343 results on '"Apyrase pharmacology"'
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2. Blocking connexin 43 hemichannel-mediated ATP release reduces communication within and between tubular epithelial cells and medullary fibroblasts in a model of diabetic nephropathy.
- Author
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Williams BM, Cliff CL, Demirel I, Squires PE, and Hills CE
- Subjects
- Humans, Adenosine Triphosphate metabolism, Adenosine Triphosphate pharmacology, Apyrase metabolism, Apyrase pharmacology, Communication, Connexin 43 metabolism, Epithelial Cells metabolism, Fibroblasts metabolism, Fibrosis, Glucose pharmacology, Diabetes Mellitus metabolism, Diabetic Nephropathies metabolism
- Abstract
Introduction: Fibrosis of renal tubules is the final common pathway in diabetic nephropathy and develops in the face of tubular injury and fibroblast activation. Aberrant connexin 43 (Cx43) hemichannel activity has been linked to this damage under euglycaemic conditions, however, its role in glycaemic injury is unknown. This study investigated the effect of a Cx43 blocker (Tonabersat) on hemichannel activity and cell-cell interactions within and between tubular epithelial cells and fibroblasts in an in vitro model of diabetic nephropathy., Methods: Human kidney (HK2) proximal tubule epithelial cells and medullary fibroblasts (TK173) were treated in low (5 mM) or high (25 mM) glucose ± transforming growth factor beta-1 (TGFβ1) ± Tonabersat in high glucose. Carboxyfluorescein dye uptake and ATPlite luminescence assessed changes in hemichannel-mediated ATP release, while immunoblotting determined protein expression. Co-incubation with the ATP-diphosphohydrolase apyrase or a P2X7R inhibitor (A438079) assessed ATP-P2X7R signalling. Indirect co-culture with conditioned media from the alternate cell type evaluated paracrine-mediated heterotypic interactions., Results: Tonabersat partially negated glucose/TGFβ1-induced increases in Cx43 hemichannel-mediated ATP release and downstream changes in adherens junction and extracellular matrix (ECM) protein expression in HK2 and TK173 cells. Apyrase and A438079 highlighted the role for ATP-P2X7R in driving changes in protein expression in TK173 fibroblasts. Indirect co-culture studies suggest that epithelial cell secretome increases Tonabersat-sensitive hemichannel-mediated dye uptake in fibroblasts and downstream protein expression., Conclusion: Tonabersat-sensitive hemichannel-mediated ATP release enhances TGFβ1-driven heterotypic cell-cell interaction and favours myofibroblast activation. The data supports the potential benefit of Cx43 inhibition in reducing tubulointerstitial fibrosis in late-stage diabetic nephropathy., (© 2022 The Authors. Diabetic Medicine published by John Wiley & Sons Ltd on behalf of Diabetes UK.)
- Published
- 2022
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3. Inhibition of Src but not Syk causes weak reversal of GPVI-mediated platelet aggregation measured by light transmission aggregometry.
- Author
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Cheung HYF, Moran LA, Sickmann A, Heemskerk JWM, Garcia Á, and Watson SP
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- Agammaglobulinaemia Tyrosine Kinase metabolism, Apyrase pharmacology, Blood Platelets metabolism, Collagen pharmacology, Eptifibatide pharmacology, Fluorescein-5-isothiocyanate metabolism, Fluorescein-5-isothiocyanate pharmacology, Humans, Indomethacin metabolism, Indomethacin pharmacology, Intracellular Signaling Peptides and Proteins, Lectins, C-Type metabolism, Ligands, Platelet Aggregation Inhibitors pharmacology, Platelet Glycoprotein GPIIb-IIIa Complex metabolism, Protein-Tyrosine Kinases, Syk Kinase metabolism, Tyrosine metabolism, Tyrosine pharmacology, src-Family Kinases metabolism, Platelet Aggregation, Platelet Membrane Glycoproteins metabolism
- Abstract
Src tyrosine kinases and spleen tyrosine kinase (Syk) have recently been shown to contribute to sustained platelet aggregation on collagen under arterial shear. In the present study, we have investigated whether Src and Syk are required for aggregation under minimal shear following activation of glycoprotein VI (GPVI) and have extended this to C-type lectin-like receptor-2 (CLEC-2) which signals through the same pathway. Aggregation was induced by the GPVI ligand collagen-related peptide (CRP) and the CLEC-2 ligand rhodocytin and monitored by light transmission aggregometry (LTA). Aggregation and tyrosine phosphorylation by both receptors were sustained for up to 50 min. The addition of inhibitors of Src, Syk or Bruton's tyrosine kinase (Btk) at 150 sec, by which time aggregation was maximal, induced rapid loss of tyrosine phosphorylation of their downstream proteins, but only Src kinase inhibition caused a weak (~10%) reversal in light transmission. A similar effect was observed when the inhibitors were combined with apyrase and indomethacin or glycoprotein IIb-IIIa (GPIIb-IIIa) antagonist, eptifibatide. On the other hand, activation of GPIIb-IIIa by GPVI in a diluted platelet suspension, as measured by binding of fluorescein isothiocyanate-labeled antibody specific for the activated GPIIb-IIIa (FITC-PAC1), was reversed on the addition of Src and Syk inhibitors showing that integrin activation is rapidly reversible in the absence of outside-in signals. The results demonstrate that Src but not Syk and Btk contribute to sustained aggregation as monitored by LTA, possibly as a result of inhibition of outside-in signaling from GPIIb-IIIa to the cytoskeleton through a Syk-independent pathway. This is in contrast to the role of Syk in supporting sustained aggregation on collagen under arterial shear.
- Published
- 2022
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4. Wood smoke particulate matter (WSPM2.5) induces pyroptosis through both Caspase-1/IL-1β/IL-18 and ATP/P2Y-dependent mechanisms in human bronchial epithelial cells.
- Author
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Fu X, Hong W, Li S, Chen Z, Zhou W, Dai J, Deng X, Zhou H, Li B, and Ran P
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- Adenosine Triphosphate metabolism, Apyrase metabolism, Apyrase pharmacology, Biofuels, Caspase 1 genetics, Caspase 1 metabolism, Caspase 1 pharmacology, Epithelial Cells, Humans, Inflammation metabolism, Interleukin-18 metabolism, Interleukin-1beta metabolism, Interleukin-6 metabolism, Interleukin-8 metabolism, Lactate Dehydrogenases metabolism, NLR Family, Pyrin Domain-Containing 3 Protein metabolism, Particulate Matter pharmacology, RNA, Small Interfering metabolism, RNA, Small Interfering pharmacology, Nicotiana metabolism, Wood metabolism, Pyroptosis genetics, Smoke adverse effects
- Abstract
Emerging evidences have linked the air pollution particulate matters, especially the fine particulate matter PM2.5, to the disease development of chronic obstructive pulmonary disease (COPD). Our previous studies reported that biofuel PM2.5 can induce devastated damage of human bronchial epithelial cells, this study aims to further investigate the underlying molecular mechanisms how biofuel PM2.5 induces bronchial epithelial cell death and dysfunction. In this study, biofuel PM2.5 extracted from wood smoke (WSPM2.5) was used according to our previous publication. A 16-HBE cell line was used as the cell model. Results showed that: Firstly, WSPM2.5 induced significant pyroptosis in 16-HBE cells, reflected by the typical changes including elevated release of lactate dehydrogenase release (LDH) and activated activity and expression of Caspase-1/IL-1β/IL-18 signaling pathway. Then, specific inhibitors for both Caspases (Z-VAD-FMK) and Caspase-1 (VX-765), as well as specific siRNA knockdown of IL-1β all effectively attenuated the WSPM2.5-induced upregulation of downstream inflammatory cytokines and chemokines (IL-6, IL-8, CXCL-1, CXCL-2, etc), respectively. Notably, WSPM2.5 caused a novel increase of intracellular-to-extracellular ATP secretion, which could also contribute to the WSPM2.5-induced pyroptosis and inflammation by activating the Caspase-1/IL-1β/IL-18 signaling pathway through possible autocrine and/or paracrine mechanisms. Antagonism of ATP (Apyrase) or specific siRNA knockdown against ATP receptors (P2Y2 and P2Y7) both significantly inhibited the WSPM2.5-induced pyroptosis and inflammation. These results add up to the current knowledge and bring up novel insights that WSPM2.5 could induce significant pyroptosis and inflammation of human bronchial epithelial cells, through both a classic NLRP3/Caspase-1/IL-1β-dependent and a novel ATP/P2Y-dependent mechanisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)
- Published
- 2022
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5. Pannexin1 inhibits autophagy of cisplatin-resistant testicular cancer cells by mediating ATP release.
- Author
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Yuan M, Yao Y, Wu D, Zhu C, Dong S, and Tong X
- Subjects
- Adenosine Triphosphate metabolism, Animals, Apoptosis, Apyrase pharmacology, Autophagy, Cell Line, Tumor, Drug Resistance, Neoplasm, Humans, Male, Mice, Neoplasms, Germ Cell and Embryonal, TOR Serine-Threonine Kinases metabolism, Antineoplastic Agents pharmacology, Cisplatin pharmacology, Connexins genetics, Nerve Tissue Proteins genetics, Testicular Neoplasms drug therapy, Testicular Neoplasms genetics
- Abstract
Pannexin1 (Panx-1) is a gap junction channel protein that mediates the release of intracellular ATP during autophagy, and thus plays an important role in tumor cell apoptosis and chemo-resistance. However, the role of Panx-1 in cisplatin-resistance of testicular cancer cells remains unclear. We found that cisplatin-resistant I-10 testicular cancer cell lines (I-10/CDDP) autophagy-associated proteins (p62, p-mTOR, mTOR and LC3) exhibited high levels of autophagy in their expression, while LC3-II expression was more significantly in the presence of lysosomal degradation blocked by chloroquine (CQ). Xenograft models using I-10/CDDP cells with knockdown ATG5 and ATG7 were established in mouse models and showed blockade of autophagic flux and inhibition of tumor growth. In addition, inhibition of Panx-1 by carbenoxolone (CBX) and probenecid (PBN), as well as shRNA-mediated knockdown promoted autophagy in the I-10/CDDP cells, which was accompanied by a decrease in the levels of extracellular ATP. In contrast, overexpression of Panx-1 decreased autophagy of I-10/CDDP cells and increased extracellular ATP levels. To further determine the effect of panx-1-mediated ATP release on the autophagy of I-10/CDDP cells, apyrase was used to hydrolyze the extracellular ATP. Apyrase promoted autophagy in I-10/CDDP cells city by decreasing extracellular ATP, regardless of Panx-1 expression. This study demonstrated for the first time that Panx-1-mediated ATP release inhibits autophagy of I-10/CDDP cells, which provides a potential therapeutic strategy for cisplatin-resistant testicular cancer.
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- 2022
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6. Apyrase decreases phage induction and Shiga toxin release from E. coli O157:H7 and has a protective effect during infection.
- Author
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Arvidsson I, Tontanahal A, Johansson K, Kristoffersson AC, Kellnerová S, Berger M, Dobrindt U, and Karpman D
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- Adenosine Triphosphate metabolism, Animals, Apyrase metabolism, Apyrase pharmacology, Humans, Lipopolysaccharides metabolism, Mice, Mice, Inbred BALB C, Shiga Toxin metabolism, Shiga Toxin pharmacology, Shiga Toxin 2 genetics, Shiga Toxin 2 metabolism, Shiga Toxin 2 pharmacology, Bacteriophages, Escherichia coli Infections drug therapy, Escherichia coli Infections microbiology, Escherichia coli Infections prevention & control, Escherichia coli O157 genetics, Gastrointestinal Microbiome
- Abstract
Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) cause gastrointestinal infection and, in severe cases, hemolytic uremic syndrome which may lead to death. There is, to-date, no therapy for this infection. Stx induces ATP release from host cells and ATP signaling mediates its cytotoxic effects. Apyrase cleaves and neutralizes ATP and its effect on Stx and EHEC infection was therefore investigated. Apyrase decreased bacterial RecA and dose-dependently decreased toxin release from E. coli O157:H7 in vitro , demonstrated by reduced phage DNA and protein levels. The effect was investigated in a mouse model of E. coli O157:H7 infection. BALB/c mice infected with Stx2-producing E. coli O157:H7 were treated with apyrase intraperitoneally, on days 0 and 2 post-infection, and monitored for 11 days. Apyrase-treated mice developed disease two days later than untreated mice. Untreated infected mice lost significantly more weight than those treated with apyrase. Apyrase-treated mice exhibited less colonic goblet cell depletion and apoptotic cells, as well as lower fecal ATP and Stx2, compared to untreated mice. Apyrase also decreased platelet aggregation induced by co-incubation of human platelet-rich-plasma with Stx2 and E. coli O157 lipopolysaccharide in the presence of collagen. Thus, apyrase had multiple protective effects, reducing RecA levels, stx2 and toxin release from EHEC, reducing fecal Stx2 and protecting mouse intestinal cells, as well as decreasing platelet activation, and could thereby delay the development of disease.
- Published
- 2022
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7. Protein Corona Hinders N-CQDs Oxidative Potential and Favors Their Application as Nanobiocatalytic System.
- Author
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Czarnecka J, Kwiatkowski M, Wiśniewski M, and Roszek K
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- A549 Cells, Animals, Apyrase chemistry, Apyrase pharmacology, Catalase chemistry, Catalase pharmacology, Cell Proliferation drug effects, Cell Survival drug effects, Cellular Microenvironment drug effects, Enzymes, Immobilized chemistry, Enzymes, Immobilized pharmacology, HeLa Cells, Humans, Rats, Reactive Oxygen Species metabolism, beta-Galactosidase chemistry, beta-Galactosidase pharmacology, Biocatalysis, Carbon chemistry, Carbon pharmacology, Nitrogen chemistry, Nitrogen pharmacology, Oxidative Stress drug effects, Protein Corona metabolism, Quantum Dots chemistry, Quantum Dots metabolism
- Abstract
The oxidative properties of nanomaterials arouse legitimate concerns about oxidative damage in biological systems. On the other hand, the undisputable benefits of nanomaterials promote them for biomedical applications; thus, the strategies to reduce oxidative potential are urgently needed. We aimed at analysis of nitrogen-containing carbon quantum dots (N-CQDs) in terms of their biocompatibility and internalization by different cells. Surprisingly, N-CQD uptake does not contribute to the increased oxidative stress inside cells and lacks cytotoxic influence even at high concentrations, primarily through protein corona formation. We proved experimentally that the protein coating effectively limits the oxidative capacity of N-CQDs. Thus, N-CQDs served as an immobilization support for three different enzymes with the potential to be used as therapeutics. Various kinetic parameters of immobilized enzymes were analyzed. Regardless of the enzyme structure and type of reaction catalyzed, adsorption on the nanocarrier resulted in increased catalytic efficiency. The enzymatic-protein-to-nanomaterial ratio is the pivotal factor determining the course of kinetic parameter changes that can be tailored for enzyme application. We conclude that the above properties of N-CQDs make them an ideal support for enzymatic drugs required for multiple biomedical applications, including personalized medical therapies.
