Angiotension receptors subtypes in the rabbit pulmonary artery

ANGIOTENSIN RECEPTOR SUBTYPES IN THE RABBIT PULMONARY ARTERY LISA TAN MAY YIN BSc (Pharm) (Hons) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLGOY NATIONAL UNIVERSITY OF SINGAPORE 2003 ACKNOWLEDGEMENTS Many people have contributed to my graduate education, as friends, colleagues and teachers. First and foremost, I would like to express my thanks to my supervisor, Associate Professor Sim Meng Kwoon, for encouraging me to embark on this journey. His invaluable insights, guidance and support have helped me to mature as a student and as a researcher. I was also very fortunate to be acquainted with Dr John W. Ferkany and Dr Terry Kenakin who, as experts in their fields, have struck me with their humility and helpfulness. I am indebted to their kindness, encouragement and generosity in sharing their scientific expertise and extensive experience with me. In my department, I was surrounded by knowledgeable and friendly people who did not hesitate to show me how to operate equipment or loan me materials that were not available in my laboratory. My thanks to Min Le, Wu Jian, Wen Qiang, Woei Shin, Wee Lee, Siew Lan, Xiao Guang, Mr Ang, Annie and Peter for their friendship and kind help. For their financial support I would like to thank the National University of Singapore for the research scholarship. Finally, I would like to thank those closest to me, especially my partner, for their absolute confidence in me and for providing me with the strength to persevere through the difficult times. Their presence, patience, understanding and love helped make the completion of my graduate work possible. ii TABLE OF CONTENTS PAGE List of publications and poster presentations List of figures List of tables List of abbreviations vi vii ix x Summary . Chapter 1: General Introduction 1.1 Introduction 1.2 The Renin Angiotensin System (RAS) - Production of Angiotensin II and Other Bioactive Angiotensin Peptides 1.2.1 Systemic and tissue RAS 1.2.2 Bioactive angiotensin fragments 1.2.3 Angiotensin III 1.2.4 Angiotensin IV 1.2.5 Angiotensin (1-7) 1.2.6 Des-Asp-angiotensin I 1.3 Angiotensin Receptors 1.3.1 Distribution in vasculature 1.3.2 AT1 receptor 1.3.3 AT2 receptor 1.3.4 AT3 receptor 1.3.5 AT4 receptor 1.4 AT1 Signaling Pathways in the Vasculature 1.4.1 Stimulation of phospholipase C and inositol triphosphate signaling 1.4.2 Increase in intracellular calcium 1.4.3 Regulation of smooth muscle contraction and relaxation 1.4.4 Activation of protein kinase C and intracellular alkalinization 1.4.5 Activation of Src family kinases 1.4.6 Phospholipase A2 activation and arachidonic acid metabolism 1.4.7 Phospholipase D activation 1.4.8 Modulation of cyclic nucleotides 1.4.9 Tyrosine kinase phosphorylation 1.5 Functional and Structural Complexity of Signal Transduction via GPCRs 1.6 Role of RAS in Primary Pulmonary Hypertension (PPH) 1.7 Conclusion 7 9 11 12 15 16 18 18 19 21 22 23 23 25 26 26 29 31 31 35 36 37 39 42 44 iii TABLE OF CONTENTS PAGE Chapter 2: Functional Studies on Isolated Rabbit Pulmonary Trunk and Artery 2.1 2.2 Introduction Materials and Methods 2.2.1 Isolation of rabbit pulmonary trunk and artery 2.2.2 Preparation of pulmonary trunk and artery 2.2.3 Direct actions of angiotensin peptides 2.2.4 Effects of angiotensin peptides on pre-contracted trunk and artery strips 2.2.5 Effects of receptor antagonists and enzyme inhibitors 2.2.6 Structure-activity relationship of angiotensin IV 2.2.7 Expression of results 2.2.8 Statistical analysis 2.3 Results 2.3.1 Direct actions of angiotensin peptides 2.3.2 Effects of angiotensin peptides on pre-contracted trunk and artery strips 2.3.3 Effects of receptor antagonists and enzyme inhibitors 2.3.4 Structure-activity