Stimulation of voltage-dependent Ca_1hn2_1hn+ channels by NO at rat myenteric ganglia [Elektronische Ressource] / eingereicht von Mabruka Sitmo
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Stimulation of voltage-dependent Ca_1hn2_1hn+ channels by NO at rat myenteric ganglia [Elektronische Ressource] / eingereicht von Mabruka Sitmo

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124 Pages
English

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2+Stimulation of voltage-dependent Ca channels by NO at rat myenteric gangliaMABRUKA S. H. SITMO ﻮﻤﺘﻴﺳ ﺪﻤﺣ ﺢﻟﺎﺻ ﺔﻛﻭﺮﺒﻣINAUGURAL-DISSERTATIONzur Erlangung des Grades eines Dr. med. vet. beim Fachbereich Veterinärmedizinder Justus-Liebig-Universität Gießenédition scientifique VVB LAUFERSWEILER VERLAGVVB LAUFERSWEILER VERLAG ISBN 3-8359-5386-9STAUFENBERGRING 15D-35396 GIESSENTel: 0641-5599888 Fax: -5599890redaktion@doktorverlag.dewww.doktorverlag.de 9 7 8 3 8 3 5 9 5 3 8 6 4édition scientifiqueVVB LAUFERSWEILER VERLAGVVBMABRUKA SITMO STIMULATION OF VDCCs BY NO AT MYENTERIC GANGLIADas Werk ist in allen seinen Teilen urheberrechtlich geschützt. Jede Verwertung ist ohne schriftliche Zustimmung des Autors oder des Verlages unzulässig. Das gilt insbesondere für Vervielfältigungen, Übersetzungen, Mikroverfilmungen und die Einspeicherung in und Verarbeitung durch elektronische Systeme.1. Auflage 2009All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the Author or the Publishers.st1 Edition 2009© 2009 by VVB LAUFERSWEILER VERLAG, GiessenPrinted in Germany édition scientifiqueVVB LAUFERSWEILER VERLAGSTAUFENBERGRING 15, D-35396 GIESSENTel: 0641-5599888 Fax: 0641-5599890 email: redaktion@doktorverlag.dewww.doktorverlag.

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2+Stimulation of voltage-dependent Ca channels
by NO at rat myenteric ganglia
MABRUKA S. H. SITMO
ﻮﻤﺘﻴﺳ ﺪﻤﺣ ﺢﻟﺎﺻ ﺔﻛﻭﺮﺒﻣ
INAUGURAL-DISSERTATION
zur Erlangung des Grades eines
Dr. med. vet.
beim Fachbereich Veterinärmedizin
der Justus-Liebig-Universität Gießen
édition scientifique
VVB LAUFERSWEILER VERLAG
VVB LAUFERSWEILER VERLAG ISBN 3-8359-5386-9
STAUFENBERGRING 15
D-35396 GIESSEN
Tel: 0641-5599888 Fax: -5599890
redaktion@doktorverlag.de
www.doktorverlag.de 9 7 8 3 8 3 5 9 5 3 8 6 4
édition scientifique
VVB LAUFERSWEILER VERLAGVVB
MABRUKA SITMO STIMULATION OF VDCCs BY NO AT MYENTERIC GANGLIADas Werk ist in allen seinen Teilen urheberrechtlich geschützt.
Jede Verwertung ist ohne schriftliche Zustimmung des Autors
oder des Verlages unzulässig. Das gilt insbesondere für
Vervielfältigungen, Übersetzungen, Mikroverfilmungen
und die Einspeicherung in und Verarbeitung durch
elektronische Systeme.
1. Auflage 2009
All rights reserved. No part of this publication may be
reproduced, stored in a retrieval system, or transmitted,
in any form or by any means, electronic, mechanical,
photocopying, recording, or otherwise, without the prior
written permission of the Author or the Publishers.
st1 Edition 2009
© 2009 by VVB LAUFERSWEILER VERLAG, Giessen
Printed in Germany
édition scientifique
VVB LAUFERSWEILER VERLAG
STAUFENBERGRING 15, D-35396 GIESSEN
Tel: 0641-5599888 Fax: 0641-5599890
email: redaktion@doktorverlag.de
www.doktorverlag.deAus dem Institut für Veterinär-Physiologie
der Justus-Liebig-Universität Gießen

Betreuer: Prof. Dr. Martin Diener



2+Stimulation of voltage-dependent Ca channels
by NO at rat myenteric ganglia





INAUGURAL-DISSERTATION
zur Erlangung des Grades eines
Dr. med. vet.
beim Fachbereich Veterinärmedizin
der Justus-Liebig-Universität Gießen





eingereicht von




Mabruka Sitmo

Tierärztin aus Elbaida-Libyen





Gießen 2009


Mit Genehmigung des Fachbereichs Veterinärmedizin
der Justus-Liebig-Universität Gießen















Dekan: Prof. Dr. Dr. habil. Georg Baljer


Gutachter: Prof. Dr. Martin Diener
Prof. Dr. Eberhard Burkhardt







Tag der Disputation: 06.02.2009



Table of Contents

ABBREVIATIONS III-IV

1. INTRODUCTION
1.1 The myenteric plexus in the small intestine 1
1.2 Classification of myenteric neurons 3
1.3 Synaptic events in myenteric neurons 5
1.4 The communication between the enteric nervous system and
the central nervous system 9
1.5 Vagal input to the enteric nervous system of the small intestine 11
1.6 Modulation of the enteric nervous system by the central nervous system 13
1.7 Sympathetic influence on gastrointestinal function 14
1.8 Nitric oxide 16
1.9 Interstitial cells of Cajal and NO 18
1.10 Calcium signaling in myenteric neurons 20
1.11 Aim of the study 27

