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Pro-Inflammatory Role of P2Y6 Receptor Signalling During Vascular Inflammation

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Pro-Inflammatory Role of P2Y6 Receptor Signalling During Vascular Inflammation Aus der Universitätsklinik für Anästhesiologie und Intensivmedizin Tuebingen Ärztlicher Direktor: Professor Dr. P. Rosenberger Pro-inflammatory role of P2Y6 receptor signalling during vascular inflammation Inaugural Dissertation zur Erlangung des Doktorgrades der Medizin der Medizinischen Fakultät der Eberhard Karls Universität zu Tübingen vorgelegt von Ann-Kathrin Riegel, geb. Stenz, aus Herrenberg 2011 Dekan: Professor Dr. I. B. Autenrieth 1. Berichterstatter: Professor Dr. H.K. Eltzschig 2. Berichterstatter: Professor Dr. Dr. K. Zacharowski 3. Berichterstatterin: Professor Dr. C.E. Mueller To my parents Helga and Dr. Rainer Stenz, to my sisters Carolin Franziska and Kerstin Julia Stenz to Jens-Jochen Riegel and Margit, Adam, Max, Christel, Dagmar and Martin, to my grandparents and to my family-in-law :-) Abbreviations 1321 N1 astrocytoma Human glial cell line from brain astrocytoma, naturally cell line lacking any known P2 receptor A1 Adenosine receptor type 1 A2a Adenosine receptor type 2a A2b Adenosine receptor type 2b A3 Adenosine receptor type 3 AC Adenylate cyclase ADP Adenosine Diphosphate AMP Adenosine Monophosphate ATP Adenosine Triphosphate BSA Bovine Serum Albumine Ca2+ Calcium cAMP Cyclic Adenosine Monophosphate CD39 Ecto-Apyrase CD73 Ecto-5’-nucleotidase CDP Cytidine diphosphate COX-2 Cyclooxygenase 2 CTP Cytidine triphosphate DNA Deoxyribonucleic Acid DMSO Dimethyl sulfoxide ERK 1/2 Extracellular signal-regulated kinases 1 and 2 = specific subset of MAPK GDP Guanosine diphosphate G-protein Guanine-nucleotide binding protein Gαq α-subunit of heterotrimeric Gq-proteins Gi Heterotrimeric G-protein that inhibits the production of cAMP from ATP. Gq/11 Family of heterotrimeric G-proteins that stimulate the membrane-bound PLC-ß and increase the intracellular concentration of IP3 and cAMP. Gs Heterotrimeric G-protein that stimulates the production of cAMP from ATP and activates the AC. GRK G-protein coupled receptor protein kinase GTP Guanosine triphosphate [H³] Tritium, a radioactive form of heavy hydrogen HPLC High performance liquid chromatography IDP Inosine diphosphate IL Interleukin IP3 Inositol-Triphosphate ITP Inosine Triphosphate MAPK Mitogen-activated protein kinase mRNA Messenger Ribonucleic Acid NDP Nucleoside diphosphate NF-κB Nuclear factor kappa B NTP Nucleoside triphosphate P2 Purinergic nucleotide receptor P2X Purinergic ionchannel receptor P2Y Purinergic / pyrimidinergic G-protein coupled receptor PBS Phosphat Buffered Saline PCR Polymerase Chain Reaction PKA Protein Kinase A PKC Phosphokinase C PLC-ß Phospholipase C beta PMN Polymorphonuclear leukocytes Pi Inorganic phosphate PPi Pyrophosphate RIPA Radio-Immuno Precipitation Assay RNA Ribonuclein Acid SDS Sodium dodecyl sulfate RT-PCR Real-Time Polymerase Chain Reaction TBS Tris Buffered Saline TRIS Tris(hydroxymethyl)aminomethane UDP Uridine Diphosphate UMP Uridine Monophosphate UTP Uridine Triphosphate Table of contents 1. INTRODUCTION .................................................................................................... 1 1.1. EXTRACELLULAR NUCLEOSIDE AND NUCLEOTIDE RECEPTORS ................................. 1 1.2. P1 RECEPTORS ..................................................................................................... 5 1.3. P2X RECEPTORS .................................................................................................. 6 1.4. P2Y RECEPTORS .................................................................................................. 8 1.5. P2Y6-RECEPTOR ................................................................................................. 12 1.5.1. TISSUE DISTRIBUTION ........................................................................................ 12 1.5.2. AGONISTS AND ANTAGONISTS ............................................................................ 14 1.5.3. CELL SIGNALLING .............................................................................................. 16 2+ 1.5.3.1. GQ/11, PLC, IP3, DAG, CA , PKC AND ERK 1/2 ............................................... 16 1.5.3.2. G12/13, AC, CAMP, PKA AND RHO ................................................................... 20 1.5.3.3. PLD .............................................................................................................. 21 1.5.3.4. PI3K ............................................................................................................. 22 1.5.3.5. NF-ΚB ........................................................................................................... 22 1.5.4. INDUCED GENES AFTER P2Y6-RECEPTOR STIMULATION ....................................... 