relationship of angiotensin IV 2.4 Discussion Chapter 3: Receptor Binding Studies 3.1 3.2 Introduction Materials and Methods 3.2.1 Membrane preparation 3.2.2 125I-Sar1-Ile8-angiotensin II competition binding experiments 3.2.3 125I-angiotensin IV association binding experiments 3.2.4 125I-angiotensin IV competition binding experiments 3.2.5 Data analysis 3.2.6 Statistical analysis 3.2.7 Drugs and reagents 3.3 Results 3.4 Discussion 45 45 46 46 47 48 48 49 50 50 51 51 51 53 53 56 56 62 62 63 63 64 65 66 66 67 67 68 72 iv TABLE OF CONTENTS PAGE Chapter 4: RNA Studies 4.1 4.2 4.3 4.4 4.5 Introduction Rationale for Experimental Design Materials and Methods 4.3.1 Isolation of primary vascular smooth muscle cells (VSMCs) – primary explant technique 4.3.2 Cell culture 4.3.3 Isolation and purification of RNA 4.3.4 Generation of probes for Northern Blot 4.3.4.1 Primers 4.3.4.2 RT-PCR and DNA cloning 4.3.5 Northern blot Results Discussion Chapter 5: Signal Transduction Studies 5.1 5.2 Introduction Rationale for Experimental Design 5.2.1 Prostaglandin studies 5.2.2 Inositol triphosphate studies 5.3 Materials and Methods 5.3.1 Prostaglandin studies 5.3.1.1 Cell culture 5.3.1.2 Measurement of PGE2 and 6-ketoPGF1α release 5.3.1.3 Expression of results and statistical analysis 5.3.2 Inositol triphosphate studies 5.3.2.1 Preparation of tissue 5.3.2.2 Agonist reaction 5.3.2.3 IP3-binding assay 5.4 Results 5.4.1 Prostaglandin studies 5.4.2 Inositol triphosphate studies 5.5 Discussion 78 78 79 81 81 82 83 85 85 85 87 88 88 93 93 95 95 96 97 97 97 98 98 99 99 99 100 101 101 105 105 Chapter 6: General Discussion 117 References 125 v LIST OF PUBLICATIONS AND CONFERENCE PAPERS Lisa MY Tan and MK Sim Actions of angiotensin peptides on the rabbit pulmonary artery. Life Sciences, 2000; 66(19): 1839-1847. Lisa MY Tan and MK Sim Actions of angiotensin peptides on the rabbit pulmonary artery. International Forum on Angiotensin II Receptor Antagonism, 27-30 January 1999, MonteCarlo vi LIST OF FIGURES TITLE FIGURE PAGE 1.1 Pulmonary trunk and artery 1.2 Metabolism of angiotensinogen 10 1.3 Angiotensin AT1 receptor-mediated signaling pathways in vascular smooth muscle cells 24 1.4 Regulation of smooth muscle contraction and relaxation 28 1.5 Modulation of the Ca sensitivity of myosin by modifying the activities of MLCK and MLCP 30 1.6 Arachidonic acid metabolism 34 2.1 Direct contractile action of angiotensin II, angiotensin III and angiotensin IV on the rabbit pulmonary trunk and artery. 52 2.2 Effect of angiotensin II, angiotensin III and angiotensin IV on the noradrenaline pre-contracted pulmonary trunk and artery 54 2.3 Influence of losartan and indomethacin on responses to angiotensin II, angiotensin III and angiotensin IV in the noradrenaline-contracted pulmonary trunk and artery 55 2.4 Influence of amastatin on responsese to angiotensin II, angiotensin III and angiotensin IV in the noradrenalinecontracted pulmonary trunk and artery 57 2.5 Influence of divalinal-angiotensin IV on responses to angiotensin IV in rabbit pulmonary trunk and artery 58 3.1 125 I-Sar1-Ile8-angiotensin II competition curves in the rabbit pulmonary trunk and artery 69 3.2 125 I-angiotensin IV competition curves in the rabbit pulmonary trunk and artery 73 4.1 Nucleotide sequence of cDNA encoding a rabbit kidney cortex angiotensin II receptor. 