EVIDENCE of GENE EXPRESSION and ENZYME ACTIVITY By
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STEROID HYDROXYLASES IN THE RAT BRAIN: EVIDENCE OF GENE EXPRESSION AND ENZYME ACTIVITY by SCOTT M. MacKENZIE B.Sc.(Hons) © Scott M. MacKenzie, March 2000 Thesis submitted for the degree of Doctor of Philosophy to the University of Glasgow Department of Medicine and Therapeutics UNIVERSITY OF GLASGOW Western Infirmary, Glasgow 1 ProQuest Number: 11007810 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 11007810 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 G IA SG O W UNIVERSITY LIBRARY , liW ^ Abstract Aldosterone and corticosterone are the main mineralocorticoid and glucocorticoid products of the rat adrenal cortex. They share a common biosynthetic pathway until the final stage where the substrate deoxycorticosterone (DOC) is converted to either aldosterone or corticosterone by the actions of the enzymes aldosterone synthase (CYP11B2) or llp-hydroxylase (CYP11B1) respectively. These enzymes are the products of highly homologous genes whose expression, along with that of other components of the corticosteroidogenic pathway, was long thought to be confined to the adrenal cortex. In recent years, however, due to the advent of more sensitive molecular biological techniques, evidence has accumulated to suggest that this is not the case and that certain extra-adrenal tissues may be capable of autonomous aldosterone and corticosterone production. In chapter 3, RT-PCR was used to detect transcripts from the CYP11B1 and CYP11B2 genes in a number of different tissue types. The transcription of genes encoding other components of corticosteroidogenesis, such as the side-chain cleavage enzyme and adrenodoxin, was also examined. Of the tissues examined, only brain tissue was found to contain transcripts from all these genes. Chapter 4 describes attempts using an SDS-polyacrylamide gel electrophoresis and immunoblotting technique to show that these transcripts are translated within brain tissue to result in the enzymes themselves. This utilised two monoclonal antibodies raised against non-homologous regions of the rat aldosterone synthase and 11 P-hydroxylase enzymes. However, no evidence of translation in extra-adrenal tissue was obtained using this technique, despite the identification of a positive band corresponding to 11 p-hydroxylase in adrenal tissue fractions. 2 In chapter 5, the same antibodies were used to detect aldosterone synthase and 11 P-hydroxylase within extra-adrenal tissues by immunostaining methods. Using adrenal tissue sections, the two antibodies were found to be highly specific for their respective antigens and apparently free of the cross-reactivity which might be expected between such highly homologous enzymes. Positive staining was also produced in brain tissue, where the enzymes were found to colocalise within the hippocampus and the cerebellum. Although previously published work had established that steroidogenesis occurs in the glial cells of the central nervous system, strong positive staining within the Purkinje cells of the cerebellum in this study presented strong evidence for neuronal expression. Rigorous control experiments confirmed the specificity of these results. Attempts to produce specific staining within heart tissue were unsuccessful. Primary cultures of rat fetal hippocampal neurons permitted the study of CYP11B1 and CYP11B2 expression within a homogenous neuronal cell type. This work is described in chapter 6. CYP11B1 and CYP11B2 transcription and translation within these neuronal cells was demonstrated by the techniques used previously. In addition, the substrate DOC was incubated with the cells for 24-hour periods. Upon extraction and partial purification, the aldosterone and corticosterone content of the cell medium was measured by radioimmunoassay and shown to be significantly higher than that of control medium incubated in the absence of DOC. In summary, this thesis provides compelling evidence of the production of aldosterone and corticosterone within the rat central nervous system. It also includes detailed information concerning the distribution of the aldosterone synthase and 11 p- hydroxylase enzymes that produce these steroids. Finally, it demonstrates that 3 neuronal cells cultured from the fetal rat hippocampus are capable of converting DOC into their respective products. These findings could have profound implications for the diverse physiological processes that are regulated by corticosterone and aldosterone. 