Response of alveolar type II pneumocytes to mechanical stimulation [Elektronische Ressource] / Giorgio Fois

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• Gesundheit

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2012
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Universität Ulm
Institut für Allgemeine Physiologie
Leitung Prof. Paul Dietl



Response of alveolar type II
pneumocytes to mechanical
stimulation



Dissertation
zur Erlangung des Doktorgrades der Humanbiologie
der Medizinischen Fakultät der Universität Ulm


Giorgio Fois
Seriate, Italien


2011

Amtierender Dekan: Prof. Dr. Thomas Wirth

1. Berichterstatter: Prof. Dr. Paul Dietl
2. Beritter: PD Dr. Petra Pandur
Tag der Promotion: 16-12-2011
To my parents and Sibilla
Table of contents
________________________________________________________________________________
Table of Contents
Table of Contents ................................................................................................................................ i
Abbreviations ..... iii
1 Introduction ............................... 1
1.1 Effects of mechanical stimulation on the lung ................................................................... 6
1.2 General mechanisms of mechanotransduction at a cellular level ... 11
1.3 The transient receptor potential channel family ............................. 17
1.4 Aims of the study ............................................................................................................. 24
2 Material and Methods ............. 25
2.1 Technical description of the devices ................ 25
2.2 Isolation of ATII cells and cultivation on silastic membranes .......................................... 33
2.3 Labeling procedures with fluorescent dyes ..................................... 35
2.4 Mechanical stimulation of the cells on the microscope .................................................. 38
2.5 Solutions and pharmacological compounds .... 42
2.6 Silencing of TRPV2 in ATII cells ......................................................................................... 43
2.7 Detection of TRPV2 with Western Blots .......................................................................... 44
2.8 Fluorescence labeling of focal adhesions, adherens junctions, TRPV2 channels and actin
cytoskeleton ................................................................. 48
2.9 Coimmunoprecipitation of TRPV2 with FAs proteins....................................................... 49
2.10 Data analysis ..................................................................................... 52
3 Results ...................................................................................................................................... 55
3.1 Characterization of the stretch devices ........... 55
3.2 Stretch induced cell injury 60
3.3 Stretch induced LB fusions ............................................................................................... 61
2+
3.4 Mechanically induced increase of [Ca ] ......................................... 62 c
2+3.5 Role of the cytoskeleton for the stretch induced [Ca ] increase ... 74 c
3.6 Temporal aspects of the stretch-induced channel gating ................................................ 79
4 Discussion ................................................................................................................................ 82
4.1 Methodological aspects of mechanical stimulation......................... 82
4.2 Stretch-induced ATII injury ............................... 86
4.3 Stretch-induced LB fusions ................................................................ 88
2+4.4 Mechanically induced increase of intracellular [Ca ] ..................... 89 c
i
Table of contents
________________________________________________________________________________
4.5 The role of the cytoskeleton for force transmission to the mechanosensitive channel . 99
5 Summary ................................................................................................................................ 102
6 References ............................. 104
7 List of figures .......................... 115
8 List of Tables ................................................................................................ 117
9 Curriculum Vitae .................... 118