- Published
- 2021
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8. Structural and functional characterization of engineered bifunctional fusion proteins of CD39 and CD73 ectonucleotidases.
- Author
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Zhong EH, Ledderose C, De Andrade Mello P, Enjyoji K, Lunderberg JM, Junger W, and Robson SC
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- 5'-Nucleotidase chemistry, 5'-Nucleotidase genetics, 5'-Nucleotidase metabolism, Adenine Nucleotides metabolism, Anti-Inflammatory Agents chemistry, Anti-Inflammatory Agents metabolism, Apyrase chemistry, Apyrase genetics, Apyrase metabolism, GPI-Linked Proteins chemistry, GPI-Linked Proteins genetics, GPI-Linked Proteins metabolism, GPI-Linked Proteins pharmacology, HEK293 Cells, Humans, Hydrogen-Ion Concentration, Hydrolysis, Kinetics, Platelet Aggregation Inhibitors chemistry, Platelet Aggregation Inhibitors metabolism, Protein Conformation, Recombinant Fusion Proteins metabolism, Signal Transduction, Structure-Activity Relationship, Substrate Specificity, 5'-Nucleotidase pharmacology, Anti-Inflammatory Agents pharmacology, Apyrase pharmacology, Platelet Aggregation drug effects, Platelet Aggregation Inhibitors pharmacology, Protein Engineering
- Abstract
Extracellular diphosphate and triphosphate nucleotides are released from activated or injured cells to trigger vascular and immune P
2 purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the monophosphate nucleoside end products occurs by surface ecto-5'-nucleotidase (NMPase) or CD73. These ectoenzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ectoenzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase-ectodomain (ALP-ECD) and human acid phosphatase-ectodomain (HAP-ECD) fusion proteins were also generated, characterized, and compared with these CD39-ECD, CD73-ECD, and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ectoenzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting triphosphate and diphosphate nucleotides into nucleosides when compared with ALP-ECD and HAP-ECD. We also note a pH sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ectoenzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge proinflammatory ATP and also generate anti-inflammatory adenosine.- Published
- 2021
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9. Extracellular release of ATP promotes systemic inflammation during acute pancreatitis.
- Author
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Dixit A, Cheema H, George J, Iyer S, Dudeja V, Dawra R, and Saluja AK
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- Acute Disease, Adenosine Triphosphate blood, Animals, Apyrase pharmacology, Arginine, Bronchoalveolar Lavage Fluid chemistry, Ceruletide, Cytokines blood, Cytokines metabolism, Inflammation chemically induced, Inflammation prevention & control, Lung metabolism, Mice, Mice, Inbred C57BL, Pancreas metabolism, Pancreatitis chemically induced, Pancreatitis prevention & control, Peroxidase metabolism, Receptors, Purinergic metabolism, Signal Transduction, Suramin pharmacology, Adenosine Triphosphate metabolism, Inflammation metabolism, Pancreatitis metabolism
- Abstract
In the current study, we explored the role of extracellular ATP (eATP) in promoting systemic inflammation during development of acute pancreatitis (AP). Release of extracellular (e)ATP was evaluated in plasma and bronchoalveolar lavage fluid (BALF) of mice with experimental acute pancreatitis (AP). Prophylactic intervention using apyrase or suramin was used to understand the role and contribution of eATP in pancreatitis-associated systemic injury. AP of varying severity was induced in C57BL/6 mice using 1-day or 2-day caerulein, caerulein + LPS and l-arginine models. eATP was measured in plasma and BALF. Mice were treated with suramin or apyrase in the caerulein and l-arginine models of AP. Plasma cytokines, lung, and pancreatic myeloperoxidase, and morphometric analysis of pancreatic and lung histology, were used to assess the severity of pancreatitis. Plasma eATP and purinergic 2 (P2) receptors in the pancreas and lungs were significantly elevated in the experimental models of AP. Blocking the effect of eATP by suramin led to reduced levels of plasma IL-6 and TNFα as well as reduced lung, and pancreatic injury. Neutralizing eATP with apyrase reduced systemic injury but did not ameliorate local injury. The results of this study support the role of eATP and P2 receptors in promoting systemic inflammation during AP. Modulating purinergic signaling during AP can be an important therapeutic strategy in controlling systemic inflammation and, thus, systemic inflammatory response syndrome during AP. NEW & NOTEWORTHY Released ATP from injured cells promotes systemic inflammation in acute pancreatitis.
- Published
- 2019
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10. Thromboelastographic platelet mapping in dogs with complicated Babesia rossi infection.
- Author
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van Rooyen LJ, Hooijberg EH, Schoeman JP, and Goddard A
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- Animals, Apyrase pharmacology, Arachidonic Acid pharmacology, Blood Platelets chemistry, Blood Platelets drug effects, Case-Control Studies, Dog Diseases parasitology, Dogs, Female, Fibrinogen analysis, Male, Babesia, Babesiosis blood, Blood Platelets physiology, Dog Diseases blood, Thrombelastography veterinary
- Abstract
Background: Dogs with Babesia rossi infection display a normocoagulable thromboelastogram, despite being markedly thrombocytopenic, which is purportedly due to large-scale platelet activation. Thromboelastographic platelet mapping (TEG-PM) evaluates individual contributions of thrombin, fibrinogen, and platelets to clot formation, and may elucidate some of the pathomechanisms of thrombocytopenia-associated hemostatic alterations., Objective: This study investigated potential differences in TEG-PM variables in dogs with complicated B rossi infection compared with healthy controls, and whether these variables correlated with platelet activation indices., Methods: The maximum amplitude (MA) following thrombin generation (MA
Thrombin ) was determined using kaolin-activated TEG. The TEG-PM variables included MA following the addition of platelet agonists arachidonic acid (MAAA ) and adenosine diphosphate (MAADP ), and MA due to fibrin alone (MAFibrin ). In addition, platelet indices and fibrinogen concentrations were determined., Results: Thirteen dogs with complicated B rossi infection and five healthy controls were included. The median MAFibrin and fibrinogen concentrations were significantly higher (P < 0.01 for both) and median platelet count was significantly lower (P < 0.01) in the babesiosis group vs the control group. No significant differences were found for MAThrombin and MAAA/ADP . maximum amplitude due to fibrin alone was positively correlated with fibrinogen concentration (r = 0.735), mean platelet volume (r = 0.517), and mean platelet mass (r = 0.498), and negatively correlated with hematocrit (r = -0.685), platelet count (r = -0.476), and plateletcrit (r = -0.479) (P < 0.05 for all)., Conclusions: This study suggests that the presence of hyperfibrinogenemia offsets the severe thrombocytopenia associated with B rossi to result in normal thromboelastograms and lack of overt clinical bleeding., (© 2018 American Society for Veterinary Clinical Pathology.)- Published
- 2019
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11. Adenylate cyclase inhibition is required for normal redistribution of platelet surface GPIb in response to PAR1 activation.
- Author
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Pouget T, Pillois X, and Fiore M
- Subjects
- Apyrase pharmacology, Blood Platelets cytology, Blood Platelets metabolism, Humans, Peptide Fragments pharmacology, Receptor, PAR-1 metabolism, Receptors, Purinergic P2Y12 metabolism, Thrombin metabolism, Adenosine Diphosphate metabolism, Adenylyl Cyclases metabolism, Blood Platelets drug effects, Platelet Glycoprotein GPIb-IX Complex metabolism, Receptor, PAR-1 agonists
- Published
- 2019
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12. Effects of storage conditions on two platelet agonists for whole blood impedance platelet aggregometry in dogs.
- Author
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Shropshire SB and Olver CS
- Subjects
- Animals, Blood Platelets drug effects, Blood Specimen Collection methods, Dogs blood, Electric Impedance, Apyrase pharmacology, Arachidonic Acid pharmacology, Blood Specimen Collection veterinary, Platelet Aggregation drug effects, Platelet Function Tests veterinary
- Abstract
Background: Whole blood impedance platelet aggregometry (Multiplate-) can be performed with different agonists to evaluate platelet function. Although the manufacturer recommends disposal of stored reagents after 1 month at -20°C or 24 hours at 4°C, reagent integrity after reconstitution under different storage conditions is unknown. If reagent integrity is stable for longer periods, assay costs could decrease dramatically., Objectives: This study aimed to determine the stability of reconstituted arachidonic acid (AA) and adenosine diphosphate (ADP) platelet agonists stored at -20°C and -80°C for up to 6 months., Methods: Aliquots of reconstituted AA and ADP were stored at -20°C and -80°C each month for a total of 6 months. Six healthy staff-owned dogs were enrolled in the study. A physical examination, CBC, diagnostic panel, urinalysis, and baseline platelet aggregometry assessment was performed on all of the dogs. Platelet aggregometry was performed using fresh and stored aliquots of AA and ADP reagents on the same day. The area under the curve (AUC) was recorded from each platelet aggregometry analysis. Repeated measures (RM) analysis (one-way ANOVA) was performed and subsequent time points (1, 2, 3, 4, 5, and 6 months) were compared with fresh AUC results., Results: All dogs were clinically healthy, and all diagnostic tests were normal. There were no differences in AUC obtained from fresh samples at any time point or either temperature for AA or ADP., Conclusions: Whole blood impedance platelet aggregometry reagents, AA and ADP, were stable for up to 6 months when stored at -20°C or -80°C, obviating the need to discard viable reagents, and decreasing assay costs., (© 2018 American Society for Veterinary Clinical Pathology.)
- Published
- 2018
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13. ATP promotes immunosuppressive capacities of mesenchymal stromal cells by enhancing the expression of indoleamine dioxygenase.
- Author
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Lotfi R, Steppe L, Hang R, Rojewski M, Massold M, Jahrsdörfer B, and Schrezenmeier H
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- Apyrase pharmacology, Cell Proliferation, Cells, Cultured, Culture Media, Conditioned chemistry, Humans, Immunomodulation, Kynurenine metabolism, Lymphocyte Activation, Tryptophan metabolism, Adenosine Triphosphate pharmacology, Immune Tolerance, Indoleamine-Pyrrole 2,3,-Dioxygenase genetics, Mesenchymal Stem Cells immunology
- Abstract
Introduction: MSCs are often found within tumors, promote cancer progression and enhance metastasis. MSCs can act as immuosuppressive cells, partially due to the expression of the enzyme indoleamine dioxygenase (IDO) which converts tryptophan to kynurenine. Decreased concentration of tryptophan and increased kynurenine, both interfere with effective immune response. Damage associated molecular patterns (DAMPs) including ATP are found within the tumor microenvironment, attract MSCs, and influence their biology., Methods: Bone marrow derived MSCs were exposed to ATP for 4 days, in the presence of 100 ng IFNγ/mL. Intracellular expression of IDO in MSCs was assessed by FACS. Conditioned media from thus stimulated MSCs was analyzed for kynurenine content and its suppressive effect on lymphocyte proliferation. Apyrase or P2 × 7-receptor antagonist (AZ 11645373) were applied in order to inhibit ATP induced effect on MSCs., Results: We demonstrate, that ATP at concentrations between 0.062 and 0.5 mM increases dose dependently the expression of IDO in MSCs with subsequent increased kynurenine concentrations within the supernatant at about 60%. This effect could be abolished completely in the presence of ATP degrading enzyme (apyrase) or when MSCs were pretreated with a P2 × 7-receptor antagonist (AZ 11645373). Consistently, supernatants from MSCs stimulated with ATP, inhibited lymphocyte proliferation from 65% to 16%., Conclusions: We characterized ATP as a DAMP family member responsible for necrosis-induced immunomodulation. Given the increased concentration of DAMPs within tumor tissue and the fact that DAMPs can act as chemotattractants to MSCs, our results have implications for therapeutic strategies targeting the tumor microenvironment., (© 2018 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.)
- Published
- 2018
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14. Extracellular ATP drives breast cancer cell migration and metastasis via S100A4 production by cancer cells and fibroblasts.
- Author
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Liu Y, Geng YH, Yang H, Yang H, Zhou YT, Zhang HQ, Tian XX, and Fang WG
- Subjects
- Animals, Apyrase pharmacology, Cancer-Associated Fibroblasts drug effects, Female, Gene Expression Profiling, Gene Knockdown Techniques, Humans, MCF-7 Cells, Mice, Mice, Nude, Niclosamide pharmacology, Oligonucleotide Array Sequence Analysis, Primary Cell Culture, RNA, Messenger metabolism, RNA, Small Interfering metabolism, S100 Calcium-Binding Protein A4 genetics, Up-Regulation, Adenosine Triphosphate metabolism, Breast Neoplasms pathology, Cancer-Associated Fibroblasts metabolism, Cell Movement drug effects, S100 Calcium-Binding Protein A4 metabolism
- Abstract
Our previous work has demonstrated that extracellular ATP is an important pro-invasive factor, and in this study, we tapped into a possible mechanism involved. We discovered that ATP could upregulate both the intracellular expression and secretion of S100A4 in breast cancer cells and fibroblasts. Apart from stimulating breast cancer cell motility via intracellular S100A4, ATP enhanced the ability of breast cancer cells to transform fibroblasts into cancer-associated fibroblast (CAF)-like cells, which in turn secreted S100A4 to further promote cancer cell motility. Both apyrase and niclosamide treatments could inhibit metastasis of inoculated tumors to lung, liver and kidney in mice model, and CAFs from these treated tumors exhibited weakened migration-stimulating capacity for breast cancer cells. Collectively, our data indicate that extracellular ATP promotes the interactions between breast cancer cells and fibroblasts, which work collaboratively via production of S100A4 to exacerbate breast cancer metastasis., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2018
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15. Mechanism of Nucleoside Triphosphate Diphosphohydrolase-1-Associated Imbalance in Adenosine Diphosphate Degradation, B-Cell Activation, and Related Injury During Acute Antibody-Mediated Rejection.
