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1.  Contact lenses: new devices for nucleotide delivery in ocular pathologies 
Purinergic Signalling  2014;10(3):419-420.
doi:10.1007/s11302-014-9422-7
PMCID: PMC4152449  PMID: 25138143
2.  Adenosine enhances progenitor cell recruitment and nerve growth via its A2B receptor during adult fin regeneration 
Purinergic Signalling  2014;10(4):595-602.
A major issue in regenerative medicine is the control of progenitor cell mobilisation. Apoptosis has been reported as playing a role in cell plasticity, and it has been recently shown that apoptosis is necessary for organ and appendage regeneration. In this context, we explore its possible mode of action in progenitor cell recruitment during adult regeneration in zebrafish. Here, we show that apoptosis inhibition impairs blastema formation and nerve growth, both of which can be restored by exogenous adenosine acting through its A2B receptor. Moreover, adenosine increases the number of progenitor cells. Purinergic signalling is therefore an early and essential event in the pathway from lesion to blastema formation and provides new targets for manipulating cell plasticity in the adult.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-014-9420-9) contains supplementary material, which is available to authorized users.
doi:10.1007/s11302-014-9420-9
PMCID: PMC4272362  PMID: 25084769
Apoptosis; Stem cells; Adenosine; A2B receptor; Nerve growth; Proliferation; Zebrafish; Regeneration
3.  P2X receptors regulate adenosine diphosphate release from hepatic cells 
Purinergic Signalling  2014;10(4):587-593.
Extracellular nucleotides act as paracrine regulators of cellular signaling and metabolic pathways. Adenosine polyphosphate (adenosine triphosphate (ATP) and adenosine diphosphate (ADP)) release and metabolism by human hepatic carcinoma cells was therefore evaluated. Hepatic cells maintain static nanomolar concentrations of extracellular ATP and ADP levels until stress or nutrient deprivation stimulates a rapid burst of nucleotide release. Reducing the levels of media serum or glucose has no effect on ATP levels, but stimulates ADP release by up to 10-fold. Extracellular ADP is then metabolized or degraded and media ADP levels fall to basal levels within 2–4 h. Nucleotide release from hepatic cells is stimulated by the Ca2+ ionophore, ionomycin, and by the P2 receptor agonist, 2′3′-O-(4-benzoyl-benzoyl)-adenosine 5′-triphosphate (BzATP). Ionomycin (10 μM) has a minimal effect on ATP release, but doubles media ADP levels at 5 min. In contrast, BzATP (10–100 μM) increases both ATP and ADP levels by over 100-fold at 5 min. Ion channel purinergic receptor P2X7 and P2X4 gene silencing with small interference RNA (siRNA) and treatment with the P2X inhibitor, A438079 (100 μM), decrease ADP release from hepatic cells, but have no effect on ATP. P2X inhibitors and siRNA have no effect on BzATP-stimulated nucleotide release. ADP release from human hepatic carcinoma cells is therefore regulated by P2X receptors and intracellular Ca2+ levels. Extracellular ADP levels increase as a consequence of a cellular stress response resulting from serum or glucose deprivation.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-014-9419-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s11302-014-9419-2
PMCID: PMC4272363  PMID: 25059924
ADP; ATP; Nucleotide release; P2X receptor; P2X4; P2X7
4.  UTP is not a biased agonist at human P2Y11 receptors 
Purinergic Signalling  2014;10(4):581-585.
Biased agonism describes a multistate model of G protein-coupled receptor activation in which each ligand induces a unique structural conformation of the receptor, such that the receptor couples differentially to G proteins and other intracellular proteins. P2Y receptors are G protein-coupled receptors that are activated by endogenous nucleotides, such as adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP). A previous report suggested that UTP may be a biased agonist at the human P2Y11 receptor, as it increased cytosolic [Ca2+], but did not induce accumulation of inositol phosphates, whereas ATP did both. The mechanism of action of UTP was unclear, so the aim of this study was to characterise the interaction of UTP with the P2Y11 receptor in greater detail. Intracellular Ca2+ was monitored in 1321N1 cells stably expressing human P2Y11 receptors using the Ca2+-sensitive fluorescent indicator, fluo-4. ATP evoked a rapid, concentration-dependent rise in intracellular Ca2+, but surprisingly, even high concentrations of UTP were ineffective. In contrast, UTP was slightly, but significantly more potent than ATP in evoking a rise in intracellular Ca2+ in 1321N1 cells stably expressing the human P2Y2 receptor, with no difference in the maximum response. Thus, the lack of response to UTP at hP2Y11 receptors was not due to a problem with the UTP solution. Furthermore, coapplying a high concentration of UTP with ATP did not inhibit the response to ATP. Thus, contrary to a previous report, we find no evidence for an agonist action of UTP at the human P2Y11 receptor, nor does UTP act as an antagonist.
doi:10.1007/s11302-014-9418-3
PMCID: PMC4272372  PMID: 25015314
Biased agonism; P2Y11 receptor; Inositol phosphates; Intracellular Ca2+
5.  Striatal adenosine A2A receptor expression is controlled by S-adenosyl-L-methionine-mediated methylation 
Purinergic Signalling  2014;10(3):523-528.
