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ATP release from activated neutrophils occurs via connexin 43 and modulates adenosine-dependent endothelial cell function [Elektronische Ressource] / vorgelegt von Natalie Daniela Küper

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57 pages
Aus der Universitätsklinik für Anaesthesiologie und Intensivmedizin Tübingen Ärztlicher Direktor: Professor Dr. K. Unertl ATP release from activated neutrophils occurs via connexin 43 and modulates adenosine-dependent endothelial cell function Inaugural-Dissertation zur Erlangung des Doktorgrades der Medizin der Medizinischen Fakultät der Eberhard Karls Universität zu Tübingen vorgelegt von Natalie Daniela Küper aus Reutlingen 2008 Dekan: Professor Dr. I. B. Autenrieth 1. Berichterstatter: Professor Dr. H. Eltzschig 2. Berichterstatter: Professor Dr. M. Duszenko 2 Gewidmet meinem Vater für seine Begeisterung und das außerordentliche Interesse bei der Entstehung dieser Arbeit 3Inhaltsverzeichnis 1. Introduction............................................................................................ 6 1.1. Structural and functional elements of the vascular barrier ...............................6 1.2. Vascular barrier during inflammation..................................................................8 1.2.1. Barrier disruptive pathways .............................................................................9 1.2.2. Barrier protective pathways ...........................................................................10 1.2.3. Effect of adenosine receptor activation on endothelial barrier function..........14 1.3.
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Aus der Universitätsklinik für Anaesthesiologie und Intensivmedizin Tübingen Ärztlicher Direktor: Professor Dr. K. Unertl     ATP release from activated neutrophils occurs via connexin 43 and modulates adenosine-dependent endothelial cell function Inaugural-Dissertation zur Erlangung des Doktorgrades der Medizin der Medizinischen Fakultät der Eberhard Karls Universität zu Tübingen vorgelegt von Natalie Daniela Küper aus Reutlingen 2008
Dekan:
1. Berichterstatter:
2. Berichterstatter:
 
Professor Dr. I. B. Autenrieth
Professor Dr. H. Eltzschig
Professor Dr. M. Duszenko
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Gewidmet meinem Vater für seine Begeisterung und das außerordentliche Interesse bei der Entstehung dieser Arbeit
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Inhaltsverzeichnis 
 
1. Introduction ............................................................................................ 6 1.1.  ............................... 6Structural and functional elements of the vascular barrier 1.2.  .................................................................. 8Vascular barrier during inflammation 1.2.1.  9Barrier disruptive pathways ............................................................................. 1.2.2.  ........................................................................... 10Barrier protective pathways 1.2.3.  14adenosine receptor activation on endothelial barrier function..........Effect of  1.3. Increased Adenosine Production during hypoxia ............................................ 14 1.4. Role of Adenosine Deaminase in vascular inflammation during hypoxia...... 15 
2. Materials and Methods ........................................................................ 18 2.1. Materials ............................................................................................................... 18 2.2. Methods................................................................................................................ 18 2.2.1. Isolation of Human PMN................................................................................ 18 2.2.2. Preparation of Activated PMN Supernatants and Measurement of ATP or myeloperoxidase (MPO) content ................................................................... 19 2.2.3. PMN Granule isolation................................................................................... 20 2.2.4. of endothelial surface enzyme activity of the ecto-apyraseMeasurement (CD39) and the 5’-ectonucleotidase (CD73).................................................. 20 2.2.5.  21Endothelial Cell Isolation and Culture............................................................ 2.2.6. Endothelial Macromolecule Paracellular Permeability Assay ........................ 21 2.2.7.  22Immunoblotting experiments ......................................................................... 2.2.8. surface expression of CD 39 and CD 73....Flowcytometric analysis of PMN  22 2.2.9.  23PMN adhesion assay..................................................................................... 2.2.10. Isolation and activation of murine PMN ......................................................... 23 
 
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3.  25Results .................................................................................................. 3.1. PMN release ATP upon activation...................................................................... 25 3.2. Mechanism of extracellular ATP metabolism.................................................... 26 3.3. Different kinetics of ATP-levels within the supernatant of activated PMN derived from cd39-null-mice ............................................................................... 29 3.4. Biologically active adenosine liberated via PMN CD 39 and endothelial ....... 29  29CD 73 .................................................................................................................... 3.5. Mechanisms of PMN ATP release ...................................................................... 30 3.6.  ...................................................... 31The role of Cx 43 in ATP release from PMN 3.7.  33Activation-dependent PMN Cx 43 dephosphorylation ..................................... 3.8. Role of Cx 43 dependent ATP release by PMN in modulating endothelial cell function.......................................................................................................... 35 3.9. Activated PMN from mice with induced deletion of Cx 43 show decreased ATP release .......................................................................................................... 37 
4. Discussion............................................................................................ 39 
5. Summary............................................................................................... 42 
6. References............................................................................................ 43 
7. Danksagung ......................................................................................... 56 
8.  57Lebenslauf ............................................................................................ 
 
  
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