- Author
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Wang X, Zhang Y, Wei G, Li Z, Tian D, and Huang W
- Subjects
- Animals, Antigens, CD genetics, Antigens, CD pharmacology, Apyrase genetics, Apyrase pharmacology, Cell Size, Graft Rejection immunology, Lymphocyte Activation physiology, Male, Mice, Mice, Nude, RNA, Messenger metabolism, Adenosine Diphosphate metabolism, Adenosine Triphosphate physiology, Antigens, CD metabolism, Apyrase metabolism, B-Lymphocytes physiology, Graft Rejection enzymology, Platelet Activation physiology, Skin Transplantation
- Abstract
Objective: The objective of this study was to investigate the effect of nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) during acute antibody-mediated rejection (AMR)., Methods: NTPDase1 overexpression, NTPDase1 knockout, and wild-type nude mice skin graft models were used to induce acute AMR. NTPDase1 expression in B cells, NTPDase1 messenger RNA expression in skin grafts, extracellular adenosine diphosphate (ADP) concentration, B-cell volume and surface antigens expression, average platelet transport rate, and ultrastructure and apoptosis of skin graft cells were investigated., Results: During acute AMR in nude mice, higher NTPDase1 expression caused lower extracellular ADP concentration, smaller increase in B-cell volume, and major histocompatibility complex II surface antigen expression, suggesting a negative correlation between them; higher NTPDase1 expression also caused slower average platelet transport rate and less severe skin graft injury, suggesting a negative correlation between them. Pretreatment with high-dose exogenous NTPDase1 inhibited platelet activation and protected skin grafts, but it resulted in prolonged bleeding time (by 51.4%) and prolonged coagulation time (by 44.1%)., Conclusion: An NTPDase1-associated imbalance in extracellular ADP degradation may contribute to B-cell activation, platelet activation, and more severe skin graft injury in nude mice. Pretreatment with high-dose exogenous NTPDase1 effectively protected skin grafts in nude mice at 1 week, but it increased the risk of bleeding., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
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16. First report of the characterization of a snake venom apyrase (Ruviapyrase) from Indian Russell's viper (Daboia russelii) venom.
- Author
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Kalita B, Patra A, Jahan S, and Mukherjee AK
- Subjects
- Animals, Anticoagulants, Antivenins pharmacology, Apyrase isolation & purification, Apyrase pharmacology, Blood Platelets drug effects, Humans, Platelet Aggregation drug effects, Antivenins chemistry, Apyrase chemistry, Daboia, Viper Venoms enzymology
- Abstract
A novel apyrase from Russell's viper venom (RVV) was purified and characterized, and it was named Ruviapyrase (Russell's viper apyrase). It is a high molecular weight (79.4 kDa) monomeric glycoprotein that contains 2.4% neutral sugars and 58.4% N-linked oligosaccharides and strongly binds to Concanavalin A. The LC-MS/MS analysis did not identify any protein in NCBI protein database, nevertheless some de novo sequences of Ruviapyrase showed putative conserved domain of apyrase superfamily. Ruviapyrase hydrolysed adenosine triphosphate (ATP) to a significantly greater extent (p < .05) as compared to adenosine diphosphate (ADP); however, it was devoid of 5'-nucleotidase and phosphodiesterase activities. The Km and Vmax values for Ruviapyrase towards ATP were 2.54 μM and 615 μM of Pi released min
-1 , respectively with a turnover number (Kcat) of 24,600 min-1 . Spectrofluorometric analysis demonstrated interaction of Ruviapyrase with ATP and ADP at Kd values of 0.92 nM and 1.25 nM, respectively. Ruviapyrase did not show cytotoxicity against breast cancer (MCF-7) cells and haemolytic activity, it exhibited marginal anticoagulant and strong antiplatelet activity, and dose-dependently reversed the ADP-induced platelet aggregation. The catalytic activity and platelet deaggregation property of Ruviapyrase was significantly inhibited by EDTA, DTT and IAA, and neutralized by commercial monovalent and polyvalent antivenom., (Copyright © 2018 Elsevier B.V. All rights reserved.)- Published
- 2018
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17. Purinergic receptor P2Y6 contributes to 1-methyl-4-phenylpyridinium-induced oxidative stress and cell death in neuronal SH-SY5Y cells.
- Author
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Qian Y, Xu S, Yang X, and Xiao Q
- Subjects
- 1-Methyl-4-phenylpyridinium pharmacology, Apyrase pharmacology, Cell Line, Tumor, Humans, Isothiocyanates pharmacology, Malondialdehyde metabolism, Neuroblastoma pathology, Nitric Oxide Synthase Type II metabolism, Purinergic P2 Receptor Agonists pharmacology, Purinergic P2 Receptor Antagonists pharmacology, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Reactive Oxygen Species metabolism, Receptors, Purinergic P2 genetics, Superoxide Dismutase-1 metabolism, Thiourea analogs & derivatives, Thiourea pharmacology, Transfection, Uridine Diphosphate pharmacology, Cell Death drug effects, Gene Expression Regulation, Neoplastic drug effects, Herbicides pharmacology, Oxidative Stress drug effects, Receptors, Purinergic P2 metabolism
- Abstract
Oxidative stress and neural degeneration have been shown to be involved in the pathogenesis of Parkinson's disease (PD). The P2Y6 purinergic receptor (P2Y6R) has been shown to participate in the activation of microglia and the production of pro-inflammatory factors induced by lipopolysaccharide to cause neuronal loss. However, the function of P2Y6R during oxidative stress in neurons is unclear. In the present study, 1-methyl-4-phenylpyridinium (MPP
+ ) treatment increased the level of UDP/P2Y6R on neuronal SH-SY5Y cells. Importantly, pharmacological inhibition of P2Y6R or knockdown of P2Y6R using a siRNA exerted an increased protective effect by preventing MPP+ -induced increases in the levels of reactive oxygen species (ROS), superoxide anion, inducible nitric oxide synthase (iNOS), and malondialdehyde (MDA) and down-regulation of superoxide dismutase 1 (SOD1) expression. UDP, an agonist of P2Y6R, enhanced the effects of MPP+ , which was also inhibited by apyrase or MRS2578. Additionally, P2Y6R knockdown also significantly reversed both the loss of cell viability and the increase in the levels of phosphorylated extracellular signal-regulated protein kinase (p-ERK1/2) and p38 (p-p38) caused by MPP+ stimulation. However, the inhibition of the ERK1/2 and p38 kinase signaling pathways had no effect on P2Y6R expression. Taken together, these results support the hypothesis that P2Y6R expressed on neuronal SH-SY5Y cell is associated with the progression of oxidative stress and cell death induced by MPP+ , suggesting that P2Y6R may play an important role in the pathogenesis of PD., (© 2017 Wiley Periodicals, Inc.)- Published
- 2018
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18. Inorganic polyphosphate induces accelerated tube formation of HUVEC endothelial cells.
- Author
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Müller WEG, Ackermann M, Wang S, Neufurth M, Muñoz-Espí R, Feng Q, Schröder HC, and Wang X
- Subjects
- Adenosine Triphosphate metabolism, Alkaline Phosphatase metabolism, Apyrase pharmacology, Cell Line, Tumor, Cells, Cultured, Human Umbilical Vein Endothelial Cells metabolism, Human Umbilical Vein Endothelial Cells physiology, Humans, Matrix Metalloproteinase 2 genetics, Matrix Metalloproteinase 2 metabolism, Matrix Metalloproteinase 9 genetics, Matrix Metalloproteinase 9 metabolism, Microscopy, Electron, Scanning, Microscopy, Fluorescence, Microvessels metabolism, Microvessels ultrastructure, Gene Expression Regulation, Enzymologic drug effects, Human Umbilical Vein Endothelial Cells drug effects, Microvessels drug effects, Polyphosphates pharmacology
- Abstract
In this study, the effect of inorganic polyphosphate (polyP) on the initial phase of angiogenesis and vascularization was investigated, applying the HUVEC cell tube formation assay. PolyP is a physiological and high energy phosphate polymer which has been proposed to act as a metabolic fuel in the extracellular space with only a comparably low ATP content. The experiments revealed that polyP accelerates tube formation of human umbilical vein endothelial cells (HUVEC), seeded onto a solidified basement membrane extract matrix which contains polyP-metabolizing alkaline phosphatase (ALP) activity. This effect is abolished by co-addition of apyrase, which degrades ATP to AMP and inorganic phosphate. The assumption that ATP, derived from polyP, activates HUVEC cells leading to tube formation was corroborated by experiments showing that addition of polyP to the cells causes a strong rise of ATP level in the culture medium. Finally, we show that at a later stage of cultivation of HUVEC cells, after 3 d, polyP causes a strong enhancement of the expression of the genes encoding for the two major matrix metalloproteinases (MMPs) released by endothelial cells during tube formation, MMP-9 and MMP-2. This stimulatory effect is again abrogated by addition of apyrase together with polyP. From these results, we propose that polyP is involved either directly or indirectly in energy supply, via ALP-mediated transfer of energy-rich phosphate under ATP formation. This ATP is utilized for the activation and oriented migration of endothelial cells and for the matrix organization during the initial phases of tube formation.
- Published
- 2018
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19. Role of Pannexin1 channels in the resistance of I-10 testicular cancer cells to cisplatin mediated by ATP/IP 3 pathway.
- Author
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Wu D, Wu J, Liu H, Yu M, Tao L, Dong S, and Tong X
- Subjects
- Animals, Apoptosis drug effects, Apyrase pharmacology, Cell Line, Tumor, Gene Knockdown Techniques, Gene Silencing drug effects, Humans, Macrocyclic Compounds pharmacology, Male, Mice, Oxazoles pharmacology, Signal Transduction drug effects, Adenosine Triphosphate metabolism, Cisplatin pharmacology, Connexins metabolism, Drug Resistance, Neoplasm drug effects, Inositol 1,4,5-Trisphosphate metabolism, Nerve Tissue Proteins metabolism, Testicular Neoplasms metabolism, Testicular Neoplasms pathology
- Abstract
Cisplatin (DDP) is the most commonly used drug in testicular cancer. However, drug resistance severely limits its clinical use and the underlying mechanisms need to be further clarified. The aim of present study was to investigate the role of ATP/IP
3 pathway mediated by pannexin1 (Panx-1) channels on DDP-induced apoptosis and to reveal the potential mechanisms of DDP-resistance in testicular cancer. We found that the expression of Panx-1 in I-10/DDP cells (DDP-resistance) was decreased compared with parental I-10 cells determined by western blotting and immunofluorescence assay. To further clarify the role of Panx-1 in DDP resistance, Panx-1 function was modulated by overexpression and knockdown of Panx-1 expression. Panx-1 overexpression increased DDP-induced apoptosis, ATP release and IP3 levels. On the contrary, Panx-1 silencing decreased DDP-induced apoptosis, ATP release and IP3 levels. Apyrase (hydrolyzing extracellular ATP) or xestospongin C (antagonizing IP3 receptor) also decreased DDP-induced apoptosis. Our findings demonstrate that Panx-1 is involved in DDP-resistance and ATP/IP3 pathway mediated by Panx-1 channels participates in DDP-induced apoptosis in testicular cancer. Panx-1 modulation may be interesting to amplify the clinical effect of DDP and reverse the resistance of testicular cancer cells to DDP., (Copyright © 2017. Published by Elsevier Masson SAS.)- Published
- 2017
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20. Underlying mechanism and specific prevention of hemolysis-induced platelet activation.
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Gremmel T, Fedrizzi S, Weigel G, Eichelberger B, and Panzer S
- Subjects
- Adult, Aged, Apyrase pharmacology, Blood Group Incompatibility pathology, Blood Platelets pathology, Clopidogrel, Female, Humans, Male, Middle Aged, Ticlopidine analogs & derivatives, Ticlopidine pharmacology, Adenosine Diphosphate metabolism, Blood Group Incompatibility metabolism, Blood Platelets metabolism, Hemolysis, Platelet Activation, Platelet Glycoprotein GPIIb-IIIa Complex metabolism, Receptors, Purinergic P2Y12 metabolism
- Abstract
Thromboembolic complications significantly impair the outcome of hemolytic disorders. We hypothesized that red cell adenosine diphosphate (ADP) release results in significant platelet activation in hemolysis and that this prothrombotic state can be prevented by inhibition of the ADP P2Y
12 receptor. In the current study, we therefore sought to investigate the mechanism and inhibition of hemolysis-induced platelet activation. The expression of activated integrin αIIbß3 was determined by flow cytometry, and platelet aggregation was assessed by multiple electrode platelet aggregometry. We demonstrate platelet activation and increased platelet aggregation by adding hemolytic blood (lysates) to whole blood, similarly to that achieved by the platelet agonist ADP. Enhanced platelet activation and reactivity in the presence of hemolytic blood were significantly abolished by apyrase, which catalyzes ADP degradation, and inhibited by blockade of the platelet ADP P2Y12 receptor with cangrelor. Platelets from patients treated with the ADP P2Y12 receptor antagonist clopidogrel showed a reduced response to lysates compared to platelets from healthy controls without antiplatelet treatment. Further, in vitro blood group ABO incompatibility induced hemolysis and led to increased platelet activation. Finally, "spontaneous" platelet aggregation seen in patients with cold agglutinin disease was completely abolished by cangrelor. In conclusion, hemolysis is associated with increased platelet activation and aggregation due to red cell derived ADP, which can be prevented by ADP receptor blockade.- Published
- 2017
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21. Nucleotide transmitters ATP and ADP mediate intercellular calcium wave communication via P2Y12/13 receptors among BV-2 microglia.
- Author
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Jiang P, Xing F, Guo B, Yang J, Li Z, Wei W, Hu F, Lee I, Zhang X, Pan L, and Xu J
- Subjects
- Animals, Apyrase pharmacology, Biomechanical Phenomena, Boron Compounds pharmacology, Calcium Signaling drug effects, Cell Communication drug effects, Cell Line, Transformed, Gene Expression, Inositol Phosphates pharmacology, Mechanotransduction, Cellular drug effects, Mice, Microglia cytology, Microglia drug effects, Molecular Imaging, Receptors, Purinergic P2 genetics, Receptors, Purinergic P2Y12 genetics, Suramin pharmacology, Thapsigargin pharmacology, Adenosine Diphosphate metabolism, Adenosine Triphosphate metabolism, Calcium metabolism, Microglia metabolism, Receptors, Purinergic P2 metabolism, Receptors, Purinergic P2Y12 metabolism
- Abstract
Nerve injury is accompanied by a liberation of diverse nucleotides, some of which act as 'find/eat-me' signals in mediating neuron-glial interplay. Intercellular Ca2+ wave (ICW) communication is the main approach by which glial cells interact and coordinate with each other to execute immune defense. However, the detailed mechanisms on how these nucleotides participate in ICW communication remain largely unclear. In the present work, we employed a mechanical stimulus to an individual BV-2 microglia to simulate localized injury. Remarkable ICW propagation was observed no matter whether calcium was in the environment or not. Apyrase (ATP/ADP-hydrolyzing enzyme), suramin (broad-spectrum P2 receptor antagonist), 2-APB (IP3 receptor blocker) and thapsigargin (endoplasmic reticulum calcium pump inhibitor) potently inhibited these ICWs, respectively, indicating the dependence of nucleotide signals and P2Y receptors. Then, we detected the involvement of five naturally occurring nucleotides (ATP, ADP, UTP, UDP and UDP-glucose) by desensitizing receptors. Results showed that desensitization with ATP and ADP could block ICW propagation in a dose-dependent manner, whereas other nucleotides had little effect. Meanwhile, the expression of P2Y receptors in BV-2 microglia was identified and their contributions were analyzed, from which we suggested P2Y12/13 receptors activation mostly contributed to ICWs. Besides, we estimated that extracellular ATP and ADP concentration sensed by BV-2 microglia was about 0.3 μM during ICWs by analyzing calcium dynamic characteristics. Taken together, these results demonstrated that the nucleotides ATP and ADP were predominant signal transmitters in mechanical stimulation-induced ICW communication through acting on P2Y12/13 receptors in BV-2 microglia.
