The effect of ablation and acute inhibition of plasma membrane calcium ATPase 4 (PMCA4) with a novel inhibitor on isolated mouse mesenteric resistance arterial contractility A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy (PhD) in the Faculty of Medical and Human Sciences Year of Submission: 2012 Sophronia Amabel Lewis School of Medicine Table of Contents Abstract ................................................................................................................ 13 Chapter 1 Introduction ........................................................................................ 27 1.1 Impact of cardiovascular diseases (CVD) in the UK .................................. 27 1.2 The importance of the resistance arteries in the cardiovascular system ...... 29 1.3 Regulation of arterial diameter .................................................................. 33 1.3.1 Mechanisms underlying modulation of VSM contractility; the role of calcium ……. ........................................................................................................ 33 1.3.2 Role of the endothelium in the regulation of arterial contractility ............. 37 1.3.3 Sources of NO and NO-mediated signalling in the vasculature ................. 39 1.3.4 Structural remodelling of resistance arteries ............................................. 41 1.4 Ca2+ mobilisation in VSM cells ................................................................. 43 1.4.1 Ca2+ mobilisation: Ca2+ entry across the plasma membrane of VSM cells 44 1.4.2 Ca2+ mobilisation: Ca2+ release from intracellular calcium stores within VSM cells .............................................................................................................. 49 2+ 1.5 The mechanisms present in VSM cells which reduce [Ca ]i ..................... 51 1.5.1 SR and the SR Ca2+ ATPase (SERCA) ..................................................... 51 1.5.2 The sodium-calcium exchanger (NCX) .................................................... 53 1.5.3 The sodium-potassium exchanger (NKA)-NCX coupling......................... 54 1.5.4 The mitochondria ..................................................................................... 55 1.6 Plasma membrane Ca2+ ATPase (PMCA) ................................................. 59 1.6.1 The structure of PMCA ............................................................................ 59 1.6.2 PMCA expression .................................................................................... 62 1.6.3 PMCA1 isoform ...................................................................................... 62 1.6.4 PMCA 2 and 3 isoforms........................................................................... 64 1.6.5 PMCA4 isoform ...................................................................................... 66 1.7 PMCA4 in the heart .................................................................................. 68 1.7.1 Role of PMCA4 in cardiac hypertrophy ................................................... 71 1 1.7.2 Role of PMCA4 in cardiac β-adrenergic response .................................... 72 1.7.3 Role of PMCA4 in long QT syndrome ..................................................... 75 1.8 PMCA4 in smooth muscle ........................................................................ 77 1.9 PMCA4 in the vasculature ........................................................................ 79 1.10 Aims ......................................................................................................... 84 1.11 Hypothesis ................................................................................................ 84 1.12 Objectives ................................................................................................. 84 Chapter 2 Methods .............................................................................................. 85 2.1 PMCA4 gene ablated mice ........................................................................ 85 2.2 Breeding and maintenance of the PMCA4 gene targeted mice colony ....... 85 2.3 Genotyping ............................................................................................... 86 2.3.1 DNA Extraction ....................................................................................... 86 2.3.2 Polymerase Chain Reaction (PCR) ........................................................... 87 2.3.3 Agarose gel electrophoresis ..................................................................... 89 2.4 Dissection of arterial tissue ....................................................................... 92 2.5 Histology .................................................................................................. 95 2.6 Immunohistochemistry ............................................................................. 96 2.7 Western blotting ..................................................................................... 100 2.7.1 Protein extraction ................................................................................... 100 2.7.2 Protein separation, membrane blotting, blocking and antibody probing .. 101 2.8 Functional studies on isolated mouse mesenteric resistance arteries in a pressure myograph ................................................................................................ 108 2.8.1 Drug preparations used in pressure myography experiments .................. 112 2.8.2 Assessment of contractile responses: ...................................................... 112 2.8.2.1 Effects of PMCA4 ablation on arterial contractility ................................ 113 2.8.2.2 Effects of Nitric oxide synthase (NOS) inhibition by NG-nitro- L-arginine (LNNA) on arterial contractility ........................................................................... 114 2.8.2.3 Effects of PMCA4 ablation on the passive arterial properties of isolated pressurised mouse mesenteric arteries .................................................................. 114 2.8.2.4 Effects of a novel PMCA4 specific inhibitor (AP2) on arterial contractility ...................... ....................................................................................................... 115 2 2.8.2.5 Effects of AP2, on arterial contractility in the presence of a non specific NOS or a specific nNOS inhibitor ........................................................................ 116 2.8.2.6 Does methyl-β-cyclodextrin (mβcd), modulate the effects of AP2 on mouse mesenteric arterial contractility? .......................................................................... 117 2.8.2.7 What are the effects of caloxin 1b1 (a commercially available PMCA4 inhibitor) on mesenteric arterial contractility? ...................................................... 117 2+ 2+ 2.9 Measurement of global intracellular Ca ([Ca ]i) signals in pressurised mouse mesenteric arteries ..................................................................................... 119 2.10 Quantitative analysis: .............................................................................. 122 2.10.1 Contractile responsiveness ..................................................................... 122 2.11 Structural mesenteric arterial assessments ............................................... 125 2+ 2+ 2.12 Indo-1 F400/F500 Ca emission ratio (taken as global [Ca ]i) in resting and vasoconstrictor stimulated mesenteric arteries ....................................................... 126 2.13 Statistical analysis ................................................................................... 128 Chapter 3 Results ............................................................................................... 129 3.1 Confirmation of the presence and absence of PMCA4 in tissues from PMCA4 WT (+/+) and KO (-/-) mice respectively ..................................................... 129 3.1.1 Brain tissues .......................................................................................... 129 3.1.2 Vascular tissues ..................................................................................... 131 3.2 Localisation of PMCA4 within the arterial wall ...................................... 135 3.3 Does ablation of PMCA4 modify the protein expression levels of other Ca2+-regulatory proteins in the mouse vasculature? ............................................... 138 3.4 Effects of PMCA4 ablation on arterial contractility in response to: ......... 140 3.4.1 High potassium solution (KPSS) ............................................................ 140 3.4.2 Noradrenaline (NA) ............................................................................... 146 3.4.3 Acetylcholine (ACh) .............................................................................. 146 3.4.4 Sodium nitroprusside (SNP)................................................................... 150 3.5 Effects of nitric oxide synthase (NOS) inhibition by LNNA on arterial contractility to: ...................................................................................................... 152 3.5.1 High potassium solution (KPSS) ............................................................ 152 3.5.2 Noradrenaline (NA) ............................................................................... 154 3 3.5.3 Acetylcholine (ACh) .............................................................................. 154 3.5.4 Sodium nitroprusside (SNP)..................................................................