P2X7 receptor antagonism prevents IL-1ß release from salivary epithelial cells and reduces inflammation in a mouse model of autoimmune exocrinopathy.

Salivary gland inflammation is a hallmark of Sjögren's syndrome (SS), a common autoimmune disease characterized by lymphocytic infiltration of the salivary gland and loss of saliva secretion, predominantly in women. The P2X7 receptor (P2X7R) is an ATP-gated nonselective cation channel that induces inflammatory responses in cells and tissues, including salivary gland epithelium. In immune cells, P2X7R activation induces the production of proinflammatory cytokines, including IL-1ß and IL-18, by inducing the oligomerization of the multiprotein complex NLRP3-type inflammasome. Here, our results show that in primary mouse submandibular gland (SMG) epithelial cells, P2X7R activation also induces the assembly of the NLRP3 inflammasome and the maturation and release of IL-1ß, a response that is absent in SMG cells isolated from mice deficient in P2X7Rs (P2X7R-/-). P2X7R-mediated IL-1ß release in SMG epithelial cells is dependent on transmembrane Na+ and/or K+ flux and the activation of heat shock protein 90 (HSP90), a protein required for the activation and stabilization of the NLRP3 inflammasome. Also, using the reactive oxygen species (ROS) scavengers N-acetyl cysteine and Mito-TEMPO, we determined that mitochondrial reactive oxygen species are required for P2X7R-mediated IL-1ß release. Lastly, in vivo administration of the P2X7R antagonist A438079 in the CD28-/-, IFNγ-/-, NOD.H-2h4 mouse model of salivary gland exocrinopathy ameliorated salivary gland inflammation and enhanced carbachol-induced saliva secretion. These findings demonstrate that P2X7R antagonism in vivo represents a promising therapeutic strategy to limit salivary gland inflammation and improve secretory function.

P2Y2 receptor modulates shear stress-induced cell alignment and actin stress fibers in human umbilical vein endothelial cells.

Endothelial cells release ATP in response to fluid shear stress, which activates purinergic (P2) receptor-mediated signaling molecules including endothelial nitric oxide (eNOS), a regulator of vascular tone. While P2 receptor-mediated signaling in the vasculature is well studied, the role of P2Y2 receptors in shear stress-associated endothelial cell alignment, cytoskeletal alterations, and wound repair remains ill defined. To address these aspects, human umbilical vein endothelial cell (HUVEC) monolayers were cultured on gelatin-coated dishes and subjected to a shear stress of 1 Pa. HUVECs exposed to either P2Y2 receptor antagonists or siRNA showed impaired fluid shear stress-induced cell alignment, and actin stress fiber formation as early as 6 h. Similarly, when compared to cells expressing the P2Y2 Arg-Gly-Asp (RGD) wild-type receptors, HUVECs transiently expressing the P2Y2 Arg-Gly-Glu (RGE) mutant receptors showed reduced cell alignment and actin stress fiber formation in response to shear stress as well as to P2Y2 receptor agonists in static cultures. Additionally, we observed reduced shear stress-induced phosphorylation of focal adhesion kinase (Y397), and cofilin-1 (S3) with receptor knockdown as well as in cells expressing the P2Y2 RGE mutant receptors. Consistent with the role of P2Y2 receptors in vasodilation, receptor knockdown and overexpression of P2Y2 RGE mutant receptors reduced shear stress-induced phosphorylation of AKT (S473), and eNOS (S1177). Furthermore, in a scratched wound assay, shear stress-induced cell migration was reduced by both pharmacological inhibition and receptor knockdown. Together, our results suggest a novel role for P2Y2 receptor in shear stress-induced cytoskeletal alterations in HUVECs.