- Published
- 2017
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22. ADPase CD39 Fused to Glycoprotein VI-Fc Boosts Local Antithrombotic Effects at Vascular Lesions.
- Author
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Degen H, Borst O, Ziegler M, Mojica Munoz AK, Jamasbi J, Walker B, Göbel S, Fassbender J, Adler K, Brandl R, Münch G, Lorenz R, Siess W, Gawaz M, and Ungerer M
- Subjects
- Animals, Antigens, CD toxicity, Apyrase pharmacokinetics, Apyrase toxicity, Carotid Artery Diseases blood, Carotid Artery Diseases pathology, Carotid Artery Injuries blood, Carotid Artery Injuries chemically induced, Carotid Artery Injuries pathology, Chlorides, Disease Models, Animal, Dose-Response Relationship, Drug, Female, Ferric Compounds, Fibrinolytic Agents pharmacokinetics, Fibrinolytic Agents toxicity, Glycoproteins pharmacokinetics, Glycoproteins toxicity, Hemorrhage chemically induced, Humans, Immunoglobulin Fc Fragments toxicity, Male, Mice, Inbred C57BL, Plaque, Atherosclerotic, Platelet Aggregation Inhibitors pharmacokinetics, Platelet Aggregation Inhibitors toxicity, Platelet Membrane Glycoproteins pharmacokinetics, Platelet Membrane Glycoproteins toxicity, Recombinant Fusion Proteins pharmacology, Thrombosis blood, Thrombosis chemically induced, Thrombosis pathology, Antigens, CD pharmacology, Apyrase pharmacology, Carotid Artery Injuries drug therapy, Fibrinolytic Agents pharmacology, Glycoproteins pharmacology, Immunoglobulin Fc Fragments pharmacology, Platelet Aggregation drug effects, Platelet Aggregation Inhibitors pharmacology, Platelet Membrane Glycoproteins pharmacology, Thrombosis prevention & control
- Abstract
Background: GPVI (Glycoprotein VI) is the essential platelet collagen receptor in atherothrombosis. Dimeric GPVI-Fc (Revacept) binds to GPVI binding sites on plaque collagen. As expected, it did not increase bleeding in clinical studies. GPVI-Fc is a potent inhibitor of atherosclerotic plaque-induced platelet aggregation at high shear flow, but its inhibition at low shear flow is limited. We sought to increase the platelet inhibitory potential by fusing GPVI-Fc to the ectonucleotidase CD39 (fusion protein GPVI-CD39), which inhibits local ADP accumulation at vascular plaques, and thus to create a lesion-directed dual antiplatelet therapy that is expected to lack systemic bleeding risks., Methods and Results: GPVI-CD39 effectively stimulated local ADP degradation and, compared with GPVI-Fc alone, led to significantly increased inhibition of ADP-, collagen-, and human plaque-induced platelet aggregation in Multiplate aggregometry and plaque-induced platelet thrombus formation under arterial flow conditions. GPVI-CD39 did not increase bleeding time in an in vitro assay simulating primary hemostasis. In a mouse model of ferric chloride-induced arterial thrombosis, GPVI-CD39 effectively delayed vascular thrombosis but did not increase tail bleeding time in vivo., Conclusions: GPVI-CD39 is a novel approach to increase local antithrombotic activity at sites of atherosclerotic plaque rupture or injury. It enhances GPVI-Fc-mediated platelet inhibition and presents a potentially effective and safe molecule for the treatment of acute atherothrombotic events, with a favorable risk-benefit ratio., (© 2017 The Authors and AdvanceCor GmbH. Published on behalf of the American Heart Association, Inc., by Wiley.)
- Published
- 2017
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23. Development of a novel strategy to target CD39 antithrombotic activity to the endothelial-platelet microenvironment in kidney ischemia-reperfusion injury.
- Author
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Sashindranath M, Dwyer KM, Dezfouli S, Selan C, Crikis S, Lu B, Yuan Y, Hickey MJ, Peter K, Robson SC, Cowan PJ, and Nandurkar HH
- Subjects
- Animals, Blood Platelets drug effects, Cellular Microenvironment physiology, Humans, Kidney blood supply, Membrane Glycoproteins pharmacology, Mice, Platelet Aggregation drug effects, Recombinant Proteins pharmacology, Antigens, CD pharmacology, Apyrase pharmacology, Endothelium, Vascular drug effects, Fibrinolytic Agents pharmacology, Kidney drug effects, Reperfusion Injury
- Abstract
Kidney ischemia-reperfusion injury (IRI) is common during transplantation. IRI is characterised by inflammation and thrombosis and associated with acute and chronic graft dysfunction. P-selectin and its ligand PSGL-1 are cell adhesion molecules that control leukocyte-endothelial and leukocyte-platelet interactions under inflammatory conditions. CD39 is the dominant vascular nucleotidase that facilitates adenosine generation via extracellular ATP/ADP-phosphohydrolysis. Adenosine signalling is protective in renal IRI, but CD39 catalytic activity is lost with exposure to oxidant stress. We designed a P-selectin targeted CD39 molecule (rsol.CD39-PSGL-1) consisting of recombinant soluble CD39 that incorporates 20 residues of PSGL-1 that bind P-selectin. We hypothesised that rsol.CD39-PSGL-1 would maintain endothelial integrity by focusing the ectonucleotidase platelet-inhibitory activity and reducing leukocyte adhesion at the injury site. The rsol.CD39-PSGL-1 displayed ADPase activity and inhibited platelet aggregation ex vivo, as well as bound with high specificity to soluble P-selectin and platelet surface P-selectin. Importantly, mice injected with rsol.CD39-PSGL-1 and subjected to renal IRI showed significantly less kidney damage both biochemically and histologically, compared to those injected with solCD39. Furthermore, the equivalent dose of rsol.CD39-PSGL-1 had no effect on tail template bleeding times. Hence, targeting recombinant CD39 to the injured vessel wall via PSGL-1 binding resulted in substantial preservation of renal function and morphology after IRI without toxicity. These studies indicate potential translational importance to clinical transplantation and nephrology.
- Published
- 2017
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24. Recombinant soluble apyrase APT102 inhibits thrombosis and intimal hyperplasia in vein grafts without adversely affecting hemostasis or re-endothelialization.
- Author
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Ji Y, Adeola O, Strawn TL, Jeong SS, Chen R, and Fay WP
- Subjects
- Adenosine chemistry, Adenosine Triphosphatases chemistry, Animals, Blood Platelets cytology, Carotid Arteries pathology, Cell Movement, Cell Proliferation, Coronary Vessels pathology, Endothelial Cells pathology, Endothelium, Vascular pathology, Hemostasis, Humans, Hyperplasia, Male, Mice, Mice, Inbred C57BL, Muscle, Smooth, Vascular pathology, Myocytes, Smooth Muscle cytology, Platelet Aggregation, Prothrombin Time, Solubility, Tunica Intima pathology, Apyrase pharmacology, Blood Vessels transplantation, Recombinant Proteins pharmacology, Thrombosis drug therapy, Tunica Intima drug effects
- Abstract
Essentials New strategies are needed to inhibit thrombosis and intimal hyperplasia (IH) in vein grafts (VG). We studied effects of apyrase (APT102) on VGs and smooth muscle and endothelial cells (SMC/EC). APT102 inhibited thrombosis, SMC migration, and IH without impairing hemostasis or EC recovery. Apyrase APT102 is a single-drug approach to inhibit multiple processes that cause VG failure., Summary: Background Occlusion of vein grafts (VGs) after bypass surgery, owing to thrombosis and intimal hyperplasia (IH), is a major clinical problem. Apyrases are enzymes that scavenge extracellular ATP and ADP, and promote adenosine formation at sites of vascular injury, and hence have the potential to inhibit VG pathology. Objectives To examine the effects of recombinant soluble human apyrase, APT102, on platelets, smooth muscle cells (SMCs) and endothelial cells (ECs) in vitro, and on thrombosis and IH in murine VGs. Methods SMC and EC proliferation and migration were studied in vitro. Inferior vena cava segments from donor mice were grafted into carotid arteries of recipient mice. Results APT102 potently inhibited ADP-induced platelet aggregation and VG thrombosis, but it did not impair surgical hemostasis. APT102 did not directly inhibit SMC or EC proliferation, but significantly attenuated the effects of ATP on SMC and EC proliferation. APT102 significantly inhibited SMC migration, but did not inhibit EC migration, which may be mediated, at least in part, by inhibition of SMC, but not EC, migration by adenosine. At 4 weeks after surgery, there was significantly less IH in VGs of APT102-treated mice than in control VGs. APT102 significantly inhibited cell proliferation in VGs, but did not inhibit re-endothelialization. Conclusions Systemic administration of a recombinant human apyrase inhibits thrombosis and IH in VGs without increasing bleeding or compromising re-endothelialization. These results suggest that APT102 has the potential to become a novel, single-drug treatment strategy to prevent multiple pathologic processes that drive early adverse remodeling and occlusion of VGs., (© 2017 International Society on Thrombosis and Haemostasis.)
- Published
- 2017
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25. Blocking microglial pannexin-1 channels alleviates morphine withdrawal in rodents.
- Author
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Burma NE, Bonin RP, Leduc-Pessah H, Baimel C, Cairncross ZF, Mousseau M, Shankara JV, Stemkowski PL, Baimoukhametova D, Bains JS, Antle MC, Zamponi GW, Cahill CM, Borgland SL, De Koninck Y, and Trang T
- Subjects
- Adenosine Triphosphate metabolism, Animals, Apyrase pharmacology, Blotting, Western, Cell Culture Techniques, Coculture Techniques, Connexins antagonists & inhibitors, Connexins metabolism, Mefloquine pharmacology, Mice, Microglia metabolism, Naloxone pharmacology, Narcotic Antagonists adverse effects, Nerve Tissue Proteins antagonists & inhibitors, Nerve Tissue Proteins metabolism, Neurons drug effects, Neurons metabolism, Nociception drug effects, Posterior Horn Cells metabolism, Probenecid pharmacology, Rats, Substance Withdrawal Syndrome etiology, Substance Withdrawal Syndrome metabolism, Behavior, Animal drug effects, Connexins genetics, Microglia drug effects, Morphine adverse effects, Narcotics adverse effects, Nerve Tissue Proteins genetics, Posterior Horn Cells drug effects, Substance Withdrawal Syndrome genetics
- Abstract
Opiates are essential for treating pain, but termination of opiate therapy can cause a debilitating withdrawal syndrome in chronic users. To alleviate or avoid the aversive symptoms of withdrawal, many of these individuals continue to use opiates. Withdrawal is therefore a key determinant of opiate use in dependent individuals, yet its underlying mechanisms are poorly understood and effective therapies are lacking. Here, we identify the pannexin-1 (Panx1) channel as a therapeutic target in opiate withdrawal. We show that withdrawal from morphine induces long-term synaptic facilitation in lamina I and II neurons within the rodent spinal dorsal horn, a principal site of action for opiate analgesia. Genetic ablation of Panx1 in microglia abolished the spinal synaptic facilitation and ameliorated the sequelae of morphine withdrawal. Panx1 is unique in its permeability to molecules up to 1 kDa in size and its release of ATP. We show that Panx1 activation drives ATP release from microglia during morphine withdrawal and that degrading endogenous spinal ATP by administering apyrase produces a reduction in withdrawal behaviors. Conversely, we found that pharmacological inhibition of ATP breakdown exacerbates withdrawal. Treatment with a Panx1-blocking peptide (
10 panx) or the clinically used broad-spectrum Panx1 blockers, mefloquine or probenecid, suppressed ATP release and reduced withdrawal severity. Our results demonstrate that Panx1-mediated ATP release from microglia is required for morphine withdrawal in rodents and that blocking Panx1 alleviates the severity of withdrawal without affecting opiate analgesia.- Published
- 2017
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26. Reduction of the foreign body response and neuroprotection by apyrase and minocycline in chronic cannula implantation in the rat brain.
- Author
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Hayn L, Deppermann L, and Koch M
- Subjects
- Animals, Brain immunology, Brain pathology, Foreign-Body Reaction immunology, Foreign-Body Reaction pathology, Forelimb drug effects, Forelimb immunology, Forelimb pathology, Male, Microglia drug effects, Microglia immunology, Microglia pathology, Motor Activity drug effects, Motor Cortex drug effects, Motor Cortex immunology, Motor Cortex pathology, Neurons drug effects, Neurons immunology, Neurons pathology, Rats, Inbred Strains, Apyrase pharmacology, Brain drug effects, Cannula adverse effects, Foreign-Body Reaction prevention & control, Minocycline pharmacology, Neuroprotective Agents pharmacology
- Abstract
Implantation of electrodes or cannulae into the brain is accompanied by a tissue response referred to as foreign body response. Adenosine triphosphate (ATP) is one of the signalling molecules released by injured cells which mediate the chemoattraction of microglial cells. The constitutive release of pro-inflammatory and cytotoxic substances by microglial cells in chronic implants exacerbates neuronal cell death and the immune response. This study aimed to interfere with the initial events of the foreign body response in order to mitigate neurotoxicity and inflammation. For this purpose, the ATP-hydrolysing enzyme apyrase and the antibiotic minocycline with a broad range of anti-inflammatory, anti-apoptotic and glutamate-antagonist properties were locally infused during cannula implantation in the caudal forelimb area of the motor cortex in Lister Hooded rats. The rats' motor performance was assessed in a skilled reaching task and the distribution of neurons and glial cells in the vicinity of the implant was examined 2 and 6 weeks post-implantation. Apyrase as well as minocycline increased the number of surviving neurons and reduced microglial activation. Moreover, minocycline improved the motor performance and, additionally, caused a temporary reduction in astrogliosis, suggesting it as a possible therapeutic candidate to improve the biocompatibility of chronic brain implants., (© 2016 John Wiley & Sons Australia, Ltd.)
- Published
- 2017
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27. Systemic Adenosine Triphosphate Impairs Neutrophil Chemotaxis and Host Defense in Sepsis.