28 1.5.4.1. INDUCTION OF IL-8 AFTER P2Y6-RECEPTOR STIMULATION ................................. 28 1.5.4.2. PATHWAYS SHOWN NOT TO BE INVOLVED IN P2Y6-RECEPTOR SIGNAL TRANSDUCTION .............................................................................................. 29 1.5.4.3. SUMMARIZED ILLUSTRATION OF P2Y6-RECEPTOR CELL SIGNALLING PATHWAYS .. 30 1.5.5. P2Y6-RECEPTOR DESENSITIZATION AND REGULATION .......................................... 30 1.5.6. P2Y6-RECEPTOR KNOCKOUT MICE...................................................................... 33 1.6. EXTRACELLULAR NUCLEOTIDES ........................................................................... 34 1.6.1. RELEASE OF URIDINE PHOSPHATES .................................................................... 40 1.6.1.1. BASAL RELEASE OF URIDINE PHOSPHATES ....................................................... 40 1.6.1.2. RELEASE OF URIDINE PHOSPHATES BECAUSE OF STRESSFUL EVENTS OR TRIGGER MOLECULES ................................................................................................... 42 1.6.1.3. RELEASE OF URIDINE PHOSPHATES BECAUSE OF CELL LYSIS ............................. 44 1.6.2. EXTRACELLULAR METABOLISM OF URIDINE PHOSPHATES ..................................... 46 1.6.2.1. ECTO-NUCLEOSIDE 5’-TRIPHOSPHATE DIPHOSPHOHYDROLASES ........................ 48 1.6.2.2. EXTRACELLULAR NUCLEOSIDE DIPHOSPHATE KINASE (NDPK) ........................... 51 1.6.2.3. EXTRACELLULAR NUCLEOTIDE PYROPHOSPHATASE / PHOSPHODIESTERASE ....... 52 1.6.2.4. ECTO-5’-NUCLEOTIDASE ................................................................................. 53 1.6.2.5. ALKALINE PHOSPHATASE ................................................................................ 54 1.6.2.6. SUMMARY METABOLISM OF NUCLEOTIDES ........................................................ 54 1.6.3. REUPTAKE OF EXTRACELLULAR URIDINE ............................................................. 55 1.7. P2Y6-RECEPTOR IN INFLAMMATION ...................................................................... 57 1.7.1. P2Y6-RECEPTOR UPREGULATION IN INFLAMMATION ............................................ 58 1.7.2. EXTRACELLULAR RELEASE AND METABOLISM OF UDP IN INFLAMMATION .............. 59 1.7.3. LPS AND TNF-Α POTENTIATE THEIR EFFECT THROUGH AUTOCRINE STIMULATION AT THE P2Y6-RECEPTOR ........................................................................................ 61 1.8. CLINICAL RELEVANCE ......................................................................................... 63 1.9. RATIONALE OF THE THESIS .................................................................................. 64 2. MATERIALS AND METHODS ............................................................................. 66 2.1. CELL CULTURE ................................................................................................... 66 2.2. INFLAMMATORY AND P2Y6-RECEPTOR ANTAGONIST STIMULATION OF CELL CULTURE CELLS ................................................................................................................ 67 2.3. RNA-ISOLATION, TRANSCRIPTION, PCR .............................................................. 69 2.4. PRIMER .............................................................................................................. 71 2.5. MICROARRAY ..................................................................................................... 82 2.6. WESTERN BLOT .................................................................................................. 88 2.7. TRANSFECTION AND REPORTER ASSAY ............................................................... 95 2.8. IMMUNOFLUORESCENCE ...................................................................................... 97 2.9. INTRAVENOUS LPS INJECTION ............................................................................. 98 2.10. STATISTICAL ANALYSIS, PROGRAMMING, ILLUSTRATIONS AND REFERENCES ...... 101 3. RESULTS .......................................................................................................... 102 3.1. THE ENDOTHELIAL P2Y6-RECEPTOR IS SELECTIVELY INDUCED AFTER TNF-Α EXPOSURE ........................................................................................................ 102 3.2. P2Y6-RECEPTOR ANTAGONIST MRS2578 DAMPENS MEDIATOR-INDUCED INFLAMMATION OF VASCULAR INFLAMMATION OF VASCULAR ENDOTHELIA IN VITRO …………………………………………………………………………………………108 3.3. P2Y6 RECEPTOR IS INDUCED IN VIVO AFTER INTRAVENOUS LPS TREATMENT ........ 112 3.4. PHARMACOLOGIC INHIBITION OR GENETIC DELETION AF THE P2Y6 RECEPTOR CONVEY PROTECTION FROM LPS-INDUCED INFLAMMATION ............................................... 112 4. DISCUSSION ..................................................................................................... 116 5. SUMMARY ........................................................................................................
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