P2Y receptors in Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia, affecting more than 10% of people over the age of 65. Age is the greatest risk factor for AD, although a combination of genetic, lifestyle and environmental factors also contribute to disease development. Common features of AD are the formation of plaques composed of beta-amyloid peptides (Aß) and neuronal death in brain regions involved in learning and memory. Although Aß is neurotoxic, the primary mechanisms by which Aß affects AD development remain uncertain and controversial. Mouse models overexpressing amyloid precursor protein and Aß have revealed that Aß has potent effects on neuroinflammation and cerebral blood flow that contribute to AD progression. Therefore, it is important to consider how endogenous signalling in the brain responds to Aß and contributes to AD pathology. In recent years, Aß has been shown to affect ATP release from brain and blood cells and alter the expression of G protein-coupled P2Y receptors that respond to ATP and other nucleotides. Accumulating evidence reveals a prominent role for P2Y receptors in AD pathology, including Aß production and elimination, neuroinflammation, neuronal function and cerebral blood flow.

P2Y2 nucleotide receptor activation enhances the aggregation and self-organization of dispersed salivary epithelial cells.

Hyposalivation resulting from salivary gland dysfunction leads to poor oral health and greatly reduces the quality of life of patients. Current treatments for hyposalivation are limited. However, regenerative medicine to replace dysfunctional salivary glands represents a revolutionary approach. The ability of dispersed salivary epithelial cells or salivary gland-derived progenitor cells to self-organize into acinar-like spheres or branching structures that mimic the native tissue holds promise for cell-based reconstitution of a functional salivary gland. However, the mechanisms involved in salivary epithelial cell aggregation and tissue reconstitution are not fully understood. This study investigated the role of the P2Y2 nucleotide receptor (P2Y2R), a G protein-coupled receptor that is upregulated following salivary gland damage and disease, in salivary gland reconstitution. In vitro results with the rat parotid acinar Par-C10 cell line indicate that P2Y2R activation with the selective agonist UTP enhances the self-organization of dispersed salivary epithelial cells into acinar-like spheres. Other results indicate that the P2Y2R-mediated response is dependent on epidermal growth factor receptor activation via the metalloproteases ADAM10/ADAM17 or the α5ß1 integrin/Cdc42 signaling pathway, which leads to activation of the MAPKs JNK and ERK1/2. Ex vivo data using primary submandibular gland cells from wild-type and P2Y2R(-/-) mice confirmed that UTP-induced migratory responses required for acinar cell self-organization are mediated by the P2Y2R. Overall, this study suggests that the P2Y2R is a promising target for salivary gland reconstitution and identifies the involvement of two novel components of the P2Y2R signaling cascade in salivary epithelial cells, the α5ß1 integrin and the Rho GTPase Cdc42.

Up-regulation and activation of the P2Y(2) nucleotide receptor mediate neurite extension in IL-1ß-treated mouse primary cortical neurons.

The pro-inflammatory cytokine interleukin-1ß (IL-1ß), 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-1ß 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-1ß-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-αv ß3/5 integrin antibodies prevent UTP-induced cofilin activation in IL-1ß-treated mPCNs, suggesting that established P2Y2R/αv ß3/5 interactions that promote G12 -dependent Rho activation lead to cofilin phosphorylation involved in neurite extension. Cofilin phosphorylation induced by UTP in IL-1ß-treated mPCNs is also decreased by inhibitors of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), suggesting a role for P2Y2R-mediated and Gq-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 receptor activation induces inflammatory responses in salivary gland epithelium.

