A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy Ibegbu, A. 2009. The effects of Hypoxia on neuronal cell signalling. PhD thesis. Queen Margaret University. Accessed from: http://etheses.qmu.ac.uk/140/ Repository Use Policy The full-text may be used and/or reproduced, and given to third parties for personal research or study, educational or not-for-profit purposes providing that: • The full-text is not changed in any way • A full bibliographic reference is made • A hyperlink is given to the original metadata page in eResearch eResearch policies on access and re-use can be viewed on our Policies page: http://eresearch.qmu.ac.uk/policies.html http://etheses.qmu.ac.uk THE EFFECTS OF HYPOXIA ON NEURONAL CELL SIGNALLING AUGUSTINE IBEGBU A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy QUEEN MARGARET UNIVERSITY 2009 Table of Contents Contents Page No Title page i Table of contents ii-ix List of figures x-xiii List of plates xiv-xv List of tables xvi-xvii List of abbreviations and definition of terms xviii-xxii List of appendices xxiii-xxiv Declaration xxv Acknowledgements xxvi Abstract xxvii-xxix 1. CHAPTER ONE: Introduction 1 1.1. General Introduction 1-4 1.2. Signalling Overview 4-7 1.2.1. Neuronal cell signalling 8-10 1.2.2. Mechanism of signalling 10-12 1.2.3. Influence of membrane lipid on signalling 12-14 1.3. Receptors 15-17 1.3.1 G proteins 17-20 1.3.2. Heterotrimeric G proteins 21-24 1.3.3. The Gα subunit 24-26 1.3.4. The Gβγ dimer 26-27 1.3.5. The function of G protein βγ subunits 27-28 1.3.6. Modulation of heterotrimeric G proteins 28-29 1.3.7. G protein and ion channels 29-30 1.3.8 G proteins and MAPK 30-31 1.4. G proteins in disease 31-32 1.5. Second Messengers 32 1.5.1. Types of second messengers 32-33 1.5.2. Mechanisms of action of secondary messengers 33-34 ii 1.6. The nervous system 34-38 1.7. Hypoxia 38-43 1.7.1. Neuronal damage in hypoxia 43-49 1.7.2 Morphological and functional changes in hypoxia 49-51 1.7.3. Hypoxic disorders 52-53 1.8. Neuroprotection in hypoxia 53-55 1.9. Opioid receptor Agonists 56-58 1.9.1. Uses of opioids 58-59 1.9.2. Opioid receptor activation 59-62 1.10. Cannabinoids 63-65 1.10.1. Therapeutic potential of cannabinoids 66-71 1.10.2. Neuroprotection by cannabinoids 71-74 1.10.3. The role of cannabinoids in neuroinflammation 74-79 1.11. The interaction of opioids and cannabinoids 79-82 1.12. B50 Cells 82-83 1.13. Aim of the study 84 1.14. Objectives and hypotheses of the study 84 2. CHAPTER TWO: Method 85 2.1. Raising stable cells in culture 85 2.1.1. Introduction 102 2.1.2. Materials 102 2.1.3. Method 85-86 2.1.4. Method for freezing B50 cells 86 2.2. Total cell count and Viability 87 2.2.1. Introduction 87 2.2.2. Materials 87 2.2.3. Method 87-89 2.3. The effect of hypoxia on B50 neuronal cells in culture using LDH assay 89 2.3.1. Introduction 89-90 2.3.2. Materials 90 2.3.3. Method 90-91 iii 2.4. The effect of culture duration on LDH release from B50 cells in normal and hypoxic cultures 92 2.4.1. Introduction 92 2.4.2. Materials 92 2.4.3. Methods 92 2.5. Neuronal cell proliferation 92 2.5.1. Introduction 92-93 2.5.2. Materials 93 2.5.3. Method 94-95 2.6. Morphological studies 96 2.6.1. Introduction 96 2.6.2. Materials 96 2.6.3. Method 96-97 2.7. Neuronal B50 cell differentiation 97 2.7.1. Introduction 97 2.7.2. Materials 97 2.7.3. Method 98 2.8. Neuronal pattern and pattern formation 98 2.8.1. Introduction 98-99 2.8.2. Materials 99 2.8.3. Method 99 2.9. Cannabinoid (CB1) and Mu opioid receptor expression in neuronal B50 cells using semi-quantitative RT-PCR Method 99 2.9.1. Introduction 99-100 2.9.2. Extraction of total RNA 101 Introduction 101 Materials 101 Method 101-102 2.9.3. Semi-Quantitative One step RT-PCR Analysis 102 Introduction 102 Materials 103 Method 103 Programming of the thermal cycler 103 Preparation of Mater Mix 103- 104 Analysis of the RT-PCR products 104 iv 2.10. The effect of cannabinoid receptor agonists on B50 neuronal cell morphology, proliferation and viability 105 2.10.1 The effect on morphology 105 Introduction 105 2.10.2. Materials 105 2.10.3. Method 105-106 2.10.2 The effect of cannabinoid receptor agonists ± antagonists treatment on cellular proliferation in B50 cells in culture. 