Adenosine A2A receptor (A2AR) is a G protein-coupled receptor enriched in the striatum for which an increased expression has been demonstrated in certain neurological diseases. Interestingly, previous in vitro studies demonstrated that A2AR expression levels are reduced after treatment with S-adenosyl-L-methionine (SAM), a methyl donor molecule involved in the methylation of important biological structures such as DNA, proteins, and lipids. However, the in vivo effects of SAM treatment on A2AR expression are still obscure. Here, we demonstrated that 2 weeks of SAM treatment produced a significant reduction in the rat striatal A2AR messenger RNA (mRNA) and protein content as well as A2AR-mediated signaling. Furthermore, when the content of 5-methylcytosine levels in the 5′UTR region of ADORA2A was analyzed, this was significantly increased in the striatum of SAM-treated animals; thus, an unambiguous correlation between SAM-mediated methylation and striatal A2AR expression could be established. Overall, we concluded that striatal A2AR functionality can be controlled by SAM treatment, an issue that might be relevant for the management of these neurological conditions that course with increased A2AR expression.
doi:10.1007/s11302-014-9417-4
PMCID: PMC4152447  PMID: 24943396
S-adenosyl-L-Methionine; SAM; ADORA2A; Adenosine A2A receptor; Methylcytosine; Striatum
6.  Schistosome apyrase SmATPDase1, but not SmATPDase2, hydrolyses exogenous ATP and ADP 
Purinergic Signalling  2014;10(4):573-580.
Schistosomes are parasitic worms that can live in the bloodstream of their vertebrate hosts for many years. It has been proposed that the worms impinge on host purinergic signalling by degrading proinflammatory molecules like ATP as well as prothrombotic mediators like ADP. This capability may help explain the apparent refractoriness of the worms to both immune elimination and thrombus formation. Three distinct ectoenzymes, expressed at the host-exposed surface of the worm’s tegument, are proposed to be involved in the catabolism of ATP and ADP. These are alkaline phosphatase (SmAP), phosphodiesterase (SmNPP-5), and ATP diphosphohydrolase (SmATPDase1). It has recently been shown that only one of these enzymes—SmATPDase1—actually degrades exogenous ATP and ADP. However, a second ATP diphosphohydrolase homolog (SmATPDase2) is located in the tegument and has been reported to be released by the worms. It is possible that this enzyme too participates in the cleavage of exogenous nucleotide tri- and di-phosphates. To test this hypothesis, we employed RNA interference (RNAi) to suppress the expression of the schistosome SmATPDase1 and SmATPDase2 genes. We find that only SmATPDase1-suppressed parasites are significantly impaired in their ability to degrade exogenously added ATP or ADP. Suppression of SmATPDase2 does not appreciably affect the worms’ ability to catabolize ATP or ADP. Furthermore, we detect no evidence for the secretion or release of an ATP-hydrolyzing activity by cultured parasites. The results confirm the role of tegumental SmATPDase1, but not SmADTPDase2, in the degradation of the exogenous proinflammatory and prothrombotic nucleotides ATP and ADP by live intravascular stages of the parasite.
doi:10.1007/s11302-014-9416-5
PMCID: PMC4272364  PMID: 24894599
Schistosoma; Trematode; Tegument; ATPase; ADPase; RNA interference
7.  Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits 
Purinergic Signalling  2014;10(4):565-572.
Stimulation of P2X receptors by ATP in vascular smooth muscle cells (VSMCs) is proposed to mediate vascular tone. However, understanding of P2X receptor-mediated actions in human blood vessels is limited, and therefore, the current work investigates the role of P2X receptors in freshly isolated small human gastro-omental arteries (HGOAs). Expression of P2X1 and P2X4 receptor subunit messenger RNA (mRNA) and protein was identified in individual HGOA VSMCs using RT-PCR and immunofluorescent analysis and using Western blot in multi-cellular preparations. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l, a selective P2X receptor agonist, evoked robust increases in [Ca2+]i in fluo-3-loaded HGOA VSMCs. Pre-incubation with 1 μmol/l NF279, a selective P2X receptor antagonist, reduced the amplitude of αβ-meATP-induced increase in [Ca2+]i by about 70 %. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l produced similar contractile responses in segments of HGOA, and these contractions were greatly reduced by 2 μmol/l NF449, a selective P2X receptor inhibitor. These data suggest that VSMCs from HGOA express P2X1 and P2X4 receptor subunits with homomeric P2X1 receptors likely serving as the predominant target for extracellular ATP.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-014-9415-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s11302-014-9415-6
PMCID: PMC4272371  PMID: 24845338
Human; Gastro-omental arteries; ATP; P2X1 receptor; NF279
8.  IL-8 and global gene expression analysis define a key role of ATP in renal epithelial cell responses induced by uropathogenic bacteria 
Purinergic Signalling  2014;10(3):499-508.
