₁R. None of the native P2YR agonists alone induced β-hexosaminidase (β-Hex) release, but some nucleotides significantly enhanced β-Hex release induced by C3a or antigen, with a rank efficacy order of ATP > UDPG ≥ ADP >> UDP, UTP. Although P2Y₁₁R and P2Y₄R are highly expressed, they did not seem to play a major role in degranulation as neither P2Y₄R agonist UTP nor P2Y₁₁R agonists ATPγS and NF546 had a substantial effect. P2Y₁R-selective agonist MRS2365 enhanced degranulation, but ~1,000-fold weaker compared to its P2Y₁R potency, and the effect of P2Y₆R agonist 3-phenacyl-UDP was negligible. The enhancement by ADP and ATP appears mediated via multiple receptors. Both UDPG and a synthetic agonist of the P2Y₁₄R, MRS2690, enhanced C3a-induced β-Hex release, which was inhibited by a P2Y₁₄R antagonist, specific P2Y₁₄R siRNA and pertussis toxin, suggesting a role of P2Y₁₄R activation in promoting human mast cell degranulation.     

Loss of P2Y2 Nucleotide Receptors Enhances Early Pathology in the TgCRND8 Mouse Model of Alzheimer's Disease

Neuroinflammation is a prominent feature in Alzheimer's disease (AD) and activation of the brain's innate immune system, particularly microglia, has been postulated to both retard and accelerate AD progression. Recent studies indicate that the G protein-coupled P2Y₂ nucleotide receptor (P2Y₂R) is an important regulator of innate immunity by assisting in the recruitment of monocytes to injured tissue, neutrophils to bacterial infections and eosinophils to allergen-infected lungs. In this study, we investigated the role of the P2Y₂R in progression of an AD-like phenotype in the TgCRND8 mouse model that expresses Swedish and Indiana mutations in amyloid precursor protein (APP). Our results indicate that P2Y ₂ R expression is upregulated in TgCRND8 mouse brain within 10 weeks of age and then decreases after 25 weeks of age, as compared to littermate controls expressing low levels of the P2Y ₂ R. TgCRND8 mice with homozygous P2Y ₂ R deletion survive less than 5 weeks, whereas mice with heterozygous P2Y ₂ R deletion survive for 12 weeks, a time point when TgCRND8 mice are fully viable. Heterozygous P2Y ₂ R deletion in TgCRND8 mice increased β-amyloid (Aβ) plaque load and soluble Aβ_1–42 levels in the cerebral cortex and hippocampus, decreased the expression of the microglial marker CD11b in these brain regions and caused neurological deficits within 10 weeks of age, as compared to age-matched TgCRND8 mice. These findings suggest that the P2Y₂R is important for the recruitment and activation of microglial cells in the TgCRND8 mouse brain and that the P2Y₂R may regulate neuroprotective mechanisms through microglia-mediated clearance of Aβ that when lost can accelerate the onset of an AD-like phenotype in the TgCRND8 mouse.     

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.     

Tumour necrosis factor alpha-induced neuronal loss is mediated by microglial phagocytosis

Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine, expressed in many brain pathologies and associated with neuronal loss. We show here that addition of TNF-α to neuronal–glial co-cultures increases microglial proliferation and phagocytosis, and results in neuronal loss that is prevented by eliminating microglia. Blocking microglial phagocytosis by inhibiting phagocytic vitronectin and P2Y₆ receptors, or genetically removing opsonin MFG-E8, prevented TNF-α induced loss of live neurons. Thus TNF-α appears to induce neuronal loss via microglial activation and phagocytosis of neurons, causing neuronal death by phagoptosis.     

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 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.     

Role of ecto-NTPDases on UDP-sensitive P2Y(6) receptor activation during osteogenic differentiation of primary bone marrow stromal cells from postmenopausal women

This study aimed at investigating the expression and function of uracil nucleotide‐sensitive receptors (P2Y₂, P2Y₄, and P2Y₆) on osteogenic differentiation of human bone marrow stromal cells (BMSCs) in culture. Bone marrow specimens were obtained from postmenopausal female patients (68 ± 5 years old, n = 18) undergoing total hip arthroplasty. UTP and UDP (100 µM) facilitated osteogenic differentiation of the cells measured as increases in alkaline phosphatase (ALP) activity, without affecting cell proliferation. Uracil nucleotides concentration‐dependently increased [Ca²⁺]_i in BMSCs; their effects became less evident with time (7 > 21 days) of the cells in culture. Selective activation of P2Y₆ receptors with the stable UDP analog, PSB 0474, mimicked the effects of both UTP and UDP, whereas UTPγS was devoid of effect. Selective blockade of P2Y₆ receptors with MRS 2578 prevented [Ca²⁺]_i rises and osteogenic differentiation caused by UDP at all culture time points. BMSCs are immunoreactive against P2Y₂, P2Y₄, and P2Y₆ receptors. While the expression of P2Y₆ receptors remained fairly constant (7～21 days), P2Y₂ and P2Y₄ became evident only in less proliferative and more differentiated cultures (7 < 21 days). The rate of extracellular UTP and UDP inactivation was higher in less proliferative and more differentiated cell populations. Immunoreactivity against NTPDase1, ‐2, and ‐3 rises as cells differentiate (7 < 21 days). Data show that uracil nucleotides are important regulators of osteogenic cells differentiation predominantly through the activation of UDP‐sensitive P2Y₆ receptors coupled to increases in [Ca²⁺]_i. Endogenous actions of uracil nucleotides may be balanced through specific NTPDases determining whether osteoblast progenitors are driven into proliferation or differentiation. J. Cell. Physiol. 227: 2694–2709, 2012. © 2011 Wiley Periodicals, Inc.