4 Declaration I declare that, unless specified otherwise in the text, the work presented in this thesis is my own. Scott M. MacKenzie 5 List of Contents Title Page 1 Abstract 2 Declaration 5 List of Contents 6 List of Figures 11 List of Tables 15 Dedication 16 Acknowledgements 16 Publications 17 Abbreviations 18 Corticosteroid Production In Adrenal And Extra-Adrenal Tissues: 21 A Review Introduction 21 Corticosteroid Biosynthesis 22 The corticosteroids 22 Cholesterol translocation 22 Cytochrome P450 enzymes 25 Biosynthetic pathways 26 The CYP1 IB Genes 32 The human CYP11B genes 32 The rat CYP1 IB genes 32 The CYP11B gene promoters and transcription factors 34 Regulation of Corticosteroid Secretion 39 Adrenocorticotrophic hormone (ACTH) 39 Angiotensin II (Angll) 44 Potassium 48 Neuroactive substances as agonists of adrenal function 48 Corticosteroid Action And Effects 51 Classical corticosteroid action 51 The mineralocorticoid and glucocorticoid receptors 52 Classical mineralocorticoid effects 55 Classical glucocorticoid effects 56 6 The 11 p-hydroxysteroid dehydrogenases 57 Nongenomic corticosteroid effects 59 Extra-Adrenal Corticosteroid Synthesis 61 Production of adrenal corticosteroids in the cardiovascular 61 system Corticosteroid Action In The Central Nervous System 65 Structure and general functions of the brain 65 Central mineralocorticoid and glucocorticoid receptors 67 11 P-Hydroxysteroid dehydrogenases in the brain 69 Corticosteroids and brain function 71 Central effects of corticosteroid deficiency and excess 81 Long-term potentiation and memory 84 Central corticosteroids and the hypothalamo-pituitary-adrenal 84 axis Stress, physiological disorders and ageing 86 Central effects of corticosteroids on blood pressure 89 Corticosteroid Production In The Central Nervous System 93 Materials And Methods 100 Reverse-Transcription Polymerase Chain Reaction (RT-PCR) And 100 Southern Blotting Of Steroidogenic Gene Transcripts RNA isolation 100 Analysis of isolated RNA 101 Reverse transcription 102 Oligonucleotide synthesis 103 PCR of first strand cDNA 103 Agarose gel preparation and electrophoresis 104 Southern blotting 105 Radioactive labelling of probes 106 Hybridisation and washes 106 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) And 107 Immunoblotting Antibody production 107 Tissue homogenisation 108 7 Determination of protein concentration 109 Polyacrylamide gel preparation 110 Precipitation of protein for electrophoresis 111 Protein transfer 112 Blocking and antibody incubation 112 Signal detection 113 Native PAGE and immunoblotting 114 Preparation And Immunostaining Of Cryosections 114 Preparation of cryosections 114 Immunostaining of cryosections (ABC protocol) 115 Preparation And Immunostaining Of Paraffin-Embedded Tissue 116 Sections Preparation of paraffin-embedded tissue sections 116 Deparaffinisation and rehydration of tissue sections 116 Antigen-retrieval pretreatments of paraffin-embedded tissue 117 sections Immunostaining of paraffin-embedded tissue sections (ABC 117 protocol) Immunostaining of paraffin-embedded tissue sections (CSA kit 118 protocol) Quenching of antibody binding by antigenic peptides 119 Preparation And Immunostaining Of Fetal Hippocampal Neurons 120 Preparation of fetal hippocampal neurons for immunostaining 120 Immunostaining of fetal hippocampal neurons 120 In Vitro Conversion Of 3H-DOC And DOC To Aldosterone And 121 Corticosterone By Fetal Hippocampal Neurons Preparation of fetal hippocampal neurons for steroid conversion 121 studies DNase treatment of RNA 121 Steroid conversion experiments 122 Measurement of tritiated steroid products 122 Measurement of nonradioactive steroid products 123 GCMS identification of aldosterone 123 8 Results: Reverse Transcription Polymerase Chain Reaction fRT- 125 PCR) And Southern Blotting Of Steroidogenic Gene Transcripts Introduction 125 Methods 126 Results 127 Adrenodoxin 127 CYP11A1 127 CYP11B1 128 CYP11B2 128 CYP11B3 128 Discussion 133 Results: SDS-Polvacrvlamide Gel Electrophoresis (SDS-PAGEI 139 And Immunoblotting Introduction 139 Methods 139 Results 140 Discussion 142 Results: Detection Of 11 P-Hvdroxvlase & Aldosterone Svnthase 148 In Adrenal And Extra-Adrenal Tissue By Immunostaining Introduction 148 Methods 148 Results 149 Discussion 151 Results: Corticosteroid Production Bv Primary Cultures Of Fetal 172 Rat Hippocampal Neurons Introduction 172 Methods 172 Results 173 RT-PCR 173 Immunostaining 173 Conversion of tritiated DOC to aldosterone and corticosterone 177 9 6.3.4 Conversion of unlabelled DOC to aldosterone and 177 corticosterone 6.3.5