Effects of H_1tn2O_1tn2 at rat myenteric neurones in culture [Elektronische Ressource] / by Pouokam Kamgne Ervice Vidal
120 Pages
English
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Effects of H_1tn2O_1tn2 at rat myenteric neurones in culture [Elektronische Ressource] / by Pouokam Kamgne Ervice Vidal

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120 Pages
English

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Effects of H O at rat myenteric neurones in 2 2 culture Inaugural Dissertation submitted to the Faculty of Veterinary Medicine in partial fulfilment of the requirements for the PhD Degree of the Faculties of Veterinary Medicine and Medicine of the Justus Liebig University Giessen Germany by Pouokam Kamgne Ervice Vidal from Douala, Cameroon Giessen 2009 From the Institute For Veterinary Physiology of the Justus Liebig University Giessen First Supervisor and Committee Member : Prof. Dr. Martin Diener Second Supervisor and Committee Member : Prof. Dr. Winfried Neuhuber Chairman of the oral panel : Prof. Dr. Norbert Weissmann Examiner : Prof. Dr. Stefan Arnhold stDate of the Doctoral Defense : March 1 , 2010. Pouokam K.E.V. Dedication i Dedication This thesis is dedicated to my parents Kamgne Jacques and Metchuiam Elise, my brothers and sisters Kamche Pelagie, Kamsu Sylvain, Feutseu Yves, Kouam Alliance, and Tameu Jacques, my nephew Mouam Gilles and the entire Taguieguem family. Pouokam K.E.V. Table of contents ii Table of contents Dedication.................................................................................................................................i Table of contents....................................................................................................................ii List of Figures............................

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Published 01 January 2009
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Effects of H O at rat myenteric neurones in 2 2
culture


Inaugural Dissertation
submitted to the
Faculty of Veterinary Medicine
in partial fulfilment of the requirements
for the PhD Degree
of the Faculties of Veterinary Medicine and Medicine
of the Justus Liebig University Giessen
Germany

by

Pouokam Kamgne Ervice Vidal

from

Douala, Cameroon


Giessen 2009



From the Institute For Veterinary Physiology
of the Justus Liebig University Giessen






First Supervisor and Committee Member : Prof. Dr. Martin Diener
Second Supervisor and Committee Member : Prof. Dr. Winfried Neuhuber
Chairman of the oral panel : Prof. Dr. Norbert Weissmann
Examiner : Prof. Dr. Stefan Arnhold





stDate of the Doctoral Defense : March 1 , 2010. Pouokam K.E.V. Dedication i


Dedication

This thesis is dedicated to my parents Kamgne Jacques and Metchuiam Elise, my
brothers and sisters Kamche Pelagie, Kamsu Sylvain, Feutseu Yves, Kouam Alliance,
and Tameu Jacques, my nephew Mouam Gilles and the entire Taguieguem family.

Pouokam K.E.V. Table of contents ii
Table of contents

Dedication.................................................................................................................................i
Table of contents....................................................................................................................ii
List of Figures........................................................................................................................v
List of Tables.......................................................................................................................iiiv
List of Abbreviations............................................................................................................ix

1 Introduction................................................................................................................1
1.1 Short description of the enteric nervous system................................................1
1.2 Types and functions of enteric neurones.............................................................4
1.3 Synaptic behavior of myenter.........................................................10
1.3.1 Fast excitatory postsynaptic potentials............................................................10
1.3.2 Slow excitatory postsynaptic potentials..........................................................10
1.3.3 Slow inhibitory postsynaptic potentials............................................................11
1.3.4 Presynaptic inhibition........................................................................12
1.4 Ionic channels and excitability in neurones......................................................12
1.4.1 Voltage-gated sodium channels........................................................................12
1.4.2 Potassium channel..............................................................................................16
1.4.3 Calcium channels...............................................................................................16
1.4.3.1 The voltage-gated calcium channels...............................................................17
2+1.4.3.2 Ca release channels: ryanodine receptors and IP receptors.....................17 3
1.4.3.3 Mitochondrial and nuclear ion channels.........................................................18
2+ 1.4.3.4 Regulation of Ca fluxes..................................................................................18
1.5 Enteric nervous system and gastrointestinal motility.....................................21
1.6 Aim of the study................................................................................................22 Pouokam K.E.V. Table of contents iii
2 Materials and Methods………………….………………....………...………..24
2.1 Materials………………………….................………………………………..24
2.1.1 Chemicals...............................................................................................................24
2.1.2 Antibiotics............................................................................................26
2.1.3 Enzymes.................................................................................................................27
2.1.4 Biological material..............................................................................................27
2.1.5 Buffers and solutions..........................................................................................27
2.1.5.1 Myenteric plexus culturing solutions................................................................27
2.1.5.2 Solutions for patch-clamping and imaging......................................................28
2.2 Methods.....................................................................................................................29
2.2.1 Isolation and culture of the myenteric neurones............................................29
2.2.2 Patch-clamp experiments..................................................................................32
2.2.2.1 Technique......................................................................................................….32
2.2.2.2 Isolation of sodium currents and effects of H O ..........................................35 2 2
2.2.3 Calcium imaging........................................................................................…...35
2.2.4 Statistics................................................................................................................38
3 Results.......................................................................................................39
2+3.1 Effects of H O on the membrane potential and the cytosolic Ca 2 2
Concentration..........................................................................................................39
+3.2 Inhibition of the Na currents...........................................................49
3.3 Alteration of the excitability................................................................................57
+3.4 Mediation of the H O effects on Na currents.................................................59 2 2
4 Discussion.................................................................................................................65
4.1 Mechanism of the hyperpolarization evoked by H O .................................65 2 2
2+4.1.1 Ca fluxes affected by H O ..........................................................................65 2 2
2+4.1.2 The role of Ca in the hyperpolarization....................................68
4.2 Alteration of the excitability................................................................................69 Pouokam K.E.V. Table of contents iv
4.3 Mediation of H O effects, implication of thiol groups and 2 2
role of the kinases/phosphatases system............................................................71
4.4 Functional significance of the H O effects and 2 2
pathophysiological consequences........................................................................76
4.5 Conclusion..............................................................................................80
5 Summary.................................................................................................83
6 Zusammenfassung................................................................................85
7 References...............................................................................................87
8 Declaration...........................................................................................103
9 Acknowledgements...............................................................................................104
Publications..........................................................................................106