- Author
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Li X, Kondo Y, Bao Y, Staudenmaier L, Lee A, Zhang J, Ledderose C, and Junger WG
- Subjects
- Animals, Apyrase pharmacology, Humans, Mice, Inbred C57BL, Mice, Knockout, Neutrophil Activation, Sepsis mortality, Suramin pharmacology, Adenosine Triphosphate physiology, Chemotaxis, Leukocyte immunology, Neutrophils physiology, Sepsis immunology
- Abstract
Objective: Sepsis remains an unresolved clinical problem. Therapeutic strategies focusing on inhibition of neutrophils (polymorphonuclear neutrophils) have failed, which indicates that a more detailed understanding of the underlying pathophysiology of sepsis is required. Polymorphonuclear neutrophil activation and chemotaxis require cellular adenosine triphosphate release via pannexin-1 channels that fuel autocrine feedback via purinergic receptors. In the current study, we examined the roles of endogenous and systemic adenosine triphosphate on polymorphonuclear neutrophil activation and host defense in sepsis., Design: Prospective randomized animal investigation and in vitro studies., Setting: Preclinical academic research laboratory., Subjects: Wild-type C57BL/6 mice, pannexin-1 knockout mice, and healthy human subjects used to obtain polymorphonuclear neutrophils for in vitro studies., Interventions: Wild-type and pannexin-1 knockout mice were treated with suramin or apyrase to block the endogenous or systemic effects of adenosine triphosphate. Mice were subjected to cecal ligation and puncture and polymorphonuclear neutrophil activation (CD11b integrin expression), organ (liver) injury (plasma aspartate aminotransferase), bacterial spread, and survival were monitored. Human polymorphonuclear neutrophils were used to study the effect of systemic adenosine triphosphate and apyrase on chemotaxis., Measurements and Main Results: Inhibiting endogenous adenosine triphosphate reduced polymorphonuclear neutrophil activation and organ injury, but increased the spread of bacteria and mortality in sepsis. By contrast, removal of systemic adenosine triphosphate improved bacterial clearance and survival in sepsis by improving polymorphonuclear neutrophil chemotaxis., Conclusions: Systemic adenosine triphosphate impairs polymorphonuclear neutrophil functions by disrupting the endogenous purinergic signaling mechanisms that regulate cell activation and chemotaxis. Removal of systemic adenosine triphosphate improves polymorphonuclear neutrophil function and host defenses, making this a promising new treatment strategy for sepsis.
- Published
- 2017
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28. Role of pannexin 1 in Clostridium perfringens beta-toxin-caused cell death.
- Author
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Seike S, Takehara M, Kobayashi K, and Nagahama M
- Subjects
- Adenosine Triphosphate metabolism, Apyrase pharmacology, Carbenoxolone pharmacology, Cell Death drug effects, Cells, Cultured, Hexokinase pharmacology, Humans, Receptors, Purinergic P2X7 physiology, Bacterial Toxins toxicity, Connexins physiology, Nerve Tissue Proteins physiology
- Abstract
Background: Beta-toxin produced by Clostridium perfringens is a key virulence factor of fatal hemorrhagic enterocolitis and enterotoxemia. This toxin belongs to a family of β-pore-forming toxins (PFTs). We reported recently that the ATP-gated P2X
7 receptor interacts with beta-toxin. The ATP-release channel pannexin 1 (Panx1) is an important contributor to P2X7 receptor signaling. Hence, we investigated the involvement of Panx1 in beta-toxin-caused cell death., Methods: We examined the effect of Panx1 in beta-toxin-induced cell death utilizing selective antagonists, knockdown of Panx1, and binding using dot-blot analysis. Localization of Panx1 and the P2X7 receptor after toxin treatment was determined by immunofluorescence staining., Results: Selective Panx1 antagonists (carbenoxolone [CBX], probenecid, and Panx1 inhibitory peptide) prevented beta-toxin-caused cell death in THP-1 cells. CBX did not block the binding of the toxin to cells. Small interfering knockdown of Panx1 blocked beta-toxin-mediated cell death through inhibiting the oligomer formation of the toxin. Beta-toxin triggered a transient ATP release from THP-1 cells, but this early ATP release was blocked by CBX. ATP scavengers (apyrase and hexokinase) inhibited beta-toxin-induced cytotoxicity. Furthermore, co-administration of ATP with beta-toxin enhanced the binding and cytotoxicity of the toxin., Conclusions: Based on our results, Panx1 activation is achieved through the interaction of beta-toxin with the P2X7 receptor. Then, ATP released by the Panx1 channel opening promotes oligomer formation of the toxin, leading to cell death., General Significance: Pannexin 1 is a novel candidate therapeutic target for beta-toxin-mediated disease., (Copyright © 2016 Elsevier B.V. All rights reserved.)- Published
- 2016
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29. Purinergic dysregulation in pulmonary hypertension.
- Author
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Visovatti SH, Hyman MC, Goonewardena SN, Anyanwu AC, Kanthi Y, Robichaud P, Wang J, Petrovic-Djergovic D, Rattan R, Burant CF, and Pinsky DJ
- Subjects
- Adenosine metabolism, Adenosine Triphosphate metabolism, Animals, Antigens, CD genetics, Antihypertensive Agents pharmacology, Apyrase deficiency, Apyrase genetics, Apyrase pharmacology, Arterial Pressure, Disease Models, Animal, Genetic Predisposition to Disease, Humans, Hydrolysis, Hypertension, Pulmonary drug therapy, Hypertension, Pulmonary genetics, Hypertension, Pulmonary physiopathology, Hypertrophy, Right Ventricular genetics, Hypertrophy, Right Ventricular metabolism, Hypertrophy, Right Ventricular physiopathology, Hypoxia complications, Lung drug effects, Mice, Inbred C57BL, Mice, Knockout, Phenotype, Pulmonary Artery drug effects, Pulmonary Artery physiopathology, Purinergic P2X Receptor Antagonists pharmacology, Receptors, Purinergic P2X1 drug effects, Severity of Illness Index, Signal Transduction, Suramin analogs & derivatives, Suramin pharmacology, Vascular Remodeling, Ventricular Remodeling, Antigens, CD metabolism, Apyrase metabolism, Hypertension, Pulmonary metabolism, Lung metabolism, Pulmonary Artery metabolism, Receptors, Purinergic P2X1 metabolism
- Abstract
Despite the fact that nucleotides and adenosine help regulate vascular tone through purinergic signaling pathways, little is known regarding their contributions to the pathobiology of pulmonary arterial hypertension, a condition characterized by elevated pulmonary vascular resistance and remodeling. Even less is known about the potential role that alterations in CD39 (ENTPD1), the ectonucleotidase responsible for the conversion of the nucleotides ATP and ADP to AMP, may play in pulmonary arterial hypertension. In this study we identified decreased CD39 expression on the pulmonary endothelium of patients with idiopathic pulmonary arterial hypertension. We next determined the effects of CD39 gene deletion in mice exposed to normoxia or normobaric hypoxia (10% oxygen). Compared with controls, hypoxic CD39(-/-) mice were found to have a markedly elevated ATP-to-adenosine ratio, higher pulmonary arterial pressures, more right ventricular hypertrophy, more arterial medial hypertrophy, and a pro-thrombotic phenotype. In addition, hypoxic CD39(-/-) mice exhibited a marked increase in lung P2X1 receptors. Systemic reconstitution of ATPase and ADPase enzymatic activities through continuous administration of apyrase decreased pulmonary arterial pressures in hypoxic CD39(-/-) mice to levels found in hypoxic CD39(+/+) controls. Treatment with NF279, a potent and selective P2X1 receptor antagonist, lowered pulmonary arterial pressures even further. Our study is the first to implicate decreased CD39 and resultant alterations in circulating purinergic signaling ligands and cognate receptors in the pathobiology of pulmonary arterial hypertension. Reconstitution and receptor blocking experiments suggest that phosphohydrolysis of purinergic nucleotide tri- and diphosphates, or blocking of the P2X1 receptor could serve as treatment for pulmonary arterial hypertension.
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- 2016
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30. Impaired P2X1 Receptor-Mediated Adhesion in Eosinophils from Asthmatic Patients.
- Author
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Wright A, Mahaut-Smith M, Symon F, Sylvius N, Ran S, Bafadhel M, Muessel M, Bradding P, Wardlaw A, and Vial C
- Subjects
- Adenosine Triphosphate analogs & derivatives, Adenosine Triphosphate pharmacology, Apyrase pharmacology, Asthma physiopathology, Benzenesulfonates pharmacology, CD11b Antigen genetics, CD11b Antigen metabolism, Eosinophils drug effects, Eosinophils immunology, Healthy Volunteers, Humans, Leukocyte Count, Purinergic P2X Receptor Agonists pharmacology, Real-Time Polymerase Chain Reaction, Receptors, Purinergic P2X1 genetics, Receptors, Purinergic P2X4 genetics, Receptors, Purinergic P2X5 genetics, Reverse Transcriptase Polymerase Chain Reaction, Asthma immunology, Cell Adhesion, Eosinophils physiology, Receptors, Purinergic P2X1 metabolism
- Abstract
Eosinophils play an important role in the pathogenesis of asthma and can be activated by extracellular nucleotides released following cell damage or inflammation. For example, increased ATP concentrations were reported in bronchoalveolar lavage fluids of asthmatic patients. Although eosinophils are known to express several subtypes of P2 receptors for extracellular nucleotides, their function and contribution to asthma remain unclear. In this article, we show that transcripts for P2X1, P2X4, and P2X5 receptors were expressed in healthy and asthmatic eosinophils. The P2X receptor agonist α,β-methylene ATP (α,β-meATP; 10 μM) evoked rapidly activating and desensitizing inward currents (peak 18 ± 3 pA/pF at -60 mV) in healthy eosinophils, typical of P2X1 homomeric receptors, which were abolished by the selective P2X1 antagonist NF449 (1 μM) (3 ± 2 pA/pF). α,β-meATP-evoked currents were smaller in eosinophils from asthmatic patients (8 ± 2 versus 27 ± 5 pA/pF for healthy) but were enhanced following treatment with a high concentration of the nucleotidase apyrase (17 ± 5 pA/pF for 10 IU/ml and 11 ± 3 pA/pF for 0.32 IU/ml), indicating that the channels are partially desensitized by extracellular nucleotides. α,β-meATP (10 μM) increased the expression of CD11b activated form in eosinophils from healthy, but not asthmatic, donors (143 ± 21% and 108 ± 11% of control response, respectively). Furthermore, α,β-meATP increased healthy (18 ± 2% compared with control 10 ± 1%) but not asthmatic (13 ± 1% versus 10 ± 0% for control) eosinophil adhesion. Healthy human eosinophils express functional P2X1 receptors whose activation leads to eosinophil αMβ2 integrin-dependent adhesion. P2X1 responses are constitutively reduced in asthmatic compared with healthy eosinophils, probably as the result of an increase in extracellular nucleotide concentration., (Copyright © 2016 by The American Association of Immunologists, Inc.)
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- 2016
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31. Microglia-derived purines modulate mossy fibre synaptic transmission and plasticity through P2X4 and A1 receptors.
- Author
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George J, Cunha RA, Mulle C, and Amédée T
- Subjects
- Adenosine analogs & derivatives, Adenosine pharmacology, Adenosine Triphosphate analogs & derivatives, Adenosine Triphosphate pharmacology, Animals, Apyrase pharmacology, Cells, Cultured, Excitatory Postsynaptic Potentials, Lipopolysaccharides pharmacology, Mice, Mice, Inbred C57BL, Microglia drug effects, Mossy Fibers, Hippocampal drug effects, Purinergic P2X Receptor Agonists pharmacology, Purines pharmacology, Adenosine metabolism, Adenosine Triphosphate metabolism, Microglia metabolism, Mossy Fibers, Hippocampal physiology, Neuronal Plasticity drug effects, Receptors, Purinergic P2X4 physiology, Synaptic Transmission drug effects
- Abstract
Recent data have provided evidence that microglia, the brain-resident macrophage-like cells, modulate neuronal activity in both physiological and pathophysiological conditions, and microglia are therefore now recognized as synaptic partners. Among different neuromodulators, purines, which are produced and released by microglia, have emerged as promising candidates to mediate interactions between microglia and synapses. The cellular effects of purines are mediated through a large family of receptors for adenosine and for ATP (P2 receptors). These receptors are present at brain synapses, but it is unknown whether they can respond to microglia-derived purines to modulate synaptic transmission and plasticity. Here, we used a simple model of adding immune-challenged microglia to mouse hippocampal slices to investigate their impact on synaptic transmission and plasticity at hippocampal mossy fibre (MF) synapses onto CA3 pyramidal neurons. MF-CA3 synapses show prominent forms of presynaptic plasticity that are involved in the encoding and retrieval of memory. We demonstrate that microglia-derived ATP differentially modulates synaptic transmission and short-term plasticity at MF-CA3 synapses by acting, respectively, on presynaptic P2X4 receptors and on adenosine A1 receptors after conversion of extracellular ATP to adenosine. We also report that P2X4 receptors are densely located in the mossy fibre tract in the dentate gyrus-CA3 circuitry. In conclusion, this study reveals an interplay between microglia-derived purines and MF-CA3 synapses, and highlights microglia as potent modulators of presynaptic plasticity., (© 2016 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)
- Published
- 2016
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32. Human β Defensin-3 Increases CD86 Expression on Monocytes by Activating the ATP-Gated Channel P2X7.
- Author
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Lioi AB, Ferrari BM, Dubyak GR, Weinberg A, and Sieg SF
- Subjects
- Apyrase metabolism, Apyrase pharmacology, B7-1 Antigen genetics, B7-1 Antigen metabolism, B7-2 Antigen genetics, Calcium metabolism, Cells, Cultured, Flow Cytometry, Gene Expression drug effects, Humans, Monocytes metabolism, Purinergic P2X Receptor Antagonists pharmacology, Receptors, Purinergic P2X7 genetics, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction drug effects, Signal Transduction genetics, Adenosine Triphosphate pharmacology, B7-2 Antigen metabolism, Monocytes drug effects, Receptors, Purinergic P2X7 metabolism, beta-Defensins pharmacology
- Abstract
Human β defensin-3 (hBD-3), an epithelial cell-derived antimicrobial peptide, mediates chemotaxis and activation of myeloid cells. In this study, we provide evidence that hBD-3 induces the costimulatory molecule CD86 on primary human monocytes by a mechanism involving autocrine activation of ionotropic P2X7 receptors (P2X7R) by ATP. Incubation of monocytes with hBD-3 resulted in increased expression of both the CD80 and CD86 costimulatory molecules. Treatment of monocytes with a selective P2X7R antagonist inhibited the ability of hBD-3 to induce expression of CD86 but not CD80. The hBD-3-dependent upregulation of CD86 was also attenuated in monocytes incubated with apyrase, a potent scavenger of extracellular ATP. Finally, direct activation of monocyte P2X7R by exogenous ATP mimicked the ability of hBD-3 to induce CD86 expression. These data suggest that hBD-3 induces monocyte activation by both P2X7-dependent (CD86 upregulation) and P2X7-independent (CD80 upregulation) signaling mechanisms and raise the possibility that activation of P2X7R could play an important role in shaping the inflammatory microenvironment in conditions where hBD-3 is highly expressed, such as psoriasis or oral carcinoma., (Copyright © 2015 by The American Association of Immunologists, Inc.)
