Comparative and Functional Analysis of Β-Adrenoceptors in Human Myometrium
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University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2018-01 Comparative and functional analysis of β-adrenoceptors in human myometrium Albadri, Randa G. A. Albadri, R. G. A. (2018). Comparative and functional analysis of β-adrenoceptors in human myometrium (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/5447 http://hdl.handle.net/1880/106366 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Comparative and functional analysis of β-adrenoceptors in human myometrium by Randa G. A. Albadri A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN MEDICAL SCIENCE CALGARY, ALBERTA JANUARY, 2018 © Randa G. A. Albadri 2018 Abstract There are three subtypes of the β-adrenoceptors (β1, β2 and β3). All the β-adrenoceptor subtypes couple to Gαs subunit of G protein. All the β-adrenoceptor subtypes couple mainly to Gαs subunit of G protein. β2-adrenoceptor agonists have been used clinically to suppress myometrial contractions during the management of preterm labour. β2-adrenoceptor agonists lose their effectiveness in a short time and their use causes several side effects. Evidence in the literature supports the expression of functional β2- and β3-adrenoceptors in the lower pregnant human myometrial tissues. However, no definitive understanding in terms of regional and temporal expression and function of the β-adrenoceptors in the uterus was obtained by reviewing the literature. Experiments of this work have confirmed the expression of the three β-adrenoceptor subtypes in term pregnant (upper and lower) human myometrial tissues by real-time RT-PCR analysis. Poor antibody specificity was an obstacle to determine and localize the β-adrenoceptor proteins in the myometrium by immunohistochemistry. β2- and β3-adrenoceptor agonists did not affect the expression of CRISPLD2, RGS2 and NA4A3 in primary MSM cells after 2 and 6 h of incubation. Further research must be done to determine and localize the expression of the β-adrenoceptors in the myometrium. ii Acknowledgements I would like to thank my supervisors Dr. Donna Slater and Dr. Mark Giembycz for their guidance, support and advice throughout my studies. I also would like to thank all my lab- mates for their help. I also would like to thank my committee members Dr. William Cole and Dr. Claudia Klein for their recommendations and feedback. I am also very grateful to Dr. Robert Newton and students in his lab for their assistance, advice and encouragement. I finally would like to thank my husband for being very patient, loving and understanding. iii Table of Contents Abstract …………………………………………………………………………….…… ii Acknowledgements …………………………………………………………………….. ііі Table of Contents ………………………………………………………………….....…. iv List of Tables ……………………………….………………………….………………. vii List of Figures ……………………………………………………….….….…………... viii List of Abbreviations ……………………………………………………….….….…….. x CHAPTER ONE: INTRODUCTION …………………….……….………………….…. 1 1.1 Significance ……………………..………………….………….…………………... 1 1.2 Preterm labour …………..…………………………...………...…………………... 1 1.3 The uterus and parturition ……………...………………………………………….. 2 1.4 Myometrial contraction-relaxation control ………………………………………… 5 1.5 The β-Adrenoceptors ………………………………………………………………. 6 1.6 β-adrenoceptors canonical signaling pathway ……………………………………... 7 1.6.1 Cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2) .. 8 1.6.2 Nuclear receptor subfamily 4 group A member 3 (NR4A3) ……………….. 9 1.6.3 Regulator of G-protein signaling (RGS2) …………………………………. 9 1.6.4 Interleukin-6 (IL-6) ………………………………………………………. 10 1.7 Other β-adrenoceptor signaling pathways ……...…………………………………. 11 1.8 The β2-adrenoceptor in the management of preterm labour (clinical observations) ……………………………………………………………….……….….……...… 12 1.9 β-adrenoceptor desensitization ……………...……………………….…………... 12 1.10 Systematic review: The β-adrenoceptors as targets for the management of preterm labour; what is the evidence? …………..………………………...………………. 15 1.10.1 Main question ……………………………………………………………. 15 1.10.2 Rationale …………………………………………………………………. 15 1.10.3 Aims ……………………………………………………………………… 15 1.10.4 Search terms ……………………………………………………….…....... 16 1.10.5 Search results and data extraction ………………………………………… 16 1.10.6 Key findings of systematic literature review ……….……………………. 17 iv 1.11 Hypothesis …………………………………………………………………….. 25 1.12 Aims ………………………………………………………………………….. 25 CHAPTER TWO: MATERIALS AND METHODS …………………………………… 29 2.1 Tissue collection …………………………………………….