2. MATERIAL AND METHODS
2.1 Animals 28
2.2 Solutions 28
2.3 Tissue preparation and cell culture 30
2.4 Imaging experiments 33
2.5 Polymerase chain reaction (PCR) 40
2.6 Immunohistochemical experiments 52
2.7 Chemicals 56
2.8 Statistics 56

3. RESULTS
2+ 3.1 Effect of NO donors on the intracellular Ca concentration
in the myenteric ganglia 57
3.2 The effect of NO donors is dependent
2+ on the presence of extracellular Ca 61
2+ 3.3 Involvement of voltage-dependent Ca channels 63
3.4 Identification of NOS expression
in cultured myenteric ganglia by RT-PCR 72
3.5 Immunohistochemical staining 76

4. DISCUSSION
2+ 4.1 Action of NO on intracellular Ca concentration of rat myenteric ganglia 81
2+ 4.2 Mechanism of action of Ca channel blockers 82
4.3 Functional role of NO in the myenteric plexus 88
4.4 Nitric oxide synthases in the my91

I
5. SUMMARY 96
6. ZUSAMMENFASSUNG 98
7. REFERENCES 100
8. ACKNOWLEDGMENTS 114
9. ERKLÄRUNG 115

II
Abbreviations
ACh Acetylcholine
AP-1 Activating protein-1
AH Afterhyperpolarization
BAPTA 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid
BSA Bovine serum albumin
2+ 2+ [Ca ] Cytosolic free Ca concentration i
cAMP Cyclic adenosine 3',5'-monophosphate
CCH Carbachol
cDNA Complementary deoxyribonucleic acid
cGMP Cyclic guanosine 3',5'- monophosphate
CNS Central nervous system
CPA Cyclopiazonic acid
DAPI 4´,6-Diamidino-2-phenylindol dilactate
DEPC-water Diethylpyrocarbonat-Aqua dest.
DHP Dihydropyridine
DMEM Dulbecco´s modified Eagle´s Medium
DMSO Dimethylsulfoxide
dNTPs Deoxynucleotide triphosphates
EGTA Ethylene glycol-bis( β-aminoethyl ether)-N,N,N’,N’-
tetraacetic acid
eNOS Endothelial nitric oxide synthase
ENS Enteric nervous system
EPSPs Excitatory postsynaptic potentials
ER Endoplasmic reticulum
FCS Fetal calf serum
Fura-2/AM Fura-2-acetoxymethylester
GAPDH Glycerinaldehyde-3-phosphate-dehydrogenase
GFAP Glial fibrillary acidic protein
GI Gastrointestinal tract
HEPES N-(2-hydroxyethyl)-piperazine-N´-(2-ethanol sulfonic acid)
ICC Interstitial cells of Cajal
IgG Immunoglobuline
IGLE Intraganglionic laminar endings
IL-1 β Interleukin-1 β
IMAs Intramuscular arrays
iNOS Inducible nitric oxide synthase
IP Inositol 1,4,5-trisphosphate 3
IPANs Intrinsic primary afferent neurons
IPSPs Inhibitory post-synaptic potentials
IP R Inositol trisphosphate receptor 3
mRNA Messenger ribonucleic acid
NADPH Nicotinamide adenine dinucleotide phosphate
NANC Non-adrenergic non-cholinergic
III
NF- κB Nuclear factor κB
NO Nitric oxide
NOS Nitric oxide synthase
nNOS Neuronal nitric oxide synthase
–O Superoxide anions 2
¯ONOO Peroxynitrite anion
PAA Phenylalkylamine
PB Phosphate buffer
PBS Phosphate-buffered saline
PBS-T Triton-X-containing phoshate-buffered saline
PFA Paraformaldehyde
PGP 9.5 Protein gene product 9.5
ROS Reactive oxygene species
rpm Rotations per minute
RT-PCR Reverse transcriptase-Polymerase chain reaction
RYR Ryanodine receptor
SEM Standard error of the mean
2+SERCA Sarcoplasmic-endoplasmic reticulum Ca ATPase
SNP Sodium nitroprusside
TAE Tris-acetate/EDTA buffer
TNF- α Tumor necrosis factor- α
TNFR Tumor necrosis factor receptor
2+VDCCs Voltage-dependent Ca channels
2+VOCCs Voltage-operated Ca
V/V Volume per Volume
W/V Weight per Volume
λ Wave length
IV1. Introduction

1.1. The myenteric plexus in the small intestine
The myenteric (Auerbach's) plexus is the outer of the two major
plexuses of the enteric nervous system (ENS), which lies within the
intestinal wall controlling several functions of the gastrointestinal tract
(Costa et al. 2000, Figure 1).
The myenteric plexus is located between the longitudinal and the
circular layers of the smooth muscle that form the gut’s muscularis
externa and regulates the motility (Gershon 1981). The plexus
continues around and along the gut wall and has a connection with the
other part of the enteric nervous system, i.e. the submucous plexus,
which controls the transport of water and electrolytes across the
intestinal epithelium (Furness and Costa 1987).

1


Figure 1.1: Schematic drawing of the enteric nervous system. Modified after
Furness and Costa (1987).

Ultrastructure of enteric ganglia
The myenteric plexus consists of a network of nerve strands and small
ganglia. The enteric ganglia, unlike other autonomic ganglia, do not
contain blood vessels, connective tissue cells, or collagen fibers. The
absence of connective tissue and the close packing of neurons and glia
gives an appearance similar to the central nervous system. The ganglia
are not encapsulated, but lie in the connective tissue between the
muscle layers (Furness and Costa 1987).
The cells of the ganglia receive nutrients by diffusion from the blood
vessels within the discontinuous basal lamina surrounding the ganglia.
The neurons and their axons in the ganglion are partly surrounded by
glial cells. The glial cells, which are histologically characterized by
2