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- 2015
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33. Drospirenone enhances GPIb-IX-V-mediated platelet activation.
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Fan X, Chen X, Wang C, Dai J, Lu Y, Wang K, Liu J, Zhang J, and Wu X
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- Adenosine Diphosphate metabolism, Apyrase pharmacology, Blood Coagulation Tests, Blood Platelets metabolism, Cyclooxygenase Inhibitors pharmacology, Humans, Indomethacin pharmacology, Membrane Proteins metabolism, Receptors, Progesterone metabolism, Thromboxane A2 metabolism, von Willebrand Factor metabolism, Androstenes pharmacology, Blood Coagulation drug effects, Blood Platelets drug effects, Contraceptive Agents, Female pharmacology, Platelet Activation drug effects, Platelet Glycoprotein GPIb-IX Complex pharmacology
- Abstract
Background: Epidemiologic studies recently revealed that using drospirenone (DRSP)-containing contraceptives is associated with an increased risk of thrombosis in women. However, the underlying causality is unclear., Objective: To study the effects of DRSP on coagulation in vitro and the probable mechanisms involved., Methods: First, the effects of DRSP on the activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FIB) were measured. Then, the effects of DRSP on platelet activation were investigated in response to low levels of collagen, adenosine 5'-diphosphate (ADP), thrombin, U46619, adrenaline and botrocetin/von Willebrand factor (VWF)., Results: DRSP has no direct effect on APTT, PT, TT, FIB and platelet aggregation induced by low levels of collagen, ADP, thrombin, U46619 or adrenaline. However, DRSP enhances botrocetin/VWF-induced platelet aggregation and VWF receptor glycoprotein Ib-IX-V (GPIb-IX-V)-mediated signaling. This enhancement can be blocked by the progesterone receptor membrane component 1 (PGRMC1) inhibitor AG205, or by the ADP scavenger apyrase and the cyclooxygenase inhibitor indomethacin., Conclusions: Although DRSP did not directly induce platelet activation, it obviously facilitated VWF receptor GPIb-IX-V-mediated platelet activation. The potential DRSP-binding protein PGRMC1 may play a role in this process. Our study also suggested that the inhibition of thromboxane A2 production and the activation of ADP receptors might prevent the side-effects of DRSP., (© 2015 International Society on Thrombosis and Haemostasis.)
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- 2015
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34. Damage/Danger Associated Molecular Patterns (DAMPs) Modulate Chlamydia pecorum and C. trachomatis Serovar E Inclusion Development In Vitro.
- Author
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Leonard CA, Schoborg RV, and Borel N
- Subjects
- 8-Bromo Cyclic Adenosine Monophosphate pharmacology, Adenine analogs & derivatives, Adenine pharmacology, Apyrase pharmacology, Benzyl Compounds pharmacology, Chlamydia growth & development, Chlamydia metabolism, Chlamydia ultrastructure, Chlamydia trachomatis growth & development, Chlamydia trachomatis metabolism, Chlamydia trachomatis ultrastructure, HeLa Cells, Host-Pathogen Interactions, Humans, Adenosine pharmacology, Adenosine Triphosphate pharmacology, Chlamydia drug effects, Chlamydia trachomatis drug effects, Cyclic AMP pharmacology
- Abstract
Persistence, more recently termed the chlamydial stress response, is a viable but non-infectious state constituting a divergence from the characteristic chlamydial biphasic developmental cycle. Damage/danger associated molecular patterns (DAMPs) are normal intracellular components or metabolites that, when released from cells, signal cellular damage/lysis. Purine metabolite DAMPs, including extracellular ATP and adenosine, inhibit chlamydial development in a species-specific manner. Viral co-infection has been shown to reversibly abrogate Chlamydia inclusion development, suggesting persistence/chlamydial stress. Because viral infection can cause host cell DAMP release, we hypothesized DAMPs may influence chlamydial development. Therefore, we examined the effect of extracellular ATP, adenosine, and cyclic AMP exposure, at 0 and 14 hours post infection, on C. pecorum and C. trachomatis serovar E development. In the absence of de novo host protein synthesis, exposure to DAMPs immediately post or at 14 hours post infection reduced inclusion size; however, the effect was less robust upon 14 hours post infection exposure. Additionally, upon exposure to DAMPs immediately post infection, bacteria per inclusion and subsequent infectivity were reduced in both Chlamydia species. These effects were reversible, and C. pecorum exhibited more pronounced recovery from DAMP exposure. Aberrant bodies, typical in virus-induced chlamydial persistence, were absent upon DAMP exposure. In the presence of de novo host protein synthesis, exposure to DAMPs immediately post infection reduced inclusion size, but only variably modulated chlamydial infectivity. Because chlamydial infection and other infections may increase local DAMP concentrations, DAMPs may influence Chlamydia infection in vivo, particularly in the context of poly-microbial infections.
- Published
- 2015
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35. Endocannabinoids regulate the activity of astrocytic hemichannels and the microglial response against an injury: In vivo studies.
- Author
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Vázquez C, Tolón RM, Pazos MR, Moreno M, Koester EC, Cravatt BF, Hillard CJ, and Romero J
- Subjects
- Adenosine Triphosphate metabolism, Amides, Amidohydrolases genetics, Amidohydrolases metabolism, Animals, Anti-Inflammatory Agents pharmacology, Apyrase pharmacology, Astrocytes drug effects, Brain drug effects, Brain metabolism, Disease Models, Animal, Ethanolamines metabolism, Flufenamic Acid pharmacology, Glycerides metabolism, Lasers, Mice, Mice, Knockout, Mice, Transgenic, Microglia drug effects, Oleic Acids metabolism, Palmitic Acids metabolism, Receptor, Cannabinoid, CB1 antagonists & inhibitors, Arachidonic Acids metabolism, Astrocytes metabolism, Brain Injuries metabolism, Connexin 43 metabolism, Endocannabinoids metabolism, Microglia metabolism, Polyunsaturated Alkamides metabolism, Receptor, Cannabinoid, CB1 metabolism
- Abstract
Anandamide (AEA) is an endocannabinoid (EC) that modulates multiple functions in the CNS and that is released in areas of injury, exerting putative neuroprotective actions. In the present study, we have used intravital microscopy to analyze the role of the EC system in the glial response against an acute insult. Our data show that AEA modulates astroglial function in vivo by increasing connexin-43 hemichannel (HC) activity. Furthermore, the genetic inactivation of the AEA-degrading enzyme, fatty acid amide hydrolase (FAAH), also increased HC activity and enhanced the microglial response against an acute injury to the brain parenchyma, effects that were mediated by cannabinoid CB1 receptors. The contribution of ATP released through an astrocytic HC was critical for the microglial response, as this was prevented by the use of the HC blocker flufenamic acid and by apyrase. As could be expected, brain concentrations of AEA, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) were elevated in FAAH-null mice, while 2-arachidonoylglycerol (2-AG) concentrations remained unaltered. In summary, these findings demonstrate that AEA modifies glial functions by promoting an enhanced pro-inflammatory glial response in the brain., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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36. Involvement of nucleotides in glial growth following scratch injury in avian retinal cell monolayer cultures.
- Author
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Silva TM, França GR, Ornelas IM, Loiola EC, Ulrich H, and Ventura AL
- Subjects
- Animals, Apyrase pharmacology, Cell Movement physiology, Cell Proliferation drug effects, Cells, Cultured, Chickens, Chromones pharmacology, Morpholines pharmacology, Neurogenesis drug effects, Neuroglia drug effects, Neurons drug effects, Neurons metabolism, Phosphatidylinositol 3-Kinases metabolism, Quinolones pharmacology, Retina injuries, Signal Transduction drug effects, Sulfones pharmacology, Suramin pharmacology, Cell Movement drug effects, Neuroglia cytology, Nucleotides metabolism, Retina drug effects
- Abstract
When retinal cell cultures were mechanically scratched, cell growth over the empty area was observed. Only dividing and migrating, 2 M6-positive glial cells were detected. Incubation of cultures with apyrase (APY), suramin, or Reactive Blue 2 (RB-2), but not MRS 2179, significantly attenuated the growth of glial cells, suggesting that nucleotide receptors other than P2Y1 are involved in the growth of glial cells. UTPγS but not ADPβS antagonized apyrase-induced growth inhibition in scratched cultures, suggesting the participation of UTP-sensitive receptors. No decrease in proliferating cell nuclear antigen (PCNA(+)) cells was observed at the border of the scratch in apyrase-treated cultures, suggesting that glial proliferation was not affected. In apyrase-treated cultures, glial cytoplasm protrusions were smaller and unstable. Actin filaments were less organized and alfa-tubulin-labeled microtubules were mainly parallel to scratch. In contrast to control cultures, very few vinculin-labeled adhesion sites could be noticed in these cultures. Increased Akt and ERK phosphorylation was observed in UTP-treated cultures, effect that was inhibited by SRC inhibitor 1 and PI3K blocker LY294002. These inhibitors and the FAK inhibitor PF573228 also decreased glial growth over the scratch, suggesting participation of SRC, PI3K, and FAK in UTP-induced growth of glial cells in scratched cultures. RB-2 decreased dissociated glial cell attachment to fibronectin-coated dishes and migration through transwell membranes, suggesting that nucleotides regulated adhesion and migration of glial cells. In conclusion, mechanical scratch of retinal cell cultures induces growth of glial cells over the empty area through a mechanism that is dependent on activation of UTP-sensitive receptors, SRC, PI3K, and FAK.
- Published
- 2015
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37. Pannexin 1 facilitates arterial relaxation via an endothelium-derived hyperpolarization mechanism.
- Author
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Gaynullina D, Shestopalov VI, Panchin Y, and Tarasova OS
- Subjects
- Adenosine Triphosphate genetics, Adenosine Triphosphate metabolism, Animals, Apyrase pharmacology, Arteries metabolism, Connexins genetics, Mice, Mice, Knockout, Nerve Tissue Proteins genetics, Nitric Oxide Synthase Type III antagonists & inhibitors, Nitric Oxide Synthase Type III genetics, Nitric Oxide Synthase Type III metabolism, Purinergic P1 Receptor Antagonists pharmacology, Receptors, Purinergic P1 genetics, Receptors, Purinergic P1 metabolism, Vasodilation drug effects, Connexins metabolism, Endothelium, Vascular metabolism, Nerve Tissue Proteins metabolism, Vasodilation physiology
- Abstract
Pannexin 1 (Panx1) is involved in endothelium-dependent vasodilation in large arteries, but the exact mechanistic role remains poorly understood. We hypothesized that Panx1 facilitates large vessel relaxations regulating endothelium-derived hyperpolarization (EDH)-like mechanisms. The EDH-like component of acetylcholine-induced relaxation of saphenous arteries studied in isometric myograph after inhibition of NO-synthase and cyclooxygenase was significantly impaired in mice with genetically ablated Panx1 (KO) relative to that in the wild type (WT) mice. Application of P1-receptor antagonist and apyrase significantly reduced this component in WT, but not in KO mice, indicating participation of ATP released via Panx1 in the EDH-like relaxation., (Copyright © 2015 Federation of European Biochemical Societies. All rights reserved.)
- Published
- 2015
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38. Differential impact of adenosine nucleotides released by osteocytes on breast cancer growth and bone metastasis.
- Author
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Zhou JZ, Riquelme MA, Gao X, Ellies LG, Sun LZ, and Jiang JX
- Subjects
- Adenosine Triphosphate analogs & derivatives, Adenosine Triphosphate pharmacology, Alendronate pharmacology, Animals, Apyrase pharmacology, Bone Density Conservation Agents pharmacology, Bone Neoplasms secondary, Breast Neoplasms genetics, Cell Line, Tumor, Cell Movement drug effects, Cell Proliferation, Female, Humans, Mice, Mice, Inbred C57BL, Mice, Nude, Neoplasm Transplantation, Osteocytes metabolism, RNA Interference, RNA, Small Interfering, Receptor, Adenosine A2A genetics, Receptors, Purinergic P2X genetics, Signal Transduction, Transplantation, Heterologous, Adenosine metabolism, Adenosine Triphosphate metabolism, Breast Neoplasms pathology, Receptor, Adenosine A2A metabolism, Receptors, Purinergic P2X metabolism
- Abstract
Extracellular ATP has been shown to either inhibit or promote cancer growth and migration; however, the mechanism underlying this discrepancy remained elusive. Here we demonstrate the divergent roles of ATP and adenosine released by bone osteocytes on breast cancers. We showed that conditioned media (CM) collected from osteocytes treated with alendronate (AD), a bisphosphonate drug, inhibited the migration of human breast cancer MDA-MB-231 cells. Removal of the extracellular ATP by apyrase in CM abolished this effect, suggesting the involvement of ATP. ATP exerted its inhibitory effect through the activation of purinergic P2X receptor signaling in breast cancer cells evidenced by the attenuation of the inhibition by an antagonist, oxidized ATP, as well as knocking down P2X7 with small interfering RNA (siRNA), and the inhibition of migration by an agonist, BzATP. Intriguingly, ATP had a biphasic effect on breast cancer cells-lower dosage inhibited but higher dosage promoted its migration. The stimulatory effect on migration was blocked by an adenosine receptor antagonist, MRS1754, ARL67156, an ecto-ATPase inhibitor, and A2A receptor siRNA, suggesting that in contrast to ATP, adenosine, a metabolic product of ATP, promoted migration of breast cancer cells. Consistently, non-hydrolyzable ATP, ATPγS, only inhibited but did not promote cancer cell migration. ATP also had a similar inhibitory effect on the Py8119 mouse mammary carcinoma cells; however, adenosine had no effect owing to the absence of the A2A receptor. Consistently, ATPγS inhibited, whereas adenosine promoted anchorage-independent growth of MDA-MB-231 cells. Our in vivo xenograft study showed a significant delay of tumor growth with the treatment of ATPγS. Moreover, the extent of bone metastasis in a mouse intratibial model was significantly reduced with the treatment of ATPγS. Together, our results suggest the distinct roles of ATP and adenosine released by osteocytes and the activation of corresponding receptors P2X7 and A2A signaling on breast cancer cell growth, migration and bone metastasis.
- Published
- 2015
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39. Defective PDI release from platelets and endothelial cells impairs thrombus formation in Hermansky-Pudlak syndrome.