………………… 29 2.2 Primary myometrial cell culture and passage …………….…………………… 29 2.3 Cell treatments …………………………………………………………………. 31 2.4 RNA isolation …………………………………………………………………. 31 2.5 cDNA synthesis ……………………………………………………………….. 32 2.6 Primer design and verification ………………………………………………… 32 2.7 Conventional reverse transcriptase - Polymerase Chain Reaction (RT-PCR) ………………………………………………………………………………………. 33 2.8 Real-time RT-PCR …………………………………………………………….. 34 2.9 Protein isolation for western blotting …………………………………………. 35 2.10 Western blotting ………………………………………………………………. 35 2.11 Immunohistochemistry ………………………………………………………... 36 2.12 Data presentation ……………………………………………………………… 37 CHAPTER THREE: SPATIAL AND TEMPORAL EXPRESSION OF β- ADRENOCEPTOR SUBTYPES IN PREGNANT HUMAN MYOMETRUM ….……. 45 3.1 β-adrenoceptor expression in the pregnant human myometrium ……………… 45 3.2 Real-time RT-PCR efficiency and specificity ………………………………… 47 3.3 Spatial and temporal expression of β-adrenoceptor subtypes in pregnant human myometrium …………………………………………………………………... 49 3.4 Localization of β2-adrenoceptors in term non-labour and term labour upper and lower myometrial tissue by immunohistochemistry …….…………………….. 51 3.5 Optimization of β2-adrenoceptor antibody working conditions ………………. 57 3.6 Assessment of β2-adrenoceptor antibody specificity by western blotting …….. 59 3.6 Assessment of β3-adrenoceptor antibody specificity ………………………….. 61 CHAPTER FOUR: ROLE OF β-ADRENOCEPTOR AGONISTS IN GENE REGULATION IN MSM CELLS ……………………………………………………… 64 v 4.1 Expression of CRISPLD2 in the uterus ………………………………………… 64 4.2 Effect of β-adrenoceptor agonists on CRISPLD2 expression in MSM cells …... 65 4.3 Effect of β-adrenoceptor agonists on NR4A3, RGS2 and IL-6 expression in MSM cells ……………………………………………………………………… 67 4.4 Effect of the passage on the expression of ADRB1, ADRB2, and ADRB3 in MSM cells (P1-P10) …………………………………………………………… 70 4.5 ADRB2 expression in MSM cells – Effect of formoterol, isoprenaline, and forskolin treated comparing to non-treated MSM cells …………….……………..……… 72 CHAPTER FIVE: DISCUSSION ……………………………………….….………..… 74 5.1 Summary of results …………….……………………………………...………… 74 5.2 The β-adrenoceptors as targets for the management of preterm labour; what is the evidence?…………….…………………………………..……………..………… 74 5.3 Spatial and temporal expression of the β-adrenoceptors in term pregnant human myometrial tissue ………………………………………………..…….………… 76 5.4 The effect of β-adrenoceptor agonists on the expression of CRISPLD2, NR4A3, RGS2, and IL-6 in human MSM cells ……………………………………..…….. 80 5.5 Strengths and limitations ……………………………….………………..….…... 82 5.6 Future directions …………………………………………….…….……….….…. 84 REFERENCES …………………………………………………………….…..…..…… 85 APPENDIX ………………………………………………………..………………….. 101 vi List of Tables Table 1.1. The β-adrenoceptor genes …………………………………………………… 27 Table 1.2. Pharmacological properties of β-adrenoceptors …………………………….. 28 Table 2.1. Summary of patient information ……………………………………………. 39 Table 2.2. Primers used in conventional and real-time RT-PCR ……………………… 40 Table 2.3. Agonists and antagonist affinities and concentrations used in cell culture experiment …………………………………………….………..………………………. 41 Table 2.4. Primary antibodies used for immunohistochemistry and western blotting ………………………………...…………………………………………..…………….. 42 Table 2.5. Secondary antibodies used in western blotting ………………………….…... 43 Table 2.6. Staining kits used in immunohistochemistry …………………….………….. 44 vii List of Figures Figure 1.1. Different Gα subunits and their effect on smooth muscles ………………….. 4 Figure 1.2. Myometrial contraction-relaxation control …………………………….……. 5 Figure 1.3. A simplified schematic of β-adrenoceptor-mediated signaling and response ……………………………………………………………………………….……….…. 10 Figure 1.4. Schematic showing the structure of β-adrenoceptors along with a heterotrimeric G protein …………………………………………………………………….………….. 14 Figure 1.5. Data extraction flow diagram ……………………………………………..... 17 Figure 2.1. Positions of the real-time RT-PCR primers in ADRB1, ADRB2 and ADRB3 ………………………………………………………………………………………...… 33 Figure 3.1. ADRB1, ADRB2 and ADRB3 gene expression in upper and lower human myometrial tissue ………………..………………………………………..…………….. 46 Figure 3.2. Measurement of PCR efficiency …………………………………………… 47 Figure 3.3. Melting curves of ADRB1, ADRB2 and ADRB3 …………………………… 48 Figure 3.4. Expression of ADRB1, ADRB2 and ADRB3 in human myometrial tissue ………………………………………………………………………………………...… 50 Figure 3.5. Immunohistochemistry localization of β2-adrenoceptors in pregnant human myometrium ………..…………………………………………….…………………….