Inflammation of the salivary gland is a well-documented aspect of salivary gland dysfunction that occurs in Sjogren's syndrome (SS), an autoimmune disease, and in γ-radiation-induced injury during treatment of head and neck cancers. Extracellular nucleotides have gained recognition as key modulators of inflammation through activation of cell surface ionotropic and metabotropic receptors, although the contribution of extracellular nucleotides to salivary gland inflammation is not well understood. In vitro studies using submandibular gland (SMG) cell aggregates isolated from wild-type C57BL/6 mice indicate that treatment with ATP or the high affinity P2X7R agonist 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) induces membrane blebbing and enhances caspase activity, responses that were absent in SMG cell aggregates isolated from mice lacking the P2X7R (P2X7R(-/-)). Additional studies with SMG cell aggregates indicate that activation of the P2X7R with ATP or BzATP stimulates the cleavage and release of α-fodrin, a cytoskeletal protein thought to act as an autoantigen in the development of SS. In vivo administration of BzATP to ligated SMG excretory ducts enhances immune cell infiltration into the gland and initiates apoptosis of salivary epithelial cells in wild-type, but not P2X7R(-/-), mice. These findings indicate that activation of the P2X7R contributes to salivary gland inflammation in vivo, suggesting that the P2X7R may represent a novel target for the treatment of salivary gland dysfunction.

Nucleotides released from Aß1₋42 -treated microglial cells increase cell migration and Aß1₋42 uptake through P2Y2 receptor activation.

Amyloid ß-protein (Aß) deposits in brains of Alzheimer's disease patients generate proinflammatory cytokines and chemokines that recruit microglial cells to phagocytose Aß. Nucleotides released from apoptotic cells activate P2Y(2) receptors (P2Y(2) Rs) in macrophages to promote clearance of dead cells. In this study, we investigated the role of P2Y(2) Rs in the phagocytosis and clearance of Aß. Treatment of mouse primary microglial cells with fibrillar (fAß(1-42) ) and oligomeric (oAß(1-42) ) Aß(1-42) aggregation solutions caused a rapid release of ATP (maximum after 10 min). Furthermore, fAß(1-42) and oAß(1-42) treatment for 24 h caused an increase in P2Y(2) R gene expression. Treatment with fAß(1-42) and oAß(1-42) aggregation solutions increased the motility of neighboring microglial cells, a response inhibited by pre-treatment with apyrase, an enzyme that hydrolyzes nucleotides. The P2Y(2) R agonists ATP and UTP caused significant uptake of Aß(1-42) by microglial cells within 30 min, which reached a maximum within 1 h, but did not increase Aß(1-42) uptake by primary microglial cells isolated from P2Y(2) R(-/-) mice. Inhibitors of α(v) integrins, Src and Rac decreased UTP-induced Aß(1-42) uptake, suggesting that these previously identified components of the P2Y(2) R signaling pathway play a role in Aß phagocytosis by microglial cells. Finally, we found that UTP treatment enhances Aß(1-42) degradation by microglial cells, but not in cells isolated from P2Y(2) R(-/-) mice. Taken together, our findings suggest that P2Y(2) Rs can activate microglial cells to enhance Aß clearance and highlight the P2Y(2) R as a therapeutic target in Alzheimer's disease.

Neuroprotective roles of the P2Y(2) receptor.

Purinergic signaling plays a unique role in the brain by integrating neuronal and glial cellular circuits. The metabotropic P1 adenosine receptors and P2Y nucleotide receptors and ionotropic P2X receptors control numerous physiological functions of neuronal and glial cells and have been implicated in a wide variety of neuropathologies. Emerging research suggests that purinergic receptor interactions between cells of the central nervous system (CNS) have relevance in the prevention and attenuation of neurodegenerative diseases resulting from chronic inflammation. CNS responses to chronic inflammation are largely dependent on interactions between different cell types (i.e., neurons and glia) and activation of signaling molecules including P2X and P2Y receptors. Whereas numerous P2 receptors contribute to functions of the CNS, the P2Y(2) receptor is believed to play an important role in neuroprotection under inflammatory conditions. While acute inflammation is necessary for tissue repair due to injury, chronic inflammation contributes to neurodegeneration in Alzheimer's disease and occurs when glial cells undergo prolonged activation resulting in extended release of proinflammatory cytokines and nucleotides. This review describes cell-specific and tissue-integrated functions of P2 receptors in the CNS with an emphasis on P2Y(2) receptor signaling pathways in neurons, glia, and endothelium and their role in neuroprotection.

