Effect of cAMP-PKA signaling mechanism on barrier function of cultured endothelial cells [Elektronische Ressource] : role of myosin light chain phosphatase / vorgelegt von Muhammad Aslam

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• Biologie
• Gesundheit

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• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2007
• Nombre de lectures: 17
• Poids de l'ouvrage: 2 Mo

Extrait

EFFECT OF CAMP/PKA SIGNALING MECHANISM
ON BARRIER FUNCTION OF CULTURED
ENDOTHELIAL CELLS:
ROLE OF MYOSIN LIGHT CHAIN PHOSPHATASE
MUHAMMAD ASLAM
édition scientifique
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VVB LAUFERSWEILER VERLAG zur Erlangung des Doktorgrades der ISBN 3-8359-5220-X
ST AU FEN BER G R I N G 1 5 Naturwissenschaften (Dr. rer. nat.)D - 3 5 3 9 6 G I E S S E N
des Fachbereichs Biologie und Chemie
Tel: [phone] Fax: -5599890
[e-mail] der Justus-Liebig-Universität Giessen
w w w . d o k t o r v e r l a g . d e 9 7 8 3 8 3 5 9 5 2 2 0 1
Human umbilical vein endothelial cells stained for actin (red)
and ß-catenin (green)
édition scientifique
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Aus dem Physiologischen Institut am Fachbereich Medizin
und
Institut für Tierphysiologie am Fachbereich Biologie und Chemie
der Justus-Liebig-Universität



Effect of cAMP/PKA signaling mechanism on
barrier function of cultured endothelial cells:

Role of myosin light chain phosphatase




INAUGURAL-DISSERTATION
zur Erlangung des Doktorgrades der
Naturwissenschaften (Dr. rer. nat.)
des Fachbereichs Biologie und Chemie
der Justus-Liebig-Universität Giessen




vorgelegt von


Muhammad Aslam

(Pharmacist, M.Phil. Molecular Biology)
aus Muridke, Pakistan



Gießen 2007 ii
Tag der Disputation:
Friday, September 28, 2007. 14:00 hrs.
Gutachter: Prof. Dr. W. Clauß (Fachbereich Biologie und Chemie)
Gutachter: Priv. Doz. Dr. T. Noll (Fachbereich Humanmedizin)iii
In the Name of Allah (God),
the Most Gracious, the Most Merciful
Proclaim! (or read!) in the name of thy Lord and Cherisher, Who
created- Created man, out of a (mere) clot of congealed blood:
Proclaim! And thy Lord is Most Bountiful,- He Who taught (the use
of) the pen,- Taught man that which he knew not.
Man We did create from a quintessence (of clay); Then We placed
him as (a drop of) sperm in a place of rest, firmly fixed; Then We
made the sperm into a clot of congealed blood; then of that clot We
made a (foetus) lump; then we made out of that lump bones and
clothed the bones with flesh; then we developed out of it another
creature. So blessed be Allah, the best to create! After that, at length
ye will die. Again, on the Day of Judgment, will ye be raised up.
(Al-Quran) iv










