The recent recognition of receptor-mediated ATP signalling as a pathway of epithelial pro-inflammatory cytokine release challenges the ubiquitous role of the TLR4 pathway during urinary tract infection. The aim of this study was to compare cellular responses of renal epithelial cells infected with uropathogenic Escherichia coli (UPEC) strain IA2 to stimulation with ATP-γ-S. A498 cells were infected or stimulated in the presence or absence of apyrase, that degrades extracellular ATP, or after siRNA-mediated knockdown of ATP-responding P2Y2 receptors. Cellular IL-8 release and global gene expression were analysed. Both IA2 and A498 cells per se released ATP, which increased during infection. IA2 and ATP-γ-S caused a ∼5-fold increase in cellular release of IL-8 and stimulations performed in the presence of apyrase or after siRNA knockdown of P2Y2 receptors resulted in attenuation of IA2-mediated IL-8 release. Microarray results show that both IA2 and ATP-γ-S induced marked changes in gene expression of renal cells. Thirty-six genes were in common between both stimuli, and many of these are key genes belonging to classical response pathways of bacterial infection. Functional analysis shows that 88 biological function-annotated cellular pathways were identical between IA2 and ATP-γ-S stimuli. Results show that UPEC-induced release of IL-8 is dependent on P2Y2 signalling and that cellular responses elicited by UPEC and ATP-γ-S have many identical features. This indicates that renal epithelial responses elicited by bacteria could be mediated by bacteria- or host-derived ATP, thus defining a key role of ATP during infection.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-014-9414-7) contains supplementary material, which is available to authorized users.
doi:10.1007/s11302-014-9414-7
PMCID: PMC4152451  PMID: 24817659
Urinary tract infection; Host response; Adenosine triphosphate; Uropathogenic E. coli; Purinergic P2Y receptors
9.  IRF8 is a transcriptional determinant for microglial motility 
Purinergic Signalling  2014;10(3):515-521.
Microglia, the resident immune cells of the central nervous system, are constitutively mobile cells that undergo rapid directional movement toward sites of tissue disruption. However, transcriptional regulatory mechanisms of microglial motility remain unknown. In the present study, we show that interferon regulatory factor-8 (IRF8) regulates microglial motility. We found that ATP and complement component, C5a, induced chemotaxis of IRF8 wild-type microglia. However, these responses were markedly suppressed in microglia lacking IRF8 (Irf8−/−). In a consistent manner, phosphorylation of Akt (which plays a crucial role in ATP-induced chemotaxis) was abolished in Irf8−/−microglia. Real-time polymerase chain reaction analysis revealed that motility-related microglial genes such as P2Y12 receptor were significantly suppressed in Irf8−/−microglia. Furthermore, Irf8−/−microglia exhibited a differential expression pattern of nucleotide-degrading enzymes compared with their wild-type counterparts. Overall, our findings suggest that IRF8 may regulate microglial motility via the control of microglial gene expression.
doi:10.1007/s11302-014-9413-8
PMCID: PMC4152453  PMID: 24798612
Microglia; Chemotaxis; IRF8; Transcription factor; ATP
10.  Purinergic signalling and immune cells 
Purinergic Signalling  2014;10(4):529-564.
This review article provides a historical perspective on the role of purinergic signalling in the regulation of various subsets of immune cells from early discoveries to current understanding. It is now recognised that adenosine 5′-triphosphate (ATP) and other nucleotides are released from cells following stress or injury. They can act on virtually all subsets of immune cells through a spectrum of P2X ligand-gated ion channels and G protein-coupled P2Y receptors. Furthermore, ATP is rapidly degraded into adenosine by ectonucleotidases such as CD39 and CD73, and adenosine exerts additional regulatory effects through its own receptors. The resulting effect ranges from stimulation to tolerance depending on the amount and time courses of nucleotides released, and the balance between ATP and adenosine. This review identifies the various receptors involved in the different subsets of immune cells and their effects on the function of these cells.
doi:10.1007/s11302-014-9427-2
PMCID: PMC4272370  PMID: 25352330
ATP ;  UTP; Lymphocytes; Neutrophils; Mast cells; Microglia; Macrophages; Purinoceptors
11.  Lack of adenosine A3 receptors causes defects in mouse peripheral blood parameters 
Purinergic Signalling  2014;10(3):509-514.