P2Y2 nucleotide receptors mediate metalloprotease-dependent phosphorylation of epidermal growth factor receptor and ErbB3 in human salivary gland cells.

The G protein-coupled receptor P2Y(2) nucleotide receptor (P2Y(2)R) has been shown to be up-regulated in a variety of tissues in response to stress or injury. Recent studies have suggested that P2Y(2)Rs 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 P2Y(2)R 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-alpha protease inhibitor or by small interfering RNA that selectively silences ADAM10 and ADAM17 expression, suggesting that ADAM metalloproteases are required for P2Y(2)R-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 P2Y(2)R 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.

Binding of the P2Y2 nucleotide receptor to filamin A regulates migration of vascular smooth muscle cells.

The functional expression of the G protein-coupled P2Y(2) nucleotide receptor (P2Y(2)R) has been associated with proliferation and migration of vascular smooth muscle cells (SMCs), two processes involved in atherosclerosis and restenosis. Activation of the P2Y(2)R causes dynamic reorganization of the actin cytoskeleton, which transmits biochemical signals and forces necessary for cell locomotion, suggesting that P2Y(2)Rs may be linked to the actin cytoskeleton. Here, we identified filamin A (FLNa) as a P2Y(2)R-interacting protein using a yeast 2-hybrid system screen with the C-terminal region of the P2Y(2)R as bait. The FLNa binding site in the P2Y(2)R is localized between amino acids 322 and 333. Deletion of this region led to selective loss of FLNa binding to the P2Y(2)R and abolished Tyr phosphorylation of FLNa induced by the P2Y(2)R agonist UTP. Using both time-lapse microscopy and the Transwell cell migration assay, we showed that UTP significantly increased SMC spreading on collagen I (6.8 fold; P < or = 0.01) and migration (3.6 fold; P < or = 0.01) of aortic SMCs isolated from wild-type mice, as compared with unstimulated SMCs. UTP-induced spreading and migration of aortic SMCs did not occur with cells isolated from P2Y(2)R knockout mice. Expression of the full-length P2Y(2)R in SMCs isolated from P2Y(2)R knockout mice restored both UTP-induced spreading and migration. In contrast, UTP-induced spreading and migration did not occur in SMCs isolated from P2Y(2)R knockout mice transfected with a mutant P2Y(2)R that does not bind FLNa. Furthermore, ex vivo studies showed that both ATP and UTP (10 micromol/L) promoted migration of SMCs out of aortic explants isolated from wild-type but not P2Y(2)R knockout mice. Thus, this study demonstrates that P2Y(2)R/FLNa interaction selectively regulates spreading and migration of vascular SMCs.

P2Y2 receptors mediate nucleotide-induced EGFR phosphorylation and stimulate proliferation and tumorigenesis of head and neck squamous cell carcinoma cell lines.

OBJECTIVES: To assess functional expression of the P2Y2 nucleotide receptor (P2Y2R) in head and neck squamous cell carcinoma (HNSCC) cell lines and define its role in nucleotide-induced epidermal growth factor receptor (EGFR) transactivation. The use of anti-EGFR therapeutics to treat HNSCC is hindered by intrinsic and acquired drug resistance. Defining novel pathways that modulate EGFR signaling could identify additional targets to treat HNSCC. MATERIALS AND METHODS: In human HNSCC cell lines CAL27 and FaDu and the mouse oral cancer cell line MOC2, P2Y2R contributions to extracellular nucleotide-induced changes in intracellular free Ca2+ concentration and EGFR and extracellular signal-regulated kinase (ERK1/2) phosphorylation were determined using the ratiometric Ca2+ indicator fura-2 and immunoblot analysis, respectively. Genetic knockout of P2Y2Rs using CRISPR technology or pharmacological inhibition with P2Y2R-selective antagonist AR-C118925 defined P2Y2R contributions to in vivo tumor growth. RESULTS: P2Y2R agonists UTP and ATP increased intracellular Ca2+ levels and ERK1/2 and EGFR phosphorylation in CAL27 and FaDu cells, responses that were inhibited by AR-C118925 or P2Y2R knockout. P2Y2R-mediated EGFR phosphorylation was also attenuated by inhibition of the adamalysin family of metalloproteases or Src family kinases. P2Y2R knockout reduced UTP-induced CAL27 cell proliferation in vitro and significantly reduced CAL27 and FaDu tumor xenograft volume in vivo. In a syngeneic mouse model of oral cancer, AR-C118925 administration reduced MOC2 tumor volume. CONCLUSION: P2Y2Rs mediate HNSCC cell responses to extracellular nucleotides and genetic or pharmacological blockade of P2Y2R signaling attenuates tumor cell proliferation and tumorigenesis, suggesting that the P2Y2R represents a novel therapeutic target in HNSCC.