ii
Abbreviations
________________________________________________________________________________
Abbreviations
- Minus/negative
% Percentage
2+[Ca ] Calcium Concentration
2+ 2+[Ca ] Cytoplasmic Ca concentration c
+ Plus/positive
± Plus-minus
® Registered trademark
° C Degrees Celsius
β2 β2-adrenergic receptor
µl Microliters
µM Micromolar
µm Micrometers
2-AG 2-arachidonylglycerol
2-APB 2-Aminoethoxydiphenyl borate
a.u. Arbitrary units
AC Adenylate cyclase
AFM Atomic force microscope
AM ester Acetoxymethyl ester
AnkR Ankyrin repeats
APS Ammonium persulfate
ARDS Acute respiratory distress syndrome
ATI Alveolar type 1 pneumocytes
ATII Alveolar type 2 pneumocytes
ATP Adenosine-5'-triphosphate
ATP-GJ blockers Combination of Suramin, Apyrase and Glycyrrhetinic acid
avg Mean
BSA Bovine serum albumin
BTS N-benzyl-p-toluene sulphonamide
C. elegans Caenorhabditis elegans
ca. Approximately
2+Ca Calcium
2+Ca Calcium entry from the extracellular space ent
2+Ca free Calcium free bath solution
iii
Abbreviations
________________________________________________________________________________
2+ 2+Ca Ca release from the intracellular stores sto
CaCl Calcium chloride 2
CaCM-PK Calcium - calmodulin-dependent protein kinase
cAMP Cyclic adenosine monophosphate
CB Composite bodies
CCD Charge-coupled device
2+ Cd Cadmium
CK Cytokeratin
cm Centimeters
2cm Square centimeters
CO Carbon dioxide 2
CSA Cell surface area
2+
Cu Copper
Cy3 Indocarbocyanine
DAGs Diacylglycerol
ddiH O Double distilled water 2
DILT Deformation Induced Lipid Trafficking
DMEM Dulbecco’s modified Eagle's medium
DNA Deoxyribonucleic acid
DPBA Diphenylboronic anhydride
DTT Dithiothreitol
e.g. Lat. Exempli gratia (For example)
ECM Extracellular matrix
EGFP Enhanced green fluorescent protein
EGFR Epidermal growth factor receptor
EGTA Ethylene glycol tetra acetic acid
ERK1/2 Extracellular-signal-regulated kinases type 1/2
ERV Expiratory reserve volume
EthD-1 Ethidium homodimer-1
F Background subtracted fluorescence intensity of fluo-4 0
F Fluorescent emission of Fura-2 excited at 340 nm 340
F Fluorescent emission of Fura-2 excited at 380 nm 380
FA Focal adhesion
FAK Focal adhesion kinases
F Fluorescent emission of background BKG
F Fluo-4 fluorescence of a cell cell
iv
Abbreviations
________________________________________________________________________________
FCS Fetal calf serum
F Fluorescence of a small area of 100 µm l
N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium
FM 1-43
dibromide
fps Frames per second
F Fluorescence increase of the all cell t
G G-proteins
g Gravitational constant
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
3+Gd Gadolinium
GdCl Gadolinium Chloride 3
GJ Gap junctions
Grb-SOS Growth factor receptor-bound protein
GTP Guanosine triphosphate
h Hours
+H Hydrogen
HBO Mercury-vapor lamp
HCl Hydrochloric acid
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
HETE Hydroxy-Eicosa Tetraenoic Acid
HMBS Hydroxymethylbilane synthase
HPETE Hydroperoxyeicosatetraenoic acid
IF Intermediate filaments
IgG Immunoglobulin G
IP Inositol trisphosphate 3
IP R IP receptor 3 3
IPF Increase pattern factor
IRV Inspiratory reserve volume
IU International units
JNK c-Jun N-terminal kinases
KCl Potassium chloride
kD Kilo Daltons
kg Kilograms
3+La Lanthanum
LaCl Lanthanum Chloride 3
lat. Latin
LB Lamellar bodies
v
Abbreviations
________________________________________________________________________________
LD objective Long distance objective
LTG Lysotracker green
M Molar
3m Cubic meters
MAPK Mitogen-activated protein kinases
mg Milligrams
2+Mg Magnesium
MgCl Magnesium chloride 2
min Minutes
mJ Millijoule
ml Milliliters
mm Millimeters
3
mm Cubic millimeters
mN Millinewton
ms Milliseconds
MscL, MscS Mechano-Sensitive channels Large and Small
MSCs Mechano sensitive channels
MVB Multivesicular bodies
MW Molecular Weight
N Number
+
Na Sodium
NaCl Sodium chloride
n Number of cells cells
ng Nanograms
n Number of isolations iso
nm Nanometers
nM Nanomolar
OEA Oleoylethanolamide
OSM-9 Osmotic avoidance abnormal family member 9
P values Probability value
P2Y2 Purinergic receptor P2Y type 2
PA Phosphatidic acid
2+Pb Lead
PBS Phosphate buffered saline
PEEP Positive end expiratory pressure
PFA Paraformaldehyde
vi
Abbreviations
________________________________________________________________________________
pH Pondus Hydrogenii (lat. pondus = weight and hydrogenium = Hydrogen)
PIP2 Phosphatidylinositol 4,5-bisphosphate
PKA