107 Introduction 107 Materials 107 Method 107-108 2.10.3. The effect of cannabinoid agonist ±antagonist on cellular viability using LDH release from neuronal B50 cells in culture. 109 Introduction 109 Materials 109 Method 109-113 2.11. The effect of opioid agonists on neuronal B50 cell morphology, proliferation and viability 114 2.11.1. The effect on morphology 114 Introduction 114 Materials 114 Method 114-115 2.11.2 The effect of opioid receptor agonists ± antagonists treatment on cellular proliferation in B50 cells in culture. 115 Introduction 115 Materials 115 Method 115-116 v 2.11.3 The effect of opioid agonist ± antagonist treatment on cellular viability using LDH release from B50 neuronal cells 116 Introduction 116 Materials 116 Method 117-119 2.12. The effect of DbcAMP on LDH release from B50 cells treated with cannabinoid and opioid agonists ± antagonists. 120 2.12.1. Introduction 120 2.12.2. Materials 120 2.12.3. Method 120-121 2.13. The effect of hypoxia on cAMP levels in cultured B50 cells: cAMP enzyme immunoassay (ELISA) method. 122 2.13.1. Introduction 122 2.13.2. Materials 122 2.13.3. Method 122-126 2.14 The effect of hypoxia on extracellular signal-regulated protein kinases (ERK) in cultured B50 neuronal cells: ERK1/2 enzyme immunoassay (ELISA) method 127 2.14.1. Introduction 127 2.14.2. Materials 127 2.14.3. Method 127-129 2.14.4 Method for phospho-ERK 1&2 assay in B50 cells 129 Well Arrangement 129 Assay incubation 129-130 Calculation of ERK concentration 130 2.15. Statistical Analysis 131 vi 3. CHAPTER THREE: Results 132 3.1. Cell Characteristics 133 3.1.1. B50 Cell culture 133 3.1.2. Morphological Studies 133 3.1.3. Pattern formation 134 3.1.4. Cell Count and Viability 139-143 3.1.5. B50 Cell Proliferation 144-146 3.1.6. Neuronal differentiation 146-147 3.1.7. LDH release from cultured B50 cells 148-149 3.2. Expression of cannabinoid CB1 and mu opioid (MOR) receptors in B50 neuronal cells using semi-quantitative RT-PCR 149 3.2.1 The effect of hypoxia on the expression of CB1 and MOR receptor mRNA in B50 cells 149-150 3.2.2. Semi quantitative RT-PCR of cannabinoid CB1 and MOR 150-156 3.3. Cannabinoid receptor agonist administration 157 3.3.1. The effect of cannabinoid treatment and pre-treatment on the morphology of cultured B50 cells 157-164 3.3.2. The effect of cannabinoid agonist treatment on cellular proliferation in normal and hypoxic cultured B50 cells 165-174 3.4. LDH release from neuronal B50 cells in culture using LDH assay. 175 3.4.1. LDH release from treatment with cannabinoid agonist/antagonist in B50 neuronal cells cultured in hypoxia. 175-181 3.4.2. LDH release from pre-treatment with cannabinoid agonist/antagonist in cultured B50 cells in hypoxia. 182-188 3.5. The effect of DbcAMP on LDH release from cultured B50 cells. 189 3.5.1 Effect of DbcAMP and cannabinoid agonist treatment ± antagonist on LDH release in cultured normal B50 cells. 189-192 vii 3.5.2 Effect of DbcAMP and cannabinoid agonists treatment and pre- treatment ± antagonist on LDH release in cultured B50 cells in hypoxia. 193-196 3.6 The concentration-dependent effect of a CB1 antagonist on cannabinoid agonist. 197 3.6.1 Concentration-dependent activity of a selective CB1 antagonist (AM251) on 10nM Win agonist treatment on LDH release in cultured B50 cells in hypoxia. 197-199 3.6.2 Concentration-dependent activity of a selective CB1 antagonist (AM251) on 10nM Win pre-treatment using LDH release in cultured B50 cells in hypoxia 200-202 3.7 Opioid receptor agonist administration 203 3.7.1. The effect of opioid treatment on the morphology of cultured B50 cells. 203-212 3.7.2. The effect of opioid agonist on cellular proliferation in normal and hypoxic cultured B50 cells. 213-220 3.8 Opioid receptor agonist effect on LDH 221 3.8.1 LDH release from treatment with opioid agonist ± antagonist in B50 neuronal cells cultured in hypoxia 221-224 3.8.2 LDH Release from opioid agonist and antagonist treated and pre- treated B50 cells cultured in hypoxia. 225-228 3.8.3. LDH Release from normal cultured B50 cells treated and pre- treated with opioid agonists. 229-231 3.9. Effect of hypoxia on signalling molecules 232 viii 3.9.1. The effect of hypoxia on cAMP levels in B50 cells treated with cannabinoid agonists 232-234 3.9.2.