Interleukin-1beta enhances nucleotide-induced and alpha-secretase-dependent amyloid precursor protein processing in rat primary cortical neurons via up-regulation of the P2Y(2) receptor.

The heterologous expression and activation of the human P2Y(2) nucleotide receptor (P2Y(2)R) in human 1321N1 astrocytoma cells stimulates alpha-secretase-dependent cleavage of the amyloid precursor protein (APP), causing extracellular release of the non-amyloidogenic protein secreted amyloid precursor protein (sAPPalpha). To determine whether a similar response occurs in a neuronal cell, we analyzed whether P2Y(2)R-mediated production of sAPPalpha occurs in rat primary cortical neurons (rPCNs). In rPCNs, P2Y(2)R mRNA and receptor activity were virtually absent in quiescent cells, whereas overnight treatment with the pro-inflammatory cytokine interleukin-1beta (IL-1beta) up-regulated both P2Y(2)R mRNA expression and receptor activity by four-fold. The up-regulation of the P2Y(2)R was abrogated by pre-incubation with Bay 11-7085, an IkappaB-alpha phosphorylation inhibitor, which suggests that P2Y(2)R mRNA transcript levels are regulated through nuclear factor-kappa-B (NFkappaB) signaling. Furthermore, the P2Y(2)R agonist Uridine-5'-triphosphate (UTP) enhanced the release of sAPPalpha in rPCNs treated with IL-1beta or transfected with P2Y(2)R cDNA. UTP-induced release of sAPPalpha from rPCNs was completely inhibited by pre-treatment of the cells with the metalloproteinase inhibitor TACE inhibitor (TAPI-2) or the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, and was partially inhibited by the MAPK/extracellular signal-regulated kinase inhibitor U0126 and the protein kinase C inhibitor GF109203. These data suggest that P2Y(2)R-mediated release of sAPPalpha from cortical neurons is directly dependent on a disintegrin and metalloproteinase (ADAM) 10/17 and PI3K activity, whereas extracellular signal-regulated kinase 1/2 and PI3K activity may indirectly regulate APP processing. These results demonstrate that elevated levels of pro-inflammatory cytokines associated with neurodegenerative diseases, such as IL-1beta, can enhance non-amyloidogenic APP processing through up-regulation of the P2Y(2)R in neurons.

Regulated catalysis of extracellular nucleotides by vascular CD39/ENTPD1 is required for liver regeneration.

