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Publié par | ruprecht-karls-universitat_heidelberg |
Publié le | 01 janvier 2009 |
Nombre de lectures | 81 |
Langue | English |
Poids de l'ouvrage | 1 Mo |
Extrait
Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto‐Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
Presented by
Hanna Korhonen (MSc)
Born in: Joensuu, Finland
Oral examination: ……………..
ANALYSIS OF G /G AND G /G MEDIATED SIGNALLING IN q 11 12 13
THE ENDOTHELIAL FUNCTIONS
Referees: Prof. Dr. Stefan Offermanns
Prof. Dr. Markus Schwaninger
TABLE OF CONTENTS
1 ABBREVIATIONS.............................................................................................. I
2 SUMMARY...................................................................................................... II
3 ZUSAMMENFASSUNG ................................................................................... III
4 INTRODUCTION 4
4.1 The G‐protein mediated activation of endothelial cells................................. 5
4.1.1 The G‐protein‐mediated signalling system.......................................... 5
4.1.2 The Gα ‐family of G‐proteins............................................................... 7 q
4.1.3 The Gα ‐family of G‐s............................................................. 8 12
4.2 Regulation of endothelial barrier permeability............................................ 10
4.2.1 Structure of interendothelial junctions............................................. 10
4.2.1.1 Adherens junctions..................................................................... 10
4.2.1.2 Tight junctions ............................................................................ 12
4.2.1.3 Gap juctions................................................................................ 12
4.2.2 Signalling mechanisms regulating endothelial barrier permeability.. 13
4.3 Role of endothelium in the regulation of vascular tone............................... 16
4.3.1 Nitric oxide (NO) 16
4.3.2 Endothelium derived hyperpolarizing factor (EDHF)......................... 18
4.4 Pathophysiology of endothelial cells............................................................ 19
4.4.1 Inflammation..................................................................................... 19
4.4.2 Anaphylactic shock............................................................................ 22
5 AIM OF THE STUDY....................................................................................... 24
6 MATERIAL AND METHODS........................................................................... 25
6.1 Reagents....................................................................................................... 25
6.2 Antibodies.................................................................................................... 29
6.3 Kits................................................................................................................ 29
6.4 Buffers and solutions.................................................................................... 30
6.5 Breeding and genotyping of mice................................................................. 32
6.5.1 Isolation of the genomic DNA............................................................ 33
6.5.2 Genotyping of mice by polymerase chain reaction (PCR).................. 33
6.5.3 Agarose gel electrophoresis .............................................................. 35
6.6 Histology and detection of β‐galactosidase activity..................................... 36
6.6.1 Preparation of tissues for cryosectioning.......................................... 36
6.6.2 X‐Gal staining of cryosections 36
6.7 Detection of protein expression by Western blotting.................................. 37
6.7.1 SDS‐polyacrylamide gel electrophoresis............................................ 37
6.7.2 Protein electrotransfer...................................................................... 37
6.8 Mouse primary endothelial cell isolation and culture.................................. 38
6.8.1 Coating of magnetic beads................................................................ 38
6.8.2 Isolation of endothelial cells.............................................................. 39
6.8.3 Purification of primary endothelial cell cultures................................ 39
6.8.4 Immunofluorescence staining of primary endothelial cells............... 40
6.9 In vitro experiments..................................................................................... 41
6.9.1 Inositol 1,4,5‐triphosphate (IP ) assay............................................... 41 3
6.9.2 RhoA activation assay........................................................................ 42
6.9.3 Detection of MLC phosphorylation in endothelial cells by Western
blot ........................................................................................................... 43
6.9.4 Determination of NO production....................................................... 43
6.10 In vivo experiments............................................................................... 44
6.10.1 Vascular permeability assay in vivo................................................... 44
6.10.2 Passive cutaneous anaphylaxis.......................................................... 44
6.10.3 Blood pressure and body temperature measurements..................... 45
6.10.4 Passive systemic anaphylaxis............................................................. 46
6.10.5 Active systemic anaphylaxis............................................................... 47
6.10.6 Intravital microscopy......................................................................... 47
6.10.6.1 Data Analysis 48
7 RESULTS........................................................................................................ 50
7.1 Purity of the primary endothelial cell culture and loss of G /G and G /G in q 11 12 13
endothelial cells................................................................................................. 50
7.2 G /G primarily mediate endothelial effects of inflammatory mediators acting q 11
via GPCRs............................................................................................................ 51
7.3 Generation and genotyping of mice with endothelial cell specific Gα /Gα and q 11
Gα /Gα deficiency.......................................................................................... 54 12 13
7.4 Gα /Gα deficiency blocks local extravasation in response to various stimuli q 11
..................................................................................................................... 58
7.5 Basal blood pressure is unchanged in Gα /Gα and Gα /Gα ‐KO mice..... 61 q 11 12 13
7.6 Systemic effects of histamine and PAF, but not of LPS, are blocked in