Cytoprotective and anti-inflammatory properties of the atrial natriuretic peptide during ischemia-reperfusion and endotoxemia [Elektronische Ressource] / Melanie Keller

Melanie Keller

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 34
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München





Cytoprotective and Anti-Inflammatory Properties
of the Atrial Natriuretic Peptide during Ischemia/Reperfusion
and Endotoxemia



Melanie Keller
aus Karlsruhe
2005

































dedicated with love to my family


In loving remembrance of my brother -
I will never forget about you.
















1. Contents
2 1 Contents


1. CONTENTS........................................................................................................1
2. INTRODUCTION................................................................................................6
2.1 BACKGROUND AND AIM OF THE STUDY ............................................................................... 7
2.2 ISCHEMIA/REPERFUSION INJURY (IRI)................................................................................... 9
2.2.1 General aspects .................................................................................................................... 9
2.2.2 Tissue damage and IRI: apoptosis or necrosis?................................................................. 10
2.3 ROLE OF THE PI3-KINASE PATHWAY IN APOPTOTIC CELL DEATH ................................ 11
2.3.1 Apoptosis and necrosis ....................................................................................................... 11
2.3.2 Involvement of PI3-kinase in anti-apoptotic signaling......................................................... 13
2.4 HEAT SHOCK PROTEINS AND CYTOSKELETON................................................................. 16
2.4.1 Heat shock proteins.............................................................................................................16
2.4.2 Influence of Hsp27 on cytoskeletal structures .................................................................... 17
2.4.3 Actin polymerization ............................................................................................................18
2.5 THE ATRIAL NATRIURETIC PEPTIDE .................................................................................... 20
2.5.1 Receptors and signaling mechanisms ................................................................................ 21
2.5.2 Effects ANP during IR and inflammatory processes........................................................... 23
2.6 SEPSIS....................................................................................................................................... 25
2.6.1 General aspects .................................................................................................................. 25
2.6.2 Pathogenetic networks in sepsis......................................................................................... 26
2.6.3 Toll-like receptors................................................................................................................ 28
2.6.4 Signaling pathways during LPS-induced sepsis ................................................................. 29
3. MATERIALS AND METHODS.........................................................................32
3.1 THE ISOLATED PERFUSED RAT LIVER ................................................................................ 33 1 Contents 3


3.1.1 Animals................................................................................................................................ 33
3.1.2 Materials and solutions........................................................................................................ 33
3.1.3 Experimental setting............................................................................................................34
3.1.4 Homogenization and fractionation of liver tissue for Western Blot analysis ....................... 35
3.1.5 Quantification of actin content in liver fractions................................................................... 35
3.2 RAT LIVER TRANSPLANTATION............................................................................................ 36
3.2.1 Animals................................................................................................................................ 36
3.2.2 Materials and solutions........................................................................................................ 36
3.2.3 Experimental setting............................................................................................................36
3.2.4 Determination of activities of serum aminotransferases and lactate dehydrogenase ........ 38
3.2.5 PKA activity assay............................................................................................................... 38
3.2.6 Caspase-3-like activity assay.............................................................................................. 38
3.3 MURINE MODEL OF LPS-INDUCED SEPSIS.......................................................................... 39
3.3.1 Animals................................................................................................................................ 39
3.3.2 Materials and solutions........................................................................................................ 40
3.3.3 Experimental setting............................................................................................................40
3.3.4 Determination of TNF- α concentrations in blood samples.................................................. 43
3.4 HISTOLOGICAL ANALYSIS OF TISSUE................................................................................. 43
3.4.1 TUNEL staining ................................................................................................................... 43
3.4.2 Hematoxylin staining 44
3.4.3 Immunohistochemistry ........................................................................................................ 44
3.5 WESTERN BLOT ANALYSIS ................................................................................................... 45
3.5.1 Sample preparation – homogenization of tissue................................................................. 45
3.5.2 Measurement of protein concentrations with Pierce assay................................................. 46
3.5.3 Immunoprecipitation............................................................................................................ 46
3.5.4 SDS- polyacrylamide gel electrophoresis (SDS-PAGE) ..................................................... 47
3.5.5 Western Blot........................................................................................................................ 48 4 1 Contents


3.6 ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA)....................................................... 52
3.6.1 Solutions.............................................................................................................................. 52
3.6.2 Extraction of nuclear protein ............................................................................................... 53
3.6.3 Measurement of protein concentration with Bradford-assay............................................... 54
3.6.4 Radioactive labeling of consensus oligonucleotides........................................................... 54
3.6.5 Binding reaction and electrophoretic separation................................................................. 54
3.7 STATISTICAL ANALYSIS......................................................................................................... 55
4. RESULTS56
4.1 EFFECTS OF ANP DURING ISCHEMIA AND REPERFUSION IN THE LIVER...................... 57
4.1.1 Effects of ANP preconditioning in the isolated perfused rat liver ........................................ 57
4.1.2 Effects of ANP preconditioning in the liver during IR in vivo............................................... 62
4.2 EFFECTS OF ANP DURING LPS-INDUCED SEPTIC SHOCK 71
4.2.1 Effect of ANP pretreatment on survival after septic shock.................................................. 71
4.2.2 Effects of ANP preconditioning on blood TNF- α levels after LPS- induced septic shock... 72
4.2.3 Effects of ANP on signaling during LPS-induced sepsis in the kidney ............................... 73
5. DISCUSSION ...................................................................................................77
5.1 EFFECTS OF ANP DURING ISCHEMIA AND REPERFUSION IN THE LIVER...................... 78
5.1.1 Ex vivo pretreatment with ANP causes p38 MAPK-dependent changes in hepatocytes
cytoskeleton....................................................................................................................................... 78
5.1.2 ANP treatment in vivo has anti-apoptotic effects mediated via the PI3-kinase pathway.... 82
5.2 EFFECTS OF ANP DURING LPS-INDUCED SEPTIC SHOCK ............................................... 88
5.2.1 ANP preconditioning maintains survival after LPS-induced septic s