BACKGROUND & AIMS: Little is known about how endothelial cells respond to injury, regulate hepatocyte turnover and reconstitute the hepatic vasculature. We aimed to determine the effects of the vascular ectonucleotidase CD39 on sinusoidal endothelial cell responses following partial hepatectomy and to dissect purinergic and growth factor interactions in this model. METHODS: Parameters of liver injury and regeneration, as well as the kinetics of hepatocellular and sinusoidal endothelial cell proliferation, were assessed following partial hepatectomy in mice that do not express CD39, that do not express ATP/UTP receptor P2Y2, and in controls. The effects of extracellular ATP on vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-6 responses were determined in vivo and in vitro. Phosphorylation of the endothelial VEGF receptor in response to extracellular nucleotides and growth factors was assessed in vitro. RESULTS: After partial hepatectomy, expression of the vascular ectonucleotidase CD39 increased on sinusoidal endothelial cells. Targeted disruption of CD39 impaired hepatocellular regeneration, reduced angiogenesis, and increased hepatic injury, resulting in pronounced vascular endothelial apoptosis, and decreased survival. Decreased HGF release by sinusoidal endothelial cells, despite high levels of VEGF, reduced paracrine stimulation of hepatocytes. Failure of VEGF receptor-2/KDR transactivation by extracellular nucleotides on CD39-null endothelial cells was associated with P2Y2 receptor desensitization. CONCLUSIONS: Regulated phosphohydrolysis of extracellular nucleotides by CD39 coordinates both hepatocyte and endothelial cell proliferation following partial hepatectomy. Lack of CD39 activity is associated with decreased hepatic regeneration and failure of vascular reconstitution.

Purinergic signaling in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by three major histopathological markers: amyloid-ß (Aß) plaques, neurofibrillary tangles and gliosis in the central nervous system (CNS). It is now accepted that neuroinflammatory events in the CNS play a crucial role in the development of AD. This review focuses on neuroinflammatory signaling mediated by purinergic receptors (P1 adenosine receptors, P2X ATP-gated ion channels and G protein-coupled P2Y nucleotide receptors) and how therapeutic modulation of purinergic signaling influences disease progression in AD patients and animal models of AD.

Low-affinity binding in cis to P2Y2R mediates force-dependent integrin activation during hantavirus infection.

Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, ß3 integrins. Previous studies have implicated a cognate cis interaction between the bent conformation ß5/ß3 integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2Y2R. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant αIIbß3 integrins and (RGD)P2Y2R expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2Y2R removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2Y2R on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing αIIbß3 integrins, virus engagement at the integrin PSI domain, recapitulates physiologic activation of the integrin as indicated by staining with the activation-specific mAB PAC1. The data also show that blocking of the Gα13 protein from binding to the cytoplasmic domain of the ß3 integrin prevents outside-in signaling and infection. We propose that the cis interaction with P2Y2R provides allosteric resistance to the membrane-normal motion associated with the switchblade model of integrin activation, where the development of tensile force yields physiological integrin activation.

Differential coupling of the P2Y1 receptor to Galpha14 and Galphaq/11 proteins during the development of the rat salivary gland.

UNLABELLED: In rat submandibular gland (SMG), the P2Y1 receptor (P2Y1R) mediates increases in the intracellular calcium concentration, [Ca2+]i that diminish as the animal ages from 1 to 4-6 weeks. However, P2Y1R mRNA levels do not change with age, suggesting that the age-dependent decrease in the [Ca2+]i response to P2Y1R agonists may be due to alterations in the activity of a component of the P2Y1R signalling pathway. OBJECTIVES: To assess whether the decrease in P2Y1R-mediated intracellular calcium signalling in SMG cells as rats age is due to a decrease in P2Y1R coupling to G proteins or to a decrease in the expression of a cognate G protein. DESIGN: SMG cells were isolated from Sprague-Dawley rats. P2Y1R function was assessed by measuring 2-MeSADP-induced increases in [Ca2+]i and ERK1/2 activation. P2Y(1)R-mediated activation of G proteins was determined by the [35S]GTPgammaS binding assay. Gq protein expression was determined by RT-PCR, Northern, and Western analysis. RESULTS: In SMG cells from 1-week-old rats, two bands (52 and 42kDa) were detected using anti-Galpha14 antibody, whereas in SMG cells from 4- to 6-week-old rats only the 42 kDa band was detected. Furthermore, 2-MeSADP-induced GTPgamma35S binding to Galpha14 and Galphaq/11 decreases in SMG cells from 4- to 6-week-old rats as compared to 1-week-old rats. CONCLUSIONS: These findings suggest that the age-dependent decrease in P2Y1R-mediated intracellular calcium signalling in rat SMG cells is due to a loss of 52 kDa Galpha14 and indicate the differential coupling of the P2Y1R to Galpha14 and Galphaq/11 as the gland develops.