The role of the adenosine A3 receptor in hematopoiesis was studied using adenosine A3 receptor knockout (A3AR KO) mice. Hematological parameters of peripheral blood and femoral bone marrow of irradiated and untreated A3AR KO mice and their wild-type (WT) counterparts were investigated. Irradiation of the mice served as a defined hematopoiesis-damaging means enabling us to evaluate contingent differences in the pattern of experimentally induced hematopoietic suppression between the A3AR KO mice and WT mice. Defects were observed in the counts and/or functional parameters of blood cells in the A3AR KO mice. These defects include statistically significantly lower values of blood neutrophil and monocyte counts, as well as those of mean erythrocyte volume, mean erythrocyte hemoglobin, blood platelet counts, mean platelet volume, and plateletcrit, and can be considered to bear evidence of the lack of a positive role played by the adenosine A3 receptor in the hematopoietic system. Statistically significantly increased values of the bone marrow parameters studied in A3AR KO mice (femoral bone marrow cellularity, granulocyte/macrophage progenitor cells, and erythrocyte progenitor cells) can probably be explained by compensatory mechanisms attempting to offset the disorders in the function of blood elements in these mice. The pattern of the radiation-induced hematopoietic suppression was very similar in A3AR KO mice and their WT counterparts.
doi:10.1007/s11302-014-9412-9
PMCID: PMC4152454  PMID: 24763970
Adenosine A3 receptor; Adenosine A3 receptor knockout mice; Hematopoiesis; Whole-body irradiation
12.  Autocrine signaling via release of ATP and activation of P2X7 receptor influences motile activity of human lung cancer cells 
Purinergic Signalling  2014;10(3):487-497.
Extracellular nucleotides, such as ATP, are released from cells and play roles in various physiological and pathological processes through activation of P2 receptors. Here, we show that autocrine signaling through release of ATP and activation of P2X7 receptor influences migration of human lung cancer cells. Release of ATP was induced by stimulation with TGF-β1, which is a potent inducer of cell migration, in human lung cancer H292 cells, but not in noncancerous BEAS-2B cells. Treatment of H292 cells with a specific antagonist of P2X7 receptor resulted in suppression of TGF-β1-induced migration. PC-9 human lung cancer cells released a large amount of ATP under standard cell culture conditions, and P2X7 receptor-dependent dye uptake was observed even in the absence of exogenous ligand, suggesting constitutive activation of P2X7 receptor in this cell line. PC-9 cells showed high motile activity, which was inhibited by treatment with ecto-nucleotidase and P2X7 receptor antagonists, whereas a P2X7 receptor agonist enhanced migration. PC-9 cells also harbor a constitutively active mutation in epidermal growth factor receptor (EGFR). Treatment with EGFR tyrosine kinase inhibitor AG1478 suppressed both cell migration and P2X7 receptor expression in PC-9 cells. Compared to control PC-9 cells, cells treated with P2X7 antagonist exhibited broadened lamellipodia around the cell periphery, while AG1478-treated cells lacked lamellipodia. These results indicate that P2X7-mediated signaling and EGFR signaling may regulate migration of PC-9 cells through distinct mechanisms. We propose that autocrine ATP-P2X7 signaling is involved in migration of human lung cancer cells through regulation of actin cytoskeleton rearrangement.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-014-9411-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s11302-014-9411-x
PMCID: PMC4152450  PMID: 24627191
P2X7 receptor; Lung cancer; Cell migration; TGF-β1; EGFR
13.  The role of activated adenosine receptors in degranulation of human LAD2 mast cells 
Purinergic Signalling  2014;10(3):465-475.
Mast cell degranulation triggers hypersensitivity reactions at the body–environment interface. Adenosine modulates degranulation, but enhancement and inhibition have both been reported. Which of four adenosine receptors (ARs) mediate modulation, and how, remains uncertain. Also uncertain is whether adenosine reaches mast cell ARs by autocrine ATP release and ecto-enzymatic conversion. Uncertainties partly reflect species and cell heterogeneity, circumvented here by focusing on homogeneous human LAD2 cells. Quantitative PCR detected expression of A2A, A2B, and A3, but not A1, ARs. Nonselective activation of ARs with increasing NECA monotonically enhanced immunologically or C3a-stimulated degranulation. NECA alone stimulated degranulation slightly. Selective AR antagonists did not affect C3a-stimulated degranulation. NECA's enhancement of C3a-triggered degranulation was partially inhibited by separate application of each selective antagonist, and abolished by simultaneous addition of antagonists to the three ARs. Only the A2A antagonist separately inhibited NECA's enhancement of immunologically stimulated degranulation, which was abolished by simultaneous addition of the three selective antagonists. Immunological or C3a activation did not stimulate ATP release. NECA also enhanced immunologically triggered degranulation of mouse bone marrow derived mast cells (BMMCs), which was partially reduced only by simultaneous addition of the three antagonists or by the nonselective antagonist CGS15943. BMMCs also expressed A2A, A2B, and A3 ARs. but not A1AR detectably. We conclude that (a) A1AR is unnecessary for LAD2 degranulation or AR enhancement; (b) A2A, A2B, and A3 ARs all contribute to pharmacologic AR enhancement of LAD2 and BMMC degranulation; and (c) LAD2 cells depend on microenvironmental adenosine to trigger AR modulation.
doi:10.1007/s11302-014-9409-4
PMCID: PMC4152452  PMID: 24595664
FcεRI; C3a; A2A; A2B; A3; ATP release
14.  Role of adenosine A2B receptor signaling in contribution of cardiac mesenchymal stem-like cells to myocardial scar formation 
Purinergic Signalling  2014;10(3):477-486.
