Signal Crosstalk between Calcium-Sensing and Prostanoid Receptors and its Role in Parathyroid Hormone Secretion A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy (PhD) in the Faculty of Biology, Medicine and Health 2017 Halah Tariq Albar School of Medical Sciences Division of Diabetes, Endocrinology and Gastroenterology Table of Contents LIST OF FIGURES .................................................................................................... 9 LIST OF TABLES .................................................................................................... 12 LIST OF ABBREVIATIONS .................................................................................. 13 ABSTRACT ............................................................................................................... 17 DECLARATION ...................................................................................................... 18 COPYRIGHT STATEMENT .................................................................................. 19 ACKNOWLEDGEMENT ........................................................................................ 20 CHAPTER 1 .............................................................................................................. 21 Introduction ............................................................................................................... 21 1.1 Introduction ................................................................................................... 22 1.2 Physiology of Calcium homeostasis ............................................................. 22 1.3 Calcium distribution between kidney, bone, intestine and extracellular fluid (ECF) ............................................................................................................ 23 1.3.1 Intestinal calcium absorption ................................................................. 23 1.3.2 Renal calcium reabsorption ................................................................... 25 1.3.3 Skeletal mineral storage ......................................................................... 26 1.4 Endocrine control of serum calcium concentration ...................................... 27 1.4.1 Parathyroid hormone ............................................................................. 27 1.4.2 Vitamin D .............................................................................................. 27 1.4.3 Calcitonin ............................................................................................... 28 1.5 Parathyroid hormone .................................................................................... 29 1.5.1 Parathyroid hormone function ............................................................... 29 1.5.2 Regulation of parathyroid hormone secretion ....................................... 30 1.5.3 Diseases of parathyroid hormone secretory dysfunction: primary and secondary hyperparathyroidism ............................................................. 31 1.6 Calcium-sensing receptor ............................................................................. 33 1.6.1 Structure ................................................................................................. 33 1.6.2 CaR pharmacology ................................................................................ 35 1.6.3 Diseases resulting from CaR mutation and dysfunction ....................... 37 1.6.4 CaR signalling ....................................................................................... 37 1.6.5 Signal bias and CaR ............................................................................... 39 1.7 Potential Gαs-coupled GPCRs signalling involved in the regulation of PTH secretion ........................................................................................................ 41 2 1.7.1 Prostanoid receptors .............................................................................. 41 1.7.1.1 Prostanoid synthesis and function ................................................. 41 1.7.1.2 Prostanoid receptors signalling ...................................................... 41 1.7.1.3 Prostanoid receptors pharmacology .............................................. 42 1.7.1.4 Prostanoids in parathyroid gland ................................................... 42 1.7.2 Histamine receptors ............................................................................... 44 1.7.2.1 Histamine synthesis and function .................................................. 44 1.7.2.2 Histamine receptors signalling ...................................................... 44 1.7.2.3 Histamine signalling in parathyroid gland .................................... 45 1.7.3 B-adrenergic receptors ........................................................................... 46 1.7.4 Calcitonin receptor-like receptor (CALCRL) ........................................ 47 1.8 Aims and objectives ...................................................................................... 47 CHAPTER 2 .............................................................................................................. 49 Materials & Methods ................................................................................................ 49 2.1 Materials ....................................................................................................... 50 2.2 Bovine parathyroid cell membrane lysate preparation ................................. 51 2.3 Immunoblotting ............................................................................................ 51 2.4 Immunoblot stripping ................................................................................... 52 2.5 DNA transformation into competent cells .................................................... 53 2.6 Cell culture .................................................................................................... 53 2.7 Transient cell transfection ............................................................................. 53 2.8 Cell assay and lysates preparation ................................................................ 54 2.9 Intracellular calcium imaging ....................................................................... 54 2.10 Total RNA extraction ................................................................................... 55 2.11 Reverse transcription polymerase chain reaction (RT-PCR) ........................ 55 2.12 Immunofluorescence ..................................................................................... 57 2.13 Fluorescence resonance energy transfer (FRET) .......................................... 57 2.14 Statistical analysis ......................................................................................... 59 CHAPTER 3 .............................................................................................................. 60 Identification of Candidate Components of an Intrinsically-Stimulated Parathyroid Hormone Secretion Mechanism ......................................................... 60 3.1 Introduction ................................................................................................... 61 3.2 Materials and Methods ................................................................................. 61 3.3 Results ........................................................................................................... 61 3 3.3.1 Expression of GPCRs and related proteins in bovine and murine PT gland as revealed by gene microarray analysis ..................................... 61 3.3.2 EP4-receptor expression in bovine parathyroid gland ........................... 64 3.3.3 EP4-receptor transfection in CaR-HEK cells ......................................... 66 3.3.4 PGE synthase 1 and 2 expression in bovine parathyroid gland ............. 68 2+ 2+ 3.3.5 PGE2 enhances CaR-induced Ca i mobilisation at subthreshold Ca o concentration in CaR-HEK cells ........................................................... 69 3.3.6 CALCRL expression in bovine PT gland tissue .................................... 71 3.3.7 Other potential cAMP modulating protein expressed in bovine parathyroid gland ................................................................................... 72 3.3.8 CALCRL transfection in CaR-HEK cells ............................................. 74 2+ 3.3.9 Effect of CGRP addition during CaR-induced Ca i mobilisation ........ 75 2+ 3.3.10 Effect of CGRP on CaR-induced Ca i mobilisation as a function of extracellular calcium concentration ....................................................... 77 3.3.11 Secondary anti-goat antibody cross-react with bovine parathyroid particulate fractions ............................................................................... 79 3.3.12 Identification of certain Gαs-coupled GPCRs in murine PT gland 2+ preincubated in high, normal or low Ca o as revealed by gene microarray analysis ................................................................................ 80 3.4 Discussion ..................................................................................................... 83 3.4.1 Identification of candidate intrinsic regulators of PTH secretion .......... 83 3.4.2 EP4-receptor expression in bovine parathyroid gland ........................... 83 3.4.3 EP4-receptor transfection in CaR-HEK cells ......................................... 84 3.4.4 PGE synthase 1 and 2 expression in bovine parathyroid gland