Metabolic trafficking between astrocytes and neurons under hyperammonemia and manganism [Elektronische Ressource] : nitrogen and carbon metabolism / vorgelegt von Touraj Shokati

Sujets

• Gesundheit

Informations

• Publié par: universitat_bremen
• Publié le: 01 janvier 2005
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Metabolic trafficking between astrocytes
and neurons under hyperammonemia and
manganism:
Nitrogen- and Carbon metabolism


DISSERTATION
Zur Erlangung des Grades eines
Doktors der Naturwissenschaften
-Dr. rer. nat-

Dem Fachbereich Biologie/Chemie der
Universität Bremen
vorgelegt von

Touraj Shokati
Bremen
Feb. 2005

1. Gutachter: Prof. Dr. Dieter Leibfritz
2. Prof. Dr. Detmar Beyersmann




This is a present for my mother









Acknowledgements

I would like to thank my supervisor Prof. Dr. Dieter Leibfritz for the excellent experimental
conditions and his support for accomplishment of my thesis. He provided me with the
opportunity to study this interesting issue and enabled the cooperation with the University of
Montreal, Canada. I am very grateful to him for his moral and professional support during my
entire study in Bremen, Germany.

I am very thankful to Dr. Claudia Zwingmann for the careful reading and review of my
scientific publications and my thesis. During 6 months research work in Montreal, Canada,
she welcomed and supported me to orientate and to acclimate.

I also like to thank Dr. Alan Hazell from the Department of Medicine, Neuroscience Research
Unit, Hopital Saint-Luc (CHUM), University of Montreal, Quebec, Canada, for his
cooperation. I thank Dr. Hazell for providing me with the opportunity to learn and perform a
variety of molecular biological techniques.

I like to thank Johannes Stelten for assistance with NMR spectroscopic problems and his
friendly explanations.

My special thanks go to my brother Iraj Shokati for his moral support and advices in my
whole life. Without him, I would have been unable to overcome the time of my study and my
Ph.D. thesis.

Thank you to my only love, my wife Elnaz Razani, who supported me during my Ph.D. work.

Finally, I would like to thank all my colleagues in the department of chemistry for their
continuous support and pleasant ambience.


ABBREVIATIONS


LDH lactate dehydrogenase α-KG α-ketoglutarate
AAT aspartate aminotransferase LGP lateral globus pallidus
ADP adenosinediphosphate MPT mitochondrial permeability transition
ATP adenosinetriphosphate myo-Ins myo-inositol
ALAT alanine aminotransferase nd not detectable
Ala alanine ns not significant
AMP NAA N-acetyl-aspartate α-amino-3-hydroxy-5-methyl-4-
isoxazol-4-propionic acid
+ Asp aspartate NAD n icotineamide adenine dinucleotide
(oxd)
BSA bovine serum albumine NADH nicotineaminucleotide
(red)
Cho choline-containing compounds NMDA N-methyl-D-aspartate
CNS central nervous system NMR nuclear magnetic resonance
Cr creatine NTP nucleosidetriphosphate
CSF cerebrospinal fluid OAA oxaloacetate
DAG diacylglycerol P inorganic phosphate i
DMEM dulbecco´s modified eagle´s PAG phosphate activated glutaminase
medium
EDTA ethylendiaminetetraacetate PC phosphocholine
FBS fetal bovine serum PCA perchloric acid
FC frontal cortex PCr phosphocreatine
FID free induction decay PE phosphoethanolamine
FT Fourier transformation PEP phosphoenolpyruvate
GABA PEPCK phosphoenolpyruvate carboxykinase γ-aminobutyric acid
GDH glutamate dehydrogenase PK pyruvatekinase
GFAP glial fibrillary acidic protein PME phosphomonoester
Glc glucose Pro proline
Gln glutamine P/S penicillin/streptomycine
Glu ate Pyr pyruvate
GLT glutathione Tau taurine
Gly glycine TCA tricarboxylic acid
GPC glycerophosphocholine TSP trimethylsilylpropionic-2,2,3,3,-d -4
acid
GPE glycerophosphoethanolamine Val valine
GS glutamine synthetase
GTP guanosinetriphosphate
HA hyperammonemia
HE hepatic encephalopathy
HTau hypotaurine
Ile isoleucine
Lac Lactate
Content