Adenosine levels increase in ischemic hearts and contribute to the modulation of that pathological environment. We previously showed that A2B adenosine receptors on mouse cardiac Sca1+CD31− mesenchymal stromal cells upregulate secretion of paracrine factors that may contribute to the improvement in cardiac recovery seen when these cells are transplanted in infarcted hearts. In this study, we tested the hypothesis that A2B receptor signaling regulates the transition of Sca1+CD31− cells, which occurs after myocardial injury, into a myofibroblast phenotype that promotes myocardial repair and remodeling. In vitro, TGFβ1 induced the expression of the myofibroblast marker α-smooth muscle actin (αSMA) and increased collagen I generation in Sca1+CD31− cells. Stimulation of A2B receptors attenuated TGFβ1-induced collagen I secretion but had no effect on αSMA expression. In vivo, myocardial infarction resulted in a rapid increase in the numbers of αSMA-positive cardiac stromal cells by day 5 followed by a gradual decline. Genetic deletion of A2B receptors had no effect on the initial accumulation of αSMA-expressing stromal cells but hastened their subsequent decline; the numbers of αSMA-positive cells including Sca1+CD31− cells remained significantly higher in wild type compared with A2B knockout hearts. Thus, our study revealed a significant contribution of cardiac Sca1+CD31− cells to the accumulation of αSMA-expressing cells after infarction and implicated A2B receptor signaling in regulation of myocardial repair and remodeling by delaying deactivation of these cells. It is plausible that this phenomenon may contribute to the beneficial effects of transplantation of these cells to the injured heart.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-014-9410-y) contains supplementary material, which is available to authorized users.
doi:10.1007/s11302-014-9410-y
PMCID: PMC4152448  PMID: 24584483
Adenosine; Receptor; Adenosine A2B; Mesenchymal stromal cells; Myofibroblasts; Myocardial infarction; Alpha-smooth muscle actin
15.  Effect of ecto-5′-nucleotidase (eN) in astrocytes on adenosine and inosine formation 
Purinergic Signalling  2014;10(4):603-609.
ATP is a gliotransmitter released from astrocytes. Extracellularly, ATP is metabolized by a series of enzymes, including ecto-5′-nucleotidase (eN; also known as CD73) which is encoded by the gene 5NTE and functions to form adenosine (ADO) from adenosine monophosphate (AMP). Under ischemic conditions, ADO levels in brain increase up to 100-fold. We used astrocytes cultured from 5NTE+/+ or 5NTE−/− mice to evaluate the role of eN expressed by astrocytes in the production of ADO and inosine (INO) in response to glucose deprivation (GD) or oxygen-glucose deprivation (OGD). We also used co-cultures of these astrocytes with wild-type neurons to evaluate the role of eN expressed by astrocytes in the production of ADO and INO in response to GD, OGD, or N-methyl-d-aspartate (NMDA) treatment. As expected, astrocytes from 5NTE+/+ mice produced adenosine from AMP; the eN inhibitor α,β-methylene ADP (AOPCP) decreased ADO formation. In contrast, little ADO was formed by astrocytes from 5NTE−/− mice and AOPCP had no significant effect. GD and OGD treatment of 5NTE+/+ astrocytes and 5NTE+/+ astrocyte-neuron co-cultures produced extracellular ADO levels that were inhibited by AOPCP. In contrast, these conditions did not evoke ADO production in cultures containing 5NTE−/− astrocytes. NMDA treatment produced similar increases in ADO in both 5NTE+/+ and 5NTE−/− astrocyte-neuron co-cultures; dipyridamole (DPR) but not AOPCP inhibited ADO production. These results indicate that eN is prominent in the formation of ADO from astrocytes but in astrocyte-neuron co-cultures, other enzymes or pathways contribute to rising ADO levels in ischemia-like conditions.
doi:10.1007/s11302-014-9421-8
PMCID: PMC4272361  PMID: 25129451
Astrocyte; Neuron; Cell culture; Ecto 5′-nucleotidase; Nucleoside transport
16.  The enteric nervous system of P2Y13 receptor null mice is resistant against high-fat-diet- and palmitic-acid-induced neuronal loss 
Purinergic Signalling  2014;10(3):455-464.