- Author
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Sharda A, Kim SH, Jasuja R, Gopal S, Flaumenhaft R, Furie BC, and Furie B
- Subjects
- Adenosine Diphosphate deficiency, Adenosine Diphosphate metabolism, Adenosine Diphosphate pharmacology, Animals, Apyrase metabolism, Apyrase pharmacology, Blood Platelets drug effects, Cell Degranulation, Disease Models, Animal, Endothelial Cells pathology, Exocytosis drug effects, Female, Fibrin biosynthesis, Hermanski-Pudlak Syndrome genetics, Human Umbilical Vein Endothelial Cells, Humans, Intracellular Signaling Peptides and Proteins antagonists & inhibitors, Intracellular Signaling Peptides and Proteins blood, Intracellular Signaling Peptides and Proteins genetics, Mice, Mice, Inbred C57BL, Mice, Knockout, Platelet Aggregation, Protein Disulfide-Isomerases blood, RNA, Small Interfering genetics, Thrombin metabolism, Vesicular Transport Proteins deficiency, Vesicular Transport Proteins genetics, Blood Platelets enzymology, Endothelial Cells enzymology, Hermanski-Pudlak Syndrome blood, Hermanski-Pudlak Syndrome enzymology, Protein Disulfide-Isomerases metabolism, Thrombosis blood, Thrombosis enzymology
- Abstract
Protein disulfide isomerase (PDI), secreted from platelets and endothelial cells after injury, is required for thrombus formation. The effect of platelet and endothelial cell granule contents on PDI-mediated thrombus formation was studied by intravital microscopy using a mouse model of Hermansky-Pudlak syndrome in which platelet dense granules are absent. Platelet deposition and fibrin generation were nearly absent, and extracellular PDI was significantly reduced in HPS6(-/-) mice after vascular injury. HPS6(-/-) platelets displayed impaired PDI secretion and impaired exocytosis of α granules, lysosomes, and T granules due to decreased sensitivity to thrombin, but these defects could be corrected by addition of subthreshold amounts of adenosine 5'-diphosphate (ADP). Human Hermansky-Pudlak syndrome platelets demonstrated similar characteristics. Infusion of wild-type platelets rescued thrombus formation in HPS6(-/-) mice. Human umbilical vein endothelial cells in which the HPS6 gene was silenced displayed impaired PDI secretion and exocytosis of Weibel-Palade bodies. Defective thrombus formation in Hermansky-Pudlak syndrome, associated with impaired exocytosis of residual granules in endothelial cells and platelets, the latter due to deficiency of ADP, is characterized by a defect in T granule secretion, a deficiency in extracellular PDI secretion, and impaired fibrin generation and platelet aggregation. Hermansky-Pudlak syndrome is an example of a hereditary disease whereby impaired PDI secretion contributes to a bleeding phenotype., (© 2015 by The American Society of Hematology.)
- Published
- 2015
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40. Paracrine stimulation of P2X7 receptor by ATP activates a proliferative pathway in ovarian carcinoma cells.
- Author
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Vázquez-Cuevas FG, Martínez-Ramírez AS, Robles-Martínez L, Garay E, García-Carrancá A, Pérez-Montiel D, Castañeda-García C, and Arellano RO
- Subjects
- Adenosine Triphosphate pharmacology, Adult, Aged, Apyrase pharmacology, Cell Line, Tumor, Cell Proliferation, Female, Humans, MAP Kinase Signaling System drug effects, Middle Aged, Paracrine Communication drug effects, Adenosine Triphosphate analogs & derivatives, Calcium metabolism, Ovarian Neoplasms metabolism, Ovarian Neoplasms pathology, Receptors, Purinergic P2X7 metabolism
- Abstract
P2X7 is a purinergic receptor-channel; its activation by ATP elicits a broad set of cellular actions, from apoptosis to signals for survival. Here, P2X7 expression and function was studied in human ovarian carcinoma (OCA) cells, and biopsies from non-cancerous and cancer patients were analyzed by immunohistochemistry. Ovarian surface epithelium in healthy tissue expressed P2X7 at a high level that was maintained throughout the cancer. The cell lines SKOV-3 and CAOV-3 were used to investigate P2X7 functions in OCA. In SKOV-3 cells, selective stimulation of P2X7 by 2'(3')-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate (BzATP) induced a dose-dependent increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) but not cell death. Instead, BzATP increased the levels of phosphorylated ERK and AKT (pERK and pAKT), with an EC(50) of 44 ± 2 and 1.27 ± 0.5 μM, respectively; 10 μM BzATP evoked a maximum effect within 15 min that lasted for 120 min. Interestingly, basal levels of pERK and pAKT were decreased in the presence of apyrase in the medium, strongly suggesting an endogenous, ATP-mediated phenomenon. Accordingly: (i) mechanically stimulated cells generated a [Ca(2+)](i) increase that was abolished by apyrase; (ii) apyrase induced a decrease in culture viability, as measured by the MTS assay for mitochondrial activity; and (iii) incubation with 10 μM AZ10606120, a specific P2X7 antagonist and transfection with the dominant negative P2X7 mutant E496A, both reduced cell viability to 70.1 ± 8.9% and to 76.5 ± 5%, respectively, of control cultures. These observations suggested that P2X7 activity was auto-induced through ATP efflux; this increased pERK and pAKT levels that generated a positive feedback on cell viability., (© 2014 Wiley Periodicals, Inc.)
- Published
- 2014
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41. Gravity loading induces adenosine triphosphate release and phosphorylation of extracellular signal-regulated kinases in human periodontal ligament cells.
- Author
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Ito M, Arakawa T, Okayama M, Shitara A, Mizoguchi I, and Takuma T
- Subjects
- Adenosine Diphosphate analogs & derivatives, Adenosine Diphosphate pharmacology, Adenosine Triphosphate antagonists & inhibitors, Apyrase pharmacology, Calcium pharmacology, Cell Culture Techniques, Cells, Cultured, Centrifugation, Extracellular Signal-Regulated MAP Kinases drug effects, Fibroblasts drug effects, Fibroblasts metabolism, Gingiva cytology, Gingiva drug effects, Gingiva metabolism, Gravitation, Humans, Periodontal Ligament cytology, Periodontal Ligament drug effects, Phosphorylation, Purinergic Agonists pharmacology, Purinergic Antagonists pharmacology, Purinergic P2X Receptor Antagonists pharmacology, Purinergic P2Y Receptor Antagonists pharmacology, Receptors, Purinergic P2 drug effects, Signal Transduction drug effects, Stress, Mechanical, Suramin pharmacology, Adenosine Triphosphate metabolism, Extracellular Signal-Regulated MAP Kinases metabolism, Periodontal Ligament metabolism
- Abstract
Aim: The periodontal ligament (PDL) receives mechanical stress (MS) from dental occlusion or orthodontic tooth movement. Mechanical stress is thought to be a trigger for remodeling of the PDL and alveolar bone, although its signaling mechanism is still unclear. So we investigated the effect of MS on adenosine triphosphate (ATP) release and extracellular signal-regulated kinases (ERK) phosphorylation in PDL cells., Methods: Mechanical stress was applied to human PDL cells as centrifugation-mediated gravity loading. Apyrase, Ca(2+)-free medium and purinergic receptor agonists and antagonists were utilized to analyze the contribution of purinergic receptors to ERK phosphorylation., Results: Gravity loading and ATP increased ERK phosphorylation by 5 and 2.5 times, respectively. Gravity loading induced ATP release from PDL cells by tenfold. Apyrase and suramin diminished ERK phosphorylation induced by both gravity loading and ATP. Under Ca(2+)-free conditions the phosphorylation by gravity loading was partially decreased, whereas ATP-induced phosphorylation was unaffected. Receptors P2Y4 and P2Y6 were prominently expressed in the PDL cells., Conclusion: Gravity loading induced ATP release and ERK phosphorylation in PDL fibroblasts, and ATP signaling via P2Y receptors was partially involved in this phosphorylation, which in turn would enhance gene expression for the remodeling of PDL tissue during orthodontic tooth movement., (© 2013 Wiley Publishing Asia Pty Ltd.)
- Published
- 2014
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42. Treatment of heterotopic ossification through remote ATP hydrolysis.
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Peterson JR, De La Rosa S, Eboda O, Cilwa KE, Agarwal S, Buchman SR, Cederna PS, Xi C, Morris MD, Herndon DN, Xiao W, Tompkins RG, Krebsbach PH, Wang SC, and Levi B
- Subjects
- Achilles Tendon enzymology, Achilles Tendon physiopathology, Achilles Tendon surgery, Adolescent, Adult, Aged, Aged, 80 and over, Animals, Burns complications, Burns enzymology, Burns genetics, Burns pathology, Case-Control Studies, Cells, Cultured, Child, Child, Preschool, Disease Models, Animal, Female, Gene Expression Regulation, Gene Regulatory Networks, Humans, Hydrolysis, Infant, Infant, Newborn, Male, Mesenchymal Stem Cells enzymology, Mice, Inbred C57BL, Middle Aged, Ossification, Heterotopic enzymology, Ossification, Heterotopic etiology, Ossification, Heterotopic genetics, Ossification, Heterotopic pathology, Phosphorylation, Signal Transduction drug effects, Smad Proteins, Receptor-Regulated metabolism, Tenotomy adverse effects, Time Factors, Young Adult, Achilles Tendon drug effects, Adenosine Triphosphate metabolism, Apyrase pharmacology, Burns drug therapy, Mesenchymal Stem Cells drug effects, Ossification, Heterotopic drug therapy, Osteogenesis drug effects
- Abstract
Heterotopic ossification (HO) is the pathologic development of ectopic bone in soft tissues because of a local or systemic inflammatory insult, such as burn injury or trauma. In HO, mesenchymal stem cells (MSCs) are inappropriately activated to undergo osteogenic differentiation. Through the correlation of in vitro assays and in vivo studies (dorsal scald burn with Achilles tenotomy), we have shown that burn injury enhances the osteogenic potential of MSCs and causes ectopic endochondral heterotopic bone formation and functional contractures through bone morphogenetic protein-mediated canonical SMAD signaling. We further demonstrated a prevention strategy for HO through adenosine triphosphate (ATP) hydrolysis at the burn site using apyrase. Burn site apyrase treatment decreased ATP, increased adenosine 3',5'-monophosphate, and decreased phosphorylation of SMAD1/5/8 in MSCs in vitro. This ATP hydrolysis also decreased HO formation and mitigated functional impairment in vivo. Similarly, selective inhibition of SMAD1/5/8 phosphorylation with LDN-193189 decreased HO formation and increased range of motion at the injury site in our burn model in vivo. Our results suggest that burn injury-exacerbated HO formation can be treated through therapeutics that target burn site ATP hydrolysis and modulation of SMAD1/5/8 phosphorylation., (Copyright © 2014, American Association for the Advancement of Science.)
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- 2014
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43. Burned to the bone.
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Forsberg JA, Davis TA, Elster EA, and Gimble JM
- Subjects
- Animals, Female, Humans, Male, Achilles Tendon drug effects, Adenosine Triphosphate metabolism, Apyrase pharmacology, Burns drug therapy, Mesenchymal Stem Cells drug effects, Ossification, Heterotopic drug therapy, Osteogenesis drug effects
- Abstract
Heterotopic ossification--a complication of severe burns, head or blast injuries, and orthopaedic trauma--can result from altered adenosine metabolism in mesenchymal stem cells in response to elevated extracellular ATP (Peterson et al., this issue)., (Copyright © 2014, American Association for the Advancement of Science.)
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- 2014
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44. Combination treatment of r-tPA and an optimized human apyrase reduces mortality rate and hemorrhagic transformation 6h after ischemic stroke in aged female rats.
- Author
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Tan Z, Li X, Turner RC, Logsdon AF, Lucke-Wold B, DiPasquale K, Jeong SS, Chen R, Huber JD, and Rosen CL
- Subjects
- Animals, Brain drug effects, Brain pathology, Brain physiopathology, Brain Edema complications, Brain Edema prevention & control, Drug Interactions, Female, Humans, Infarction, Middle Cerebral Artery pathology, Infarction, Middle Cerebral Artery physiopathology, Rats, Time Factors, Apyrase pharmacology, Cerebral Hemorrhage complications, Cerebral Hemorrhage prevention & control, Infarction, Middle Cerebral Artery complications, Infarction, Middle Cerebral Artery mortality, Recombinant Proteins pharmacology, Tissue Plasminogen Activator pharmacology
- Abstract
Recombinant tissue plasminogen activator (r-tPA) is the only FDA-approved drug treatment for ischemic stroke and must be used within 4.5h. Thrombolytic treatment with r-tPA has deleterious effects on the neurovascular unit that substantially increases the risk of intracerebral hemorrhage if administered too late. These therapeutic shortcomings necessitate additional investigation into agents that can extend the therapeutic window for safe use of thrombolytics. In this study, combination of r-tPA and APT102, a novel form of human apyrase/ADPase, was investigated in a clinically-relevant aged-female rat embolic ischemic stroke model. We propose that successfully extending the therapeutic window of r-tPA administration would represent a significant advance in the treatment of ischemic stroke due to a significant increase in the number of patients eligible for treatment. Results of our study showed significantly reduced mortality from 47% with r-tPA alone to 16% with co-administration of APT102 and r-tPA. Co-administration decreased cortical (47 ± 5% vs. 29 ± 5%), striatal (50 ± 2%, vs. 40 ± 3%) and total (48 ± 3%vs. 33 ± 4%) hemispheric infarct volume compared to r-tPA alone. APT102 improved neurological outcome (8.9±0.6, vs. 6.8 ± 0.8) and decreased hemoglobin extravasation in cortical tissue (1.9 ± 0.1mg/dl vs. 1.4 ± 0.1mg/dl) striatal tissue (2.1 ± 0.3mg/dl vs. 1.4 ± 0.1mg/dl) and whole brain tissue (2.0 ± 0.2mg/dl vs. 1.4 ± 0.1mg/dl). These data suggest that APT102 can safely extend the therapeutic window for r-tPA mediated reperfusion to 6h following experimental stroke without increased hemorrhagic transformation. APT102 offers to be a viable adjunct therapeutic option to increase the number of clinical patients eligible for thrombolytic treatment after ischemic stroke., (Copyright © 2014 Elsevier B.V. All rights reserved.)
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- 2014
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45. Inhibition of UDP/P2Y6 purinergic signaling prevents phagocytosis of viable neurons by activated microglia in vitro and in vivo.