Chapter 1

1 SUMMARY 9
1 ZUSAMMENFASSUNG 13

Chapter 2

2 INTRODUCTION 17

2.1 Nervous system 17
2.1.1 Brain cells 17
2.1.1.1 Neurons 18 2.1.1.2 Glial cells

2.1.2 Neuronal signalling 19

2.1.2.1 Neurotransmission and neurotransmitters 19
2.1.2.2 Glutamine-glutamate cycle 20
2.1.3 Brain energy metabolism 21

2.1.3.1 Introduction 21 2.1.3.2 Astrocytic and neuronal role in energy metabolism 22
+ + 2.1.3.3 Na /K -ATPase and its role in cellular energy metabolism 24
2.1.3.4 Glucose and alternative energy substrates for brain 25

2.2 Ammonia 25
2.2.1 Introduction 25 2.2.2 Ammonia intoxication and hyperammonemia (HA) 27
2.2.3 Hyperammonemia and brain energy metabolism 27
2.2.4 Hyperammonemia and cell volume 28

2.3 Manganese neurotoxicity (manganism) 29

2.3.1 Introduction 29
2.3.2 Manganism and astrocytic pathogenesis 29
2.3.3 Manganism and cerebral energy metabolism 30
2.3.4 Manganism and synaptic neurotransmission 31

2.4 Aims of the study 32
Chapter 3

3 RESULTS AND DISCUSSION 33

3.1 Effect of hyperammonemia on glial and neuronal metabolism 33

3.1.1 Introduction 33 3.1.2 Glucose and energy metabolism in cortical astrocytes and neurons 34

3.1.2.1 Alterations of amino acids glutamate and glutamine 34
13 3.1.2.2 C-isotopomer pattern in glutamine 36
3.1.2.3 Alterations of the amino acids aspartate and alanine 37
3.1.2.4 Effect of ammonia on lactate synthesis 40
3.1.2.5 Effect of ammonia on the cellular energy status 41
3.1.2.6 Alterations of intracellular osmolytes 44

3.1.3 Analysis of GS and GFAP protein expression 46

3.1.3.1 Effect of ammonia on GS expression 46
3.1.3.2 Effect of ammonia on GFAP expression 47

3.1.4 Summary 49
3.2 Impact of exogenous glutamine on glial and neuronal metabolism 50

3.2.1 Introduction 50 3.2.2 Effect of glutamine on glucose- an energy metabolism
in cortical astrocytes and neurons 51

3.2.2.1 Alterations of TCA cycle-related amino acids 51
3.2.2.2 Ammonia detoxification 55
3.2.2.3 Effect of glutamine on lactate synthesis 56
3.2.2.4 Effeine on the cellular energy status 57
3.2.2.5 Alterations of intracellular osmolytes 60

3.2.3 Analysis of astrocytic GS and GFAP protein expression 61

3.2.3.1 Effect of glutamine on GS expression 61
3.2.3.2 Effeine on GFAP expression 62

3.2.4 Summary 63

133.3 Metabolism of [U- C ] glutamine in neural cells 64 5

3.3.1 Introduction 64
3.3.2 Metabolic pathways of glutamine 65

133.3.2.1 Analysis of [U- C ]glutamine-derived isotopomers 65 5
3.3.2.2 Alterations of glutamine-derived metabolites 67

3.3.3 Energy status 74

3.3.3.1 Alteration of energy-status under hypoglycemia 74

3.3.4 Summary 76 3.4 Immunohistochemical studies on GS and LDH-1 in LGP and FC 76

3.4.1 Introduction 76
3.4.2 Effect of manganese on GS in LGP and FC 77
3.4.3 Effect of manganese on LDH-1 in LGP and FC 78
3.4.4 Summary 80

3.5 Western blot- and PCR-analysis of GS- and LDH-1 expression
in LGP and FC 80
3.5.1 Effect of manganese on GS expression in LGP and FC 80
3.5.2 Effect of maLDH-1 expression in LGP and FC 82
3.5.3 Summary 82

3.6 Conclusion and perspectives 83


4 EXPERIMENTAL METHODS 87

4.1 Cell cultures 87

4.1.1 Introduction 87
4.1.2 Primary cell cultures 88

4.1.2.1 Introduction 88
4.1.2.2 Preparation of cortical astrocytes 88
4.1.2.3 Preparation of cortical neurons (GABAergic neurons) 89
4.1.2.4 Preparation of co-cultures 91
4.1.2.5 Test of culture purity 91

4.2 Analytical Methods 92

4.2.1 Introduction 92
4.2.2 Incubation conditions 92
4.2.3 Extraction methods 92

4.2.3.1 Perchloric acid extraction (PCA) 92
4.2.3.2 Protein extraction using RIPA 93
4.2.3.3 RNA extraction using TRI reagent 94

4.2.4 Protein determination methods 95

4.2.4.1 UV/VIS spectroscopy 95
4.2.4.2 Lowry method 95