Expression and role of serotonergic and nicotinic acetylcholine receptors in alveolar macrophages [Elektronische Ressource] / vorgelegt von Zbigniew Mikulski

Mikulski, Fiema Zbigniew

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• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2010
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 3 Mo

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Expression and role of serotonergic and nicotinic
acetylcholine receptors in alveolar macrophages
Zbigniew Mikulski
INAUGURALDISSERTATION
zur Erlangung des Grades eines
Doktors der Humanbiologie
des Fachbereichs Medizinédition scientifique
VVB LAUFERSWEILER VERLAG der Justus-Liebig-Universität Gießen
VVB LAUFERSWEILER VERLAG
STAUFENBERGRING 15 ISBN: 978-3-8359-5533-2
D-35396 GIESSEN
Tel: [phone] Fax: -5599890
[e-mail]
www.doktorverlag.de 9 7 8 3 8 3 5 9 5 5 3 3 2
édition scientifique
VVB LAUFERSWEILER VERLAGVVB
NACHR AND 5-HTR IN ALVEOLAR MACROPHAGES ZBIGNIEW MIKULSKI Das Werk ist in allen seinen Teilen urheberrechtlich geschützt.
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édition scientifique
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Expression and role of serotonergic and nicotinic
acetylcholine receptors in alveolar macrophages




INAUGURALDISSERTATION
zur Erlangung des Grades eines
Doktors der Humanbiologie
des Fachbereichs Medizin der
Justus-Liebig-Universität Gießen





vorgelegt von



Zbigniew Mikulski
aus Augustów, Poland


Gießen 2009
Aus dem Institut für Anatomie und Zellbiologie
des Fachbereichs Medizin der Justus-Liebig-Universität Gießen
Leiter/Direktor: Prof. Dr. Wolfgang Kummer

















Gutachter: Prof. Dr. Wolfgang Kummer
Gutachter: Prof. Dr. Ardeschir Ghofrani

Tag der Disputation: 27.04.2010 I
____________________________________________________________________________
I. Table of contents
I. Table of contents......................................................................................................................I
II. List of Figures......................................................................................................................IV
III. List of Tables........... V
IV. Abbreviations.....................................................................................................................VI
1 Introduction........................................................................................................................ 1
1.1 Macrophages1
1.2 Origin of alveolar macrophages ................................................................................. 2
1.3 Function of alveolar m ............................................................................. 2
1.4 Paracrine and autocrine signaling in macrophages .................................................... 4
1.4.1 Ligand-gated ion channels 5
1.4.2 G-protein-coupled receptors...............................................................................5
2+1.5 Role of Ca in regulation of macrophage function ................................................... 7
1.5.1 Calcium homeostasis in immune cells ............................................................... 7
1.5.2 Macrophage immune functions dependent on calcium.................................... 10
1.6 The nACh receptors in macrophages ....................................................................... 11
1.6.1 Acetylcholine synthesis, transport and degradation......................................... 11
1.6.2 Nicotinic acetylcholine receptors ..................................................................... 12
1.6.3 Expression and function of nAChR in macrophages ....................................... 12
1.6.4 “Cholinergic anti-inflammatory pathway”....................................................... 13
1.7 The role of 5-HT in macrophage biology................................................................. 14
1.7.1 Synthesis and regulated release of 5-HT.......................................................... 14
1.7.2 5-HT receptors..................................................................................................15
1.8 Aim of the study....................................................................................................... 16
2 Materials and methods.....................................................................................................17
2.1 Materials................................................................................................................... 17
2.1.1 Animals............................................................................................................17
2.1.2 Equipment........................................................................................................17
2.1.3 Kits...................................................................................................................18
2.1.4 Reagents...........................................................................................................18
2.2 Methods.................................................................................................................... 21
2.2.1 Genotyping.......................................................................................................21
2.2.2 Isolation of rat alveolar macrophages .............................................................. 22 II
____________________________________________________________________________
2.2.3 Isolation of mouse alveolar macrophages ........................................................ 22
2.2.4 NR8383 cell culture ......................................................................................... 22
2.2.5 Flow cytometric analysis.................................................................................. 22
2.2.6 RNA isolation and cDNA synthesis................................................................. 23
2.2.7 RT-PCR............................................................................................................25
2.2.8 Immunofluorescence........................................................................................27
2.2.9 SDS polyacrylamide gel electrophoresis and immunoblotting for nAChR α7
and α10 subunits detection............................................................................... 29
2.2.10 Intracellular calcium concentration measurements .......................................... 31
2.2.11 Drugs................................................................................................................32
2.2.12 Dot-blot based mouse cytokine antibody array................................................ 32
2.2.13 Statistical analysis............................................................................................33
2.3 Experimental protocols33
2.3.1 Rat alveolar macrophages................................................................................33
2.3.2 Mouse alveolar m........................................................................... 33
3 Results.............................................................................................................................. 34
3.1 Expression of nAChR on freshly isolated rat alveolar macrophages....................... 34
3.1.1 Purity of isolation............................................................................................. 34
3.1.2 Expression of nAChR analyzed by RT-PCR ................................................... 34
3.1.3 Immunocytochemistry for nAChR................................................................... 35
3.2 Functional analysis of nAChR in rat alveolar macrophages .................................... 41
2+3.2.1 ATP-triggered increase in Ca derives from intracellular stores .................... 41
2+3.2.2 Nicotine modulates ATP-induced rise in [Ca ] .............................................. 41 i
3.2.3 Nicotinic modulation is not dependent on extracellular calcium..................... 44
3.3 Expression of 5-HT receptors and nAChR on freshly isolated mouse alveolar
macrophages............................................................................................................. 45
3.3.1 Purity of isolated mouse alveolar macrophages............................................... 45
3.3.2 Expression of 5-HT receptors assessed by RT-PCR........................................ 45
3.3.3 Expression of nAChR analyzed by RT-PCR ................................................... 46
3.3.4 Suitability of the 5-HT receptor antibody for immunohistochemistry.......... 47 2C
3.4 Function of 5-HT receptors and nAChR on freshly isolated mouse alveolar
macrophages 47
2+3.4.1 5-HT potently stimulates [Ca ] rise ............