Gastrointestinal symptoms have a major impact on the quality of life and are becoming more prevalent in the western population. The enteric nervous system (ENS) is pivotal in regulating gastrointestinal functions. Purinergic neurotransmission conveys a range of short and long-term cellular effects. This study investigated the role of the ADP-sensitive P2Y13 receptor in lipid-induced enteric neuropathy. Littermate P2Y13+/+ and P2Y13−/− mice were fed with either a normal diet (ND) or high-fat diet (HFD) for 6 months. The intestines were analysed for morphological changes as well as neuronal numbers and relative numbers of vasoactive intestinal peptide (VIP)- and neuronal nitric oxide synthase (nNOS)-containing neurons. Primary cultures of myenteric neurons from the small intestine of P2Y13+/+ or P2Y13−/− mice were exposed to palmitic acid (PA), the P2Y13 receptor agonist 2meSADP and the antagonist MRS2211. Neuronal survival and relative number of VIP-containing neurons were analysed. In P2Y13+/+, but not in P2Y13−/− mice, HFD caused a significant loss of myenteric neurons in both ileum and colon. In colon, the relative numbers of VIP-containing submucous neurons were significantly lower in the P2Y13−/− mice compared with P2Y13+/+ mice. The relative numbers of nNOS-containing submucous colonic neurons increased in P2Y13+/+ HFD mice. HFD also caused ileal mucosal thinning in P2Y13+/+ and P2Y13−/− mice, compared to ND fed mice. In vitro PA exposure caused loss of myenteric neurons from P2Y13+/+ mice while neurons from P2Y13−/− mice were unaffected. Presence of MRS2211 prevented PA-induced neuronal loss in cultures from P2Y13+/+ mice. 2meSADP caused no change in survival of cultured neurons. P2Y13 receptor activation is of crucial importance in mediating the HFD- and PA-induced myenteric neuronal loss in mice. In addition, the results indicate a constitutive activation of enteric neuronal apoptosis by way of P2Y13 receptor stimulation.
doi:10.1007/s11302-014-9408-5
PMCID: PMC4152455  PMID: 24510452
Palmitic acid; Enteric nervous system; Purinergic receptors; P2Y13 knockout mice; Primary cell culture; High-fat diet
17.  Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells 
Purinergic Signalling  2014;10(3):431-440.
ATP is released from cells in response to various stimuli. Our previous studies on pancreas indicated that pancreatic acini could be major stores of secreted ATP. In the present study, our aim was to establish the role of the vesicular nucleotide transporter (VNUT), SLC17A9, in storage and release of ATP. Freshly prepared acini from mice and AR42J rat acinar cells were used in this study. We illustrate that in AR42J cells, quinacrine (an ATP store marker) and Bodipy ATP (a fluorescent ATP analog) co-localized with VNUT-mCherry to vesicles/granules. Furthermore, in acini and AR42J cells, a marker of the zymogen granule membranes, Rab3D, and VNUT co-localized. Dexamethasone treatment of AR42J cells promoted formation of acinar structures, paralleled by increased amylase and VNUT expression, and increased ATP release in response to cholinergic stimulation. Mechanical stimulus (pressure) and cell swelling also induced ATP release, but this was not influenced by dexamethasone, most likely indicating different non-zymogen-related release mechanism. In conclusion, we propose that VNUT-dependent ATP release pathway is associated with agonist-induced secretion process and downstream purinergic signalling in pancreatic ducts.
doi:10.1007/s11302-014-9406-7
PMCID: PMC4152456  PMID: 24488439
Pancreas; ATP release; VNUT; SLC17A9; AR42J; Pancreatitis; Mechanical stress
18.  A yeast screening method to decipher the interaction between the adenosine A2B receptor and the C-terminus of different G protein α-subunits 
Purinergic Signalling  2014;10(3):441-453.
The expression of human G protein-coupled receptors (GPCRs) in Saccharomyces cerevisiae containing chimeric yeast/mammalian Gα subunits provides a useful tool for the study of GPCR activation. In this study, we used a one-GPCR-one-G protein yeast screening method in combination with molecular modeling and mutagenesis studies to decipher the interaction between GPCRs and the C-terminus of different α-subunits of G proteins. We chose the human adenosine A2B receptor (hA2BR) as a paradigm, a typical class A GPCR that shows promiscuous behavior in G protein coupling in this yeast system. The wild-type hA2BR and five mutant receptors were expressed in 8 yeast strains with different humanized G proteins, covering the four major classes: Gαi, Gαs, Gαq, and Gα12. Our experiments showed that a tyrosine residue (Y) at the C-terminus of the Gα subunit plays an important role in controlling the activation of GPCRs. Receptor residues R1033.50 and I1073.54 are vital too in G protein-coupling and the activation of the hA2BR, whereas L213IL3 is more important in G protein inactivation. Substitution of S2356.36 to alanine provided the most divergent G protein-coupling profile. Finally, L2366.37 substitution decreased receptor activation in all G protein pathways, although to a different extent. In conclusion, our findings shed light on the selectivity of receptor/G protein coupling, which may help in further understanding GPCR signaling.
doi:10.1007/s11302-014-9407-6
PMCID: PMC4152457  PMID: 24464644
G protein-coupled receptor; G protein coupling; Yeast screening; Adenosine A2B receptor; DRY motif
19.  NTPDase3 and ecto-5′-nucleotidase/CD73 are differentially expressed during mouse bladder cancer progression 
Purinergic Signalling  2014;10(3):421-430.