- Author
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Neher JJ, Neniskyte U, Hornik T, and Brown GC
- Subjects
- Amyloid beta-Peptides toxicity, Animals, Apyrase pharmacology, Cell Death drug effects, Cell Death physiology, Cells, Cultured, Corpus Striatum drug effects, Corpus Striatum immunology, Isothiocyanates pharmacology, Lipopolysaccharides toxicity, Male, Microglia drug effects, Neurons drug effects, Neuroprotective Agents pharmacology, Peptide Fragments toxicity, Peroxynitrous Acid toxicity, Phagocytosis drug effects, Purinergic P2 Receptor Antagonists pharmacology, Rats, Wistar, Signal Transduction drug effects, Teichoic Acids toxicity, Thiourea analogs & derivatives, Thiourea pharmacology, Triazines pharmacology, Microglia physiology, Neurons immunology, Phagocytosis physiology, Receptors, Purinergic P2 metabolism, Uridine Diphosphate metabolism
- Abstract
Microglia activated through Toll-like receptor (TLR)-2 or -4 can cause neuronal death by phagocytosing otherwise-viable neurons-a form of cell death called "phagoptosis." UDP release from neurons has been shown to provoke microglial phagocytosis of neurons via microglial P2Y6 receptors, but whether inhibition of this process affects neuronal survival is unknown. We tested here whether inhibition of P2Y6 signaling could prevent neuronal death in inflammatory conditions, and whether UDP signaling can induce phagoptosis of stressed but viable neurons. We find that delayed neuronal loss and death in mixed neuronal/glial cultures induced by the TLR ligands lipopolysaccharide (LPS) or lipoteichoic acid was prevented by: apyrase (to degrade nucleotides), Reactive Blue 2 (to inhibit purinergic signaling), or MRS2578 (to specifically block P2Y6 receptors). In each case, inflammatory activation of microglia was not affected, and the rescued neurons remained viable for at least 7 days. Blocking P2Y6 receptors with MRS2578 also prevented phagoptosis of neurons induced by 250 nM amyloid beta 1-42, 5 μM peroxynitrite, or 50 μM 3-morpholinosydnonimine (which releases reactive oxygen and nitrogen species). Furthermore, the P2Y6 receptor agonist UDP by itself was sufficient to stimulate microglial phagocytosis and to induce rapid neuronal loss that was prevented by eliminating microglia or inhibiting phagocytosis. In vivo, injection of LPS into rat striatum induced microglial activation and delayed neuronal loss and blocking P2Y6 receptors with MRS2578 prevented this neuronal loss. Thus, blocking UDP/P2Y6 signaling is sufficient to prevent neuronal loss and death induced by a wide range of stimuli that activate microglial phagocytosis of neurons., (© 2014 The Authors. Glia Published by Wiley Periodicals, Inc.)
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- 2014
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46. Optimizing human apyrase to treat arterial thrombosis and limit reperfusion injury without increasing bleeding risk.
- Author
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Moeckel D, Jeong SS, Sun X, Broekman MJ, Nguyen A, Drosopoulos JH, Marcus AJ, Robson SC, Chen R, and Abendschein D
- Subjects
- Adenosine Diphosphate pharmacology, Animals, Apyrase adverse effects, Apyrase pharmacology, Clopidogrel, Coronary Circulation drug effects, Dogs, Fibrinolysis drug effects, Hemorrhage physiopathology, Humans, Mice, Inbred C57BL, Myocardial Infarction complications, Myocardial Infarction drug therapy, Myocardial Reperfusion Injury physiopathology, Piperazines pharmacology, Platelet Aggregation drug effects, Prasugrel Hydrochloride, Risk Factors, Thiophenes pharmacology, Thrombosis physiopathology, Ticlopidine analogs & derivatives, Ticlopidine pharmacology, Time Factors, Tissue Plasminogen Activator, Treatment Outcome, Vascular Patency drug effects, Apyrase therapeutic use, Hemorrhage etiology, Myocardial Reperfusion Injury complications, Myocardial Reperfusion Injury drug therapy, Thrombosis complications, Thrombosis drug therapy
- Abstract
In patients with acute myocardial infarction undergoing reperfusion therapy to restore blood flow through blocked arteries, simultaneous inhibition of platelet P2Y12 receptors with the current standard of care neither completely prevents recurrent thrombosis nor provides satisfactory protection against reperfusion injury. Additionally, these antiplatelet drugs increase the risk of bleeding. To devise a different strategy, we engineered and optimized the apyrase activity of human nucleoside triphosphate diphosphohydrolase-3 (CD39L3) to enhance scavenging of extracellular adenosine diphosphate, a predominant ligand of P2Y12 receptors. The resulting recombinant protein, APT102, exhibited greater than four times higher adenosine diphosphatase activity and a 50 times longer plasma half-life than did native apyrase. Treatment with APT102 before coronary fibrinolysis with intravenous recombinant human tissue-type plasminogen activator in conscious dogs completely prevented thrombotic reocclusion and significantly decreased infarction size by 81% without increasing bleeding time. In contrast, clopidogrel did not prevent coronary reocclusion and increased bleeding time. In a murine model of myocardial reperfusion injury caused by transient coronary artery occlusion, APT102 also decreased infarct size by 51%, whereas clopidogrel was not effective. These preclinical data suggest that APT102 should be tested for its ability to safely and effectively maximize the benefits of myocardial reperfusion therapy in patients with arterial thrombosis., (Copyright © 2014, American Association for the Advancement of Science.)
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- 2014
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47. Apyrase protects against allergic airway inflammation by decreasing the chemotactic migration of dendritic cells in mice.
- Author
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Li P, Cao J, Chen Y, Wang W, and Yang J
- Subjects
- Adenosine Triphosphate pharmacology, Aluminum Hydroxide, Animals, Antigens, CD genetics, Antigens, CD immunology, Apyrase genetics, Apyrase immunology, Asthma chemically induced, Asthma immunology, Asthma pathology, Bronchoalveolar Lavage Fluid chemistry, Cells, Cultured, Chickens, Cytokines biosynthesis, Cytokines metabolism, Dendritic Cells immunology, Dendritic Cells pathology, Epithelial Cells drug effects, Epithelial Cells immunology, Epithelial Cells pathology, Female, GATA3 Transcription Factor genetics, GATA3 Transcription Factor immunology, Gene Expression, Lung immunology, Lung pathology, Mice, Mice, Inbred C57BL, Ovalbumin, Signal Transduction, Anti-Asthmatic Agents pharmacology, Apyrase pharmacology, Asthma drug therapy, Chemotaxis drug effects, Dendritic Cells drug effects, Lung drug effects
- Abstract
Recent studies have demonstrated that extracellular adenosine 5'-triphosphate (eATP) is involved in allergic airway inflammation by activating purinergic receptors. eATP can be hydrolyzed by ectonucleotidases, such as CD39. In this study, we investigated the expression and distribution of CD39 in the lungs of mice, as well as the effects of apyrase on airway inflammation and the chemotactic migration of dendritic cells (DCs). A mouse model of asthma was developed with chicken ovalbumin (OVA)/aluminum hydroxide using female C57BL/6 mice. Apyrase was administered to OVA-sensitized mice prior to each challenge by intraperitoneal injection. The distribution of CD39 was detected by immunofluorescence. The mRNA and protein expression of CD39 was determined by quantitative PCR and western blot analysis, respectively. The levels of Th2 cytokines in the bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA). The effect of apyrase on the chemotactic migration of DCs towards ATP was explored by migration assay in vitro. In the lungs, CD39 was primarily located in the cytoplasm and cytomembrane of bronchial epithelial cells and CD39 expression was reduced in mice with allergic asthma. Treatment with apyrase markedly attenuated OVA-induced airway inflammation, including peribronchial eosinophilic inflammation and reduced the number of inflammatory cells, as well as the levels of cytokines in BALF. Furthermore, apyrase also markedly reduced the expression of GATA binding protein 3 (GATA3) and decreased the chemotactic migration of DCs towards ATP.Our data demonstrate that a reduction in CD39 expression may be associated with the development of allergic airway inflammation and that apyrase alleviates airway inflammation by decreasing the chemotactic migration of DCs towards eATP. Therefore, targeting at eATP or ectonucleotidases may provide a novel therapeutic approach for allergic asthma.
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- 2014
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48. Shear stress-dependent regulation of apical endocytosis in renal proximal tubule cells mediated by primary cilia.
- Author
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Raghavan V, Rbaibi Y, Pastor-Soler NM, Carattino MD, and Weisz OA
- Subjects
- Adenosine Triphosphate pharmacology, Albumins metabolism, Animals, Apyrase metabolism, Apyrase pharmacology, Biological Transport drug effects, Biological Transport physiology, Calcium metabolism, Cell Line, Cells, Cultured, Clathrin metabolism, Dextrans metabolism, Dogs, Dynamins metabolism, Egtazic Acid analogs & derivatives, Egtazic Acid pharmacology, Kidney Tubules, Proximal cytology, Kidney Tubules, Proximal metabolism, LLC-PK1 Cells, Madin Darby Canine Kidney Cells, Signal Transduction drug effects, Stress, Mechanical, Swine, Cilia physiology, Endocytosis physiology, Hydrodynamics, Kidney Tubules, Proximal physiology
- Abstract
The kidney has an extraordinary ability to maintain stable fractional solute and fluid reabsorption over a wide range of glomerular filtration rates (GFRs). Internalization of filtered low molecular weight proteins, vitamins, hormones, and other small molecules is mediated by the proximal tubule (PT) multiligand receptors megalin and cubilin. Changes in GFR and the accompanying fluid shear stress (FSS) modulate acute changes in PT ion transport thought to be mediated by microvillar bending. We found that FSS also affects apical endocytosis in PT cells. Exposure of immortalized PT cell lines to physiologically relevant levels of FSS led to dramatically increased internalization of the megalin-cubilin ligand albumin as well as the fluid phase marker dextran. FSS-stimulated apical endocytosis was initiated between 15 and 30 min postinduction of FSS, occurred via a clathrin- and dynamin-dependent pathway, and was rapidly reversed upon removing the FSS. Exposure to FSS also caused a rapid elevation in intracellular Ca(2+) [Ca(2+)]i, which was not observed in deciliated cells, upon treatment with BAPTA-AM, or upon inclusion of apyrase in the perfusion medium. Strikingly, deciliation, BAPTA-AM, and apyrase also blocked the flow-dependent increase in endocytosis. Moreover, addition of ATP bypassed the need for FSS in enhancing endocytic capacity. Our studies suggest that increased [Ca(2+)]i and purinergic signaling in response to FSS-dependent ciliary bending triggers a rapid and reversible increase in apical endocytosis that contributes to the efficient retrieval of filtered proteins in the PT.
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- 2014
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49. Extracellular ATP drives systemic inflammation, tissue damage and mortality.
- Author
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Cauwels A, Rogge E, Vandendriessche B, Shiva S, and Brouckaert P
- Subjects
- Animals, Anti-Inflammatory Agents pharmacology, Apoptosis, Apyrase pharmacology, Cytokines metabolism, Disease Models, Animal, Dose-Response Relationship, Drug, Endotoxins, Female, Hydrolysis, Inflammasomes immunology, Inflammasomes metabolism, Mice, Mice, Inbred C57BL, Mitochondria immunology, Mitochondria metabolism, Mitochondria pathology, Purinergic P2 Receptor Antagonists pharmacology, Signal Transduction, Suramin pharmacology, Systemic Inflammatory Response Syndrome chemically induced, Systemic Inflammatory Response Syndrome immunology, Systemic Inflammatory Response Syndrome pathology, Systemic Inflammatory Response Syndrome prevention & control, Time Factors, Adenosine Triphosphate metabolism, Inflammation Mediators metabolism, Systemic Inflammatory Response Syndrome metabolism
- Abstract
Systemic inflammatory response syndromes (SIRS) may be caused by both infectious and sterile insults, such as trauma, ischemia-reperfusion or burns. They are characterized by early excessive inflammatory cytokine production and the endogenous release of several toxic and damaging molecules. These are necessary to fight and resolve the cause of SIRS, but often end up progressively damaging cells and tissues, leading to life-threatening multiple organ dysfunction syndrome (MODS). As inflammasome-dependent cytokines such as interleukin-1β are critically involved in the development of MODS and death in SIRS, and ATP is an essential activator of inflammasomes in vitro, we decided to analyze the ability of ATP removal to prevent excessive tissue damage and mortality in a murine LPS-induced inflammation model. Our results indeed indicate an important pro-inflammatory role for extracellular ATP. However, the effect of ATP is not restricted to inflammasome activation at all. Removing extracellular ATP with systemic apyrase treatment not only prevented IL-1β accumulation but also the production of inflammasome-independent cytokines such as TNF and IL-10. In addition, ATP removal also prevented systemic evidence of cellular disintegration, mitochondrial damage, apoptosis, intestinal barrier disruption and even mortality. Although blocking ATP receptors with the broad-spectrum P2 purinergic receptor antagonist suramin imitated certain beneficial effects of apyrase treatment, it could not prevent morbidity or mortality at all. We conclude that removal of systemic extracellular ATP could be a valuable strategy to dampen systemic inflammatory damage and toxicity in SIRS.
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- 2014
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50. Purinergic control of vascular tone in the retina.
- Author
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Kur J and Newman EA
- Subjects
- Adenosine Triphosphate metabolism, Animals, Apyrase pharmacology, Arterioles drug effects, Arterioles metabolism, Arterioles physiology, Male, Rats, Rats, Long-Evans, Retinal Artery drug effects, Retinal Artery physiology, Purinergic P2X Receptor Agonists pharmacology, Purinergic P2X Receptor Antagonists pharmacology, Retinal Artery metabolism, Vasodilation
- Abstract
Purinergic control of vascular tone in the CNS has been largely unexplored. This study examines the contribution of endogenous extracellular ATP, acting on vascular smooth muscle cells, in controlling vascular tone in the in vivo rat retina. Retinal vessels were labelled by i.v. injection of a fluorescent dye and imaged with scanning laser confocal microscopy. The diameters of primary arterioles were monitored under control conditions and following intravitreal injection of pharmacological agents. Apyrase (500 units ml(-1)), an ATP hydrolysing enzyme, dilated retinal arterioles by 40.4 ± 2.8%, while AOPCP (12.5 mm), an ecto-5'-nucleotidase inhibitor that increases extracellular ATP levels, constricted arterioles by 58.0 ± 3.8% (P < 0.001 for both), demonstrating the importance of ATP in the control of basal vascular tone. Suramin (500 μm), a broad-spectrum P2 receptor antagonist, dilated retinal arterioles by 50.9 ± 3.7% (P < 0.001). IsoPPADS (300 μm) and TNP-ATP (50 μm), more selective P2X antagonists, dilated arterioles by 41.0 ± 5.3% and 55.2 ± 6.1% respectively (P < 0.001 for both). NF023 (50 μm), a potent antagonist of P2X1 receptors, dilated retinal arterioles by 32.1 ± 2.6% (P < 0.001). A438079 (500 μm) and AZ10606120 (50 μm), P2X7 antagonists, had no effect on basal vascular tone (P = 0.99 and P = 1.00 respectively). In the ex vivo retina, the P2X1 receptor agonist α,β-methylene ATP (300 nm) evoked sustained vasoconstrictions of 18.7 ± 3.2% (P < 0.05). In vivo vitreal injection of the gliotoxin fluorocitrate (150 μm) dilated retinal vessels by 52.3 ± 1.1% (P < 0.001) and inhibited the vasodilatory response to NF023 (50 μm, 7.9 ± 2.0%; P < 0.01). These findings suggest that vascular tone in rat retinal arterioles is maintained by tonic release of ATP from the retina. ATP acts on P2X1 receptors, although contributions from other P2X and P2Y receptors cannot be ruled out. Retinal glial cells are a possible source of the vasoconstricting ATP.
- Published
- 2014
- Full Text
- View/download PDF
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