P2 receptors in health and disease.

In the past ten years since the discovery and cloning of members of the P2 receptor family, rapid progress has been made in the field regarding the function and pharmacology of different P2 receptor subtypes. This research resulted in identifying these receptors as important players in the pathology of atherosclerosis, cystic fibrosis, neurodegenerative and autoimmune diseases, among other disorders. The signalling mechanisms whereby P2 receptors mediate pathogenesis are not clear in most cases. Future studies in this field will focus on the integration of signalling pathways coupled to P2 receptors and the generation of specific agonists/antagonists for each receptor subtype to provide strategies for the treatment of a variety of diseases.

Purinergic receptors as potential therapeutic targets in Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of memory and cognitive ability and is a serious cause of mortality. Many of the pathological characteristics associated with AD are revealed post-mortem, including amyloid-ß plaque deposition, neurofibrillary tangles containing hyperphosphorylated tau proteins and neuronal loss in the hippocampus and cortex. Although several genetic mutations and risk factors have been associated with the disease, the causes remain poorly understood. Study of disease-initiating mechanisms and AD progression in humans is inherently difficult as most available tissue specimens are from late-stages of disease. Therefore, AD researchers rely on in vitro studies and the use of AD animal models where neuroinflammation has been shown to be a major characteristic of AD. Purinergic receptors are a diverse family of proteins consisting of P1 adenosine receptors and P2 nucleotide receptors for ATP, UTP and their metabolites. This family of receptors has been shown to regulate a wide range of physiological and pathophysiological processes, including neuroinflammation, and may contribute to the pathogenesis of neurodegenerative diseases like Parkinson's disease, multiple sclerosis and AD. Experimental evidence from human AD tissue has suggested that purinergic receptors may play a role in AD progression and studies using selective purinergic receptor agonists and antagonists in vitro and in AD animal models have demonstrated that purinergic receptors represent novel therapeutic targets for the treatment of AD. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.

Functional P2Y2 nucleotide receptors mediate uridine 5'-triphosphate-induced intimal hyperplasia in collared rabbit carotid arteries.

BACKGROUND: Extracellular uridine 5'-triphosphate (UTP) induces mitogenic activation of smooth muscle cells (SMCs) through binding to P2Y2 nucleotide receptors. P2Y2 receptor mRNA is upregulated in intimal lesions of rat aorta, but it is unclear how this G-protein-coupled receptor contributes to development of intimal hyperplasia. METHODS AND RESULTS: This study used a silicone collar placed around rabbit carotid arteries to induce vascular injury and intimal thickening. Collar placement caused rapid upregulation of P2Y2 receptor mRNA in medial SMCs before appearance of neointima. Fura-2 digital imaging of single SMCs was used to measure changes in myoplasmic calcium concentration (Ca(m)) in response to P2Y receptor agonists. In contrast to UDP, activation by UTP or adenosine 5'-triphosphate (ATP) greatly increased Ca(m), which indicates upregulation of functional P2Y2 receptors at which UTP and ATP are equipotent agonists. The number of responsive cells was significantly greater for freshly dispersed SMCs from collared arteries than for controls. Perivascular infusion of UTP (100 micromol/L) within the collar significantly enhanced neointimal development. Intimas that resulted from UTP exposure were infiltrated by macrophages. Moreover, increased expression of osteopontin occurred in response to in situ application of UTP. ATP or UTP also stimulated osteopontin expression in cultured SMCs in a dose-dependent manner. Furthermore, P2Y2 antisense oligonucleotide inhibited osteopontin expression induced by UTP. CONCLUSIONS: These findings indicate for the first time a role for the UTP/ATP receptor, P2Y2, in development of intimal hyperplasia associated with atherosclerosis and restenosis.