Pouokam K.E.V. List of Figures v
List of Figures

Figure 1.1A: Schematic drawing of the enteric nervous system showing the
muscle layers and the plexuses…………………………………………3

Figure 1.1B: Drawing depicting the communication between the ENS and
the parasympathetic and the sympathetic systems……………………..3

Figure 1.2: Electrophysiological properties and classification of enteric
neurones…………………………………………………………....…..6

Figure 1.3: Pharmacological properties and classification of myenteric
neurones…………………………………………………………….…7

Figure 1. 4 Molecular structure of voltage-gated sodium channels……….……..15

Figure 1.5A: Overview of regulation of calcium fluxes in cells…………………...20

Figure 1.5B: Regulation of calcium fluxes in neurones…………………………....20

Figure 2.1: Isolation of myenteric plexus………………………………………...31

Figure 2.2: Schematic representation of the patch-clamp
technique …………………………………………………………....34

Figure 2.3: Fast sodium inward current in neurones…………………………......34

Figure 2.4: Voltage-clamp on a myenteric neurone by 30 ms pulses………....….35

Figure 2.5: Picture of myenteric neurones loaded with fura-2……………..……37 Pouokam K.E.V. List of Figures vi
2+Figure 2.6: Excitation spectra of fura-2 at a high Ca concentration
2+ and at low Ca concentration…………………...…………………………37

Figure 3.1: Hydrogen peroxide hyperpolarizes the membrane…………………...40

Figure 3.2A: Hydrogen peroxide caused an increase in the fura-2 ratio
signal at rat myenteric neurones………………...…………...…..…...42

Figure 3.2B: Effect of different concentrations of H O on the fura-2 2 2
ratio signal…………………………………………………………...42

2+Figure 3.3A: Hydrogen peroxide induces the release of Ca from internal stores
2+ and an influx of extracellular Ca …………………………………..43

Figure 3.3B: Depletion of internal stores by cyclopiazonic acid also
reduced the effect of H O ……………………………..……..……..43 2 2

Figure 3.4: Effects of H O on the fura-2 ratio in the absence of any 2 2
inhibitors, or in the combined presence of cyclopiazonic acid
3+and Gd , in the presence of ruthenium red or in the
presence of 2-APB…………….…………………………….…….…46

Figure 3.5A: The hyperpolarization evoked by H O was suppressed in the 2 2
presence of tetrapentylammonium……………………………………...48

2+ +Figure 3.5B: Inhibition of Ca -dependent K channels by paxilline prevented
the hyperpolarization………………………..……………………..….48

Figure 3.6: Fast sodium inward currents measured in the voltage-clamp
mode under control condition……………………………………….50 Pouokam K.E.V. List of Figures vii

Figure 3.7: Hydrogen peroxide inhibits the tetrodotoxin-sensitive sodium
Currents…………………………………………………………………...52

+Figure 3.8: Specificity of the H O -inhibited current for Na ……………………54 2 2

+Figure 3.9A: Effects of H O on Na currents at spherical, isolated myenteric 2 2
neurones……………………………………………………………..56

+Figure 3.9B: Effects of H O on Na currents on intact ganglionic cells after 2 2
+ 2+ blockade of K currents with Ba ……………………………...…..56

Figure 3.10 Change in action potential generation induced by H O measured 2 2
in the current-clamp mode……………………………………….....58

Figure 3.11A: A low concentration of H O failed to inhibit the inward 2 2
sodium currents …………………………………………………....61

2+Figure 3.11B: Enhancing the production of hydroxyl radical from H O by Fe 2 2
potentiated the action of a low concentration of H O ……….........61 2 2

Figure 4.1: Hypothetical mechanism underlying the inhibition of sodium
currents by H O ……………………………………………….…..81 2 2

Figure 4.2: The hypothetical “in vivo” pathophysiological consequences
of H O ………………………………………………………….…82 2 2
Pouokam K.E.V. List of Tables viii
List of Tables

Table 1.1: Overview of the main neurotransmitters in the gastrointestinal
tract and their main effects……………………………………………...9

Table 1.2: Myenteric synapses and transmitters……………………………………..11

Table 2.1: List of chemicals used…………………………………………………24

Table 2.2: List of antibiotics used in the cell culture………………………………..26

Table 2.3: List of enzymes used………………………………………………......27
.
Table 2.4: List of buffer solutions for plexus isolation and cell culture……….....27

Table 2.5: List of buffer solutions used for patch-clamp and
imaging experiments…………………………………………………..28

Table 3.1: Changes in the fura-2 ratio evoked by H O ……………………….....44 2 2

Table 3.2: Changes in sodium currents observed with different drugs…………....60