To
My Mother
v
List of Abbreviations 1
1 Introduction 4
1.1 Barrier function of the vascular endothelium 4
1.2 Endothelial actomyosin cytoskeleton 6
1.3 Endothelial contractile machinery 7
1.3.1 Myosin light chains 7
1.3.2 Mysin light chain kinase 8
1.3.3 Myosin light chain phosphatase 9
1.3.4 Regulation of myosin light chain phosphatase activity
via MYPT1 phosphorylation 10
1.3.5 Regulation of myosin light chain phosphatase activity by
CPI-17 11
1.4 cAMP and endothelial permeability 12
1.5 Aims and objectives of the study 14
2 Methods 16
2.1 Cell culture 16
2.2 General incubation conditions 17
2.3 Protein analysis 18
2.4 Determination of myosin light chain phosphorylation 22
2.5 Co-immunoprecipitation of proteins 23
2.6 Protein phosphatase assay 24
2.7 Detection of activated RhoA 25
2.8 Measurement of RhoA translocation 26
2.9 Measurement of endothelial monolayer permeability 26
2.10 Downregulation of endogenous CPI-17 27
2.11 Statistical analysis 28
3. Results 29
3.1 Effect of forskolin on endothelial permeability 29
3.2 Effect of forskolin on myosin light chain phosphorylation 30
3.3 Effect of forskolin on MLC phosphatase complex formation
and activity 34
3.4 Effect of forskolin on MYPT 1 phosphorylation 38vi
3.5 Effect of forskolin on RhoA translocation 40
3.6 Effect of forskolin on CPI-17 phosphorylation and
co-immunoprecipitation with PP1 42
3.7 Effect of CPI-17 downregulation on endothelial barrier 48
4 Discussion 50
4.1 Main findings 50
4.2 MLC phosphatase holoenzyme complex formation and activation 51
4.3 Forskolin induced MLC phosphatase complex formation is
independent of RhoA/Rock inhibition 52
4.4 MYPT1 dephosphorylation 54
4.5 cAMP/PKA signaling inactivates CPI-17 55
4.6 CPI-17 as a mediator of thrombin induced barrier failure 57
4.7 Future perspective 59
5 References 60
6 Summary 76
7 Zusammenfassung 77
8 Erklärung 78
9 Appendix 79
9.1 Chemicals and consumables 79
9.2 Antibodies 82
9.3 Laboratory instruments 83
9.4 Solutions 84
10 Acknowledgment 87
11 Curriculum vitae 88
12 Publications 89
13 Published abstracts 90 1
LIST OF ABBREVIATIONS

AC Adenylyl cyclase
app. pproximately
APS mmonium per sulfate
ATP Adenosine-5-triphosphate
bFGF Basic fibroblast growth factor
BSA ovine serum albumin
CaCl Calcium chloride 2
2+CaMKII Ca /calmodulin-dependent protein kinase II
cAMP 3'-5'-cyclic adenosine monophosphate
cGMP -5'-cycliguanosinm
CPI-17 PKC-potentiated inhibitor 17-kDa protein
DMSO Dimethyl sulfoxide
DTT Dithiothreitol
EC Endothelial cells
ECGS ndothelial cell growth supplement
ECL nhanced chemiluminescence
EC-MLCK Endothelial cell myosin light chain kinase
EDTA Ethylene diamine tetraacetic acid
EGTA thylene glycol-bis(2-aminoethylether)-N,N,N',N'-
tetraacetic acid
F-actin Filamentous actin
FCS Fetal calf serum
FSK orskolin
GPCR G-protein-coupled receptors
G-actin Globular actin
HBSS Hanks' balanced salt solution
hEGF uman epidermal growth factor
HEPES 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic
acid
HUVEC Human umbilical vein endothelial cells
IU International unit
KCl Potassium chloride
2
KH PO Potassium dihydrogen phosphate 2 4
ILK Integrin linked kinase
kDa Kilo dalton
MgCl Magnesium chloride 2
min inutes
MLC yosin light chain
MLC~P Monophosphorylated myosin light chain
MLC~PP Diphosphorylated myosin light chain
MLCK yosin light chain kinase
MLCP Myosin lichaiphosphatase
MnCl anganese chloride 2
MYPT1 Myosin phosphatase targeting subunit 1
NaCl Sodium chloride
NaF Sodium fluoride
Na HPO Di-sodium hydrogen phosphate 2 4
NaH PO odium dihydrogen phosphate 2 4
Na-orthovanadate Sodium orthovanadate
NP-40 Nonidet P-40
PAK p21-activated kinase
PAR1 Protease-activated receptor-1
PBS hosphate-buffered saline
+pH Negative log of H concentration
PKA Protein kinase A
PKC roteikiC
PKI rotein kinase A inhibitor
PMA Phorbol-12-myristate-13-acetate
PMSF henylmethylsulfonyl fluoride
PP1 rotein phosphatase 1
rec. I-2 Recombinant inhibitor 2
Rhotekin-RBD Rho-binding domain of rhotekin
Rock hoA-dependent kinase
S19 Serine 19
SDS odium dodecyl sulfate
SMC mooth muscle cells