According to the World Health Organization, bladder cancer is the seventh most common cancer among men in the world. The current treatments for this malignancy are not efficient to prevent the recurrence and progression of tumors. Then, researches continue looking for better therapeutic targets which can end up in new and more efficient treatments. One of the recent findings was the identification that the purinergic system was involved in bladder tumorigenesis. The ectonucleotidases, mainly ecto-5′-nucleotidase/CD73 have been revealed as new players in cancer progression and malignity. In this work, we investigated the NTPDase3 and ecto-5′-nucleotidase/CD73 expression in cancer progression in vivo. Bladder tumor was induced in mice by the addition of 0.05 % of N-butyl-N-(hydroxybutyl)-nitrosamine (BBN) in the drinking water for 4, 8, 12, 18, and 24 weeks. After this period, mice bladders were removed for histopathology analysis and immunofluorescence assays. The bladder of animals which has received BBN had alterations, mainly inflammation, in initial times of tumor induction. After 18 weeks, mice’s bladder has developed histological alterations similar to human transitional cell carcinoma. The cancerous urothelium, from mice that received BBN for 18 and 24 weeks, presented a weak immunostaining to NTPDase3, in contrast to an increased expression of ecto-5′-nucleotidase/CD73. The altered expression of NTPDase3 and ecto-5′-nucleotidase/CD73 presented herein adds further evidence to support the idea that alterations in ectonucleotidases are involved in bladder tumorigenesis and reinforce the ecto-5′-nucleotidase/CD73 as a future biomarker and/or a target for pharmacological therapy of bladder cancer.
doi:10.1007/s11302-014-9405-8
PMCID: PMC4152458  PMID: 24464643
Bladder cancer; BBN; Purinergic signaling; NTPDase3; Ecto-5′-nucleotidase/CD73
20.  UTP-induced ATP release is a fine-tuned signalling pathway in osteocytes 
Purinergic Signalling  2013;10(2):337-347.
Osteocytes reside as a cellular network throughout the mineralised matrix of bone and are considered the primary mechanosensors of this tissue. They sense mechanical stimulation such as fluid flow and are able to regulate osteoblast and osteoclast functions on the bone surface. Previously, we found that ATP is released load-dependently from osteocytes from the onset of mechanical stimulation. Therefore, the aim of the present study was to investigate whether and how ATP release can be evoked in osteocytes via purinergic receptor activation. ATP release was quantified by real-time determination using the luciferin-luciferase assay and the release pathway was investigated using pharmacological inhibition. The P2Y receptor profile was analysed using gene expression analysis by reverse transcription polymerase chain reaction, while functional testing was performed using measurements of intracellular calcium responses to P2 receptor agonists. These investigations demonstrated that MLO-Y4 osteocytes express functional P2Y2, P2Y4, P2Y12 and P2Y13 receptors in addition to the previously reported P2X receptors. Further, we found that osteocytes respond to nucleotides such as ATP, UTP and ADP by increasing the intracellular calcium concentration and that they release ATP dose-dependently upon stimulation with 1–10 μM UTP. In addition to this, osteocytes release large amounts of ATP upon cell rupture, which might also be a source for other nucleotides, such as UTP. These findings indicate that mechanically induced ATP signals may be propagated by P2 receptor activation and further ATP release in the osteocyte network and implicate purinergic signalling as a central signalling pathway in osteocyte mechanotransduction.
doi:10.1007/s11302-013-9404-1
PMCID: PMC4040174  PMID: 24374572
Osteocyte; ATP release; UTP; P2 receptors; Vesicles; Mechanotransduction
21.  Investigation of the functional expression of purine and pyrimidine receptors in porcine isolated pancreatic arteries 
Purinergic Signalling  2013;10(2):241-249.
Receptors for purines and pyrimidines are expressed throughout the cardiovascular system. This study investigated their functional expression in porcine isolated pancreatic arteries. Pancreatic arteries (endothelium intact or denuded) were prepared for isometric tension recording and preconstricted with U46619, a thromboxane A2 mimetic; adenosine-5′-diphosphate (ADP), uridine-5′-triphosphate (UTP) and MRS2768, a selective P2Y2 agonist, were applied cumulatively, while adenosine-5′-triphosphate (ATP) and αβ-methylene-ATP (αβ-meATP) response curves were generated from single concentrations per tissue segment. Antagonists/enzyme inhibitors were applied prior to U46619 addition. ATP, αβ-meATP, UTP and MRS2768 induced vasoconstriction, with a potency order of αβ-meATP > MRS2768 > ATP ≥ UTP. Contractions to ATP and αβ-meATP were blocked by NF449, a selective P2X1 receptor antagonist. The contraction induced by ATP, but not UTP, was followed by vasorelaxation. Endothelium removal and DUP 697, a cyclooxygenase-2 inhibitor, had no significant effect on contraction to ATP but attenuated that to UTP, indicating actions at distinct receptors. MRS2578, a selective P2Y6 receptor antagonist, had no effect on contractions to UTP. ADP induced endothelium-dependent vasorelaxation which was inhibited by MRS2179, a selective P2Y1 receptor antagonist, or SCH58261, a selective adenosine A2A receptor antagonist. The contractions to ATP and αβ-meATP were attributed to actions at P2X1 receptors on the vascular smooth muscle, whereas it was shown for the first time that UTP induced an endothelium-dependent vasoconstriction which may involve P2Y2 and/or P2Y4 receptors. The relaxation induced by ADP is mediated by P2Y1 and A2A adenosine receptors. Porcine pancreatic arteries appear to lack vasorelaxant P2Y2 and P2Y4 receptors.
doi:10.1007/s11302-013-9403-2
PMCID: PMC4040170  PMID: 24310605
αβ-meATP; ATP; UTP; ADP; MRS2578; P2Y1; P2Y2; P2X1; A2A adenosine receptors; Vasoconstriction; Vasorelaxation; Endothelium
22.  Purinergic signalling in the gastrointestinal tract and related organs in health and disease 
Purinergic Signalling  2013;10(1):3-50.
Purinergic signalling plays major roles in the physiology and pathophysiology of digestive organs. Adenosine 5′-triphosphate (ATP), together with nitric oxide and vasoactive intestinal peptide, is a cotransmitter in non-adrenergic, non-cholinergic inhibitory neuromuscular transmission. P2X and P2Y receptors are widely expressed in myenteric and submucous enteric plexuses and participate in sympathetic transmission and neuromodulation involved in enteric reflex activities, as well as influencing gastric and intestinal epithelial secretion and vascular activities. Involvement of purinergic signalling has been identified in a variety of diseases, including inflammatory bowel disease, ischaemia, diabetes and cancer. Purinergic mechanosensory transduction forms the basis of enteric nociception, where ATP released from mucosal epithelial cells by distension activates nociceptive subepithelial primary afferent sensory fibres expressing P2X3 receptors to send messages to the pain centres in the central nervous system via interneurons in the spinal cord. Purinergic signalling is also involved in salivary gland and bile duct secretion.
doi:10.1007/s11302-013-9397-9
PMCID: PMC3944042  PMID: 24307520
Gastrointestinal muscle; Enteric plexuses; Epithelial secretion; Irritable bowel syndrome; Pain; Cancer; Salivary gland
23.  Purinergic signalling in the liver in health and disease 
Purinergic Signalling  2013;10(1):51-70.
Purinergic signalling is involved in both the physiology and pathophysiology of the liver. Hepatocytes, Kupffer cells, vascular endothelial cells and smooth muscle cells, stellate cells and cholangiocytes all express purinoceptor subtypes activated by adenosine, adenosine 5′-triphosphate, adenosine diphosphate, uridine 5′-triphosphate or UDP. Purinoceptors mediate bile secretion, glycogen and lipid metabolism and indirectly release of insulin. Mechanical stress results in release of ATP from hepatocytes and Kupffer cells and ATP is also released as a cotransmitter with noradrenaline from sympathetic nerves supplying the liver. Ecto-nucleotidases play important roles in the signalling process. Changes in purinergic signalling occur in vascular injury, inflammation, insulin resistance, hepatic fibrosis, cirrhosis, diabetes, hepatitis, liver regeneration following injury or transplantation and cancer. Purinergic therapeutic strategies for the treatment of these pathologies are being explored.
doi:10.1007/s11302-013-9398-8
PMCID: PMC3944046  PMID: 24271096
Purinoceptors; Diabetes; Cirrhosis; Hepatitis; Cancer; Fibrosis
24.  Purinergic signalling in the reproductive system in health and disease 
Purinergic Signalling  2013;10(1):157-187.
There are multiple roles for purinergic signalling in both male and female reproductive organs. ATP, released as a cotransmitter with noradrenaline from sympathetic nerves, contracts smooth muscle via P2X1 receptors in vas deferens, seminal vesicles, prostate and uterus, as well as in blood vessels. Male infertility occurs in P2X1 receptor knockout mice. Both short- and long-term trophic purinergic signalling occurs in reproductive organs. Purinergic signalling is involved in hormone secretion, penile erection, sperm motility and capacitation, and mucous production. Changes in purinoceptor expression occur in pathophysiological conditions, including pre-eclampsia, cancer and pain.
doi:10.1007/s11302-013-9399-7
PMCID: PMC3944041  PMID: 24271059
Vas deferens; Testes; Penile erection; Ovary; Uterus; Placenta
25.  Program and Abstracts of the 5th Joint Italian – German Purine Club Meeting 
Purinergic Signalling  2013;10(2):369-417.
doi:10.1007/s11302-013-9389-9
PMCID: PMC4040166

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