80 vii FIGURE TITLE PAGE 4.2 AT1 receptor mRNA in rabbit pulmonary trunk and artery vascular smooth muscle cells (VSMCs) 5.1 Stimulation of PGE2 and 6-keto-PGF1α production in rabbit pulmonary trunk and artery smooth muscle cells by angiotensin II 102 5.2 Stimulation of PGE2 and 6-keto-PGF1α production in rabbit pulmonary trunk and artery smooth muscle cells by angiotensin III 103 5.3 Stimulation of PGE2 and 6-keto-PGF1α production in rabbit pulmonary trunk and artery smooth muscle cells by angiotensin IV 104 5.4 Stimulation of inositol triphosphate production in rabbit pulmonary trunk and artery tissue by angiotensin II over time 106 5.5 Stimulation of inositol triphosphate production in rabbit pulmonary trunk and artery tissue by angiotensin IV over time 107 5.6 Scheme demonstrating mechanisms whereby angiotensin II and angiotensin IV stimulation of the AT1 receptor can elicit different cellular responses. 111 89 viii LIST OF TABLES TABLE TITLE PAGE Competition binding constants for 125I-Sar1-Ile8angiotensin II and 125I-angiotensin IV binding to rabbit pulmonary trunk and artery membranes 71 Binding constants for AT4 receptors in various species and tissues (adapted from de Gasparo et al., 2000) 76 ix LIST OF ABBREVIATIONS 6-keto-PGF1α AA APA APN BSA CaM kinase cAMP cGMP DAG DAP GAP GEF GPCR HETE IP3 KCa Kd Ki LT MAPK MBS MLC20 MLCK MLCP mM NEP nM NO cNOS PA PACAP PASMCs PBS PC PDGF PG PI PIP2 PKA PKC PKG PLA2 PLC PLD 6-keto-prostaglandin F1α Arachidonic acid Aminopeptidase A Aminopeptidase N Bovine serum albumin Ca2+/Calmodulin-dependent kinase Adenosine 3’,5’-cyclic monophosphate Guanosine 3’,5’-cyclic monophosphate Diacylglycerol Dipeptidylaminopeptidase GTPase activating protein Guanosine exchange factor G protein-coupled receptor Hydroxyeicosatetraenoic acid Inositol triphosphate Ca2+-activated K+ channels Dissociation constant Inhibition constant Leukotriene Myosin-activated protein kinase Myosin binding unit Regulatory light chains of myosin Myosin light chain kinase Myosin light chain phosphatase Millimolar Neutral endopeptidase Nanomolar Nitric oxide Constitutive nitric oxide synthase Phosphatidic acid Pituitary adenylate cyclase-activating polypeptide Pulmonary artery smooth muscle cells Phosphate-buffered saline Phosphatidylcholine Platelet-derived growth factor Prostaglandin Phosphatidylinositol Phosphatidylinositol-4,5-biphosphate cAMP-dependent kinase / protein kinase A protein kinase C cGMP-dependent kinase / protein kinase G Phospholipase A2 Phospholipase C Phospholipase D x Giaid A, Saleh D: Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. 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Proc.Natl.Acad.Sci.U.S.A 1996, 93:11968-119 146 [...]... comparing the angiotensin II-, angiotensin III- and angiotensin IV-induced prostaglandin and inositol triphospate signaling in tissues of the rabbit pulmonary vasculature Angiotensin II significantly increased the production of both prostaglandin E2 and PGI2 in the rabbit pulmonary trunk and artery VSMCs This increase was inhibited by losartan but not by PD123319, implicating the role of AT1 receptors in. .. no effect Indomethacin, a cyclooxygenase inhibitor, blocked only the vasodilator response induced by angiotensin III and angiotensin IV in the trunk, indicating that vasodilator prostaglandins were probably involved in mediating this response Amastatin, a selective aminopeptidase A and N inhibitor, blocked the angiotensin III-induced vasoconstrictor and vasodilator response in the pulmonary artery and... angiotensin II and its immediate metabolites, angiotensin III, angiotensin IV, angiotensin (4-8) and angiotensin (3-7) on the regulation of pulmonary blood flow in the rabbit We investigated the actions of these angiotensin peptides on isolated endothelium-denuded pulmonary trunk and artery tissue, and characterised the angiotensin receptor subtypes in vascular smooth muscle cells isolated from the rabbit pulmonary. .. but further contracted pre-contracted pulmonary artery strips Both des-Asp-angiotensin I-induced opposing actions were inhibited by losartan, but not by PD123319; only the des-Asp-angiotensin I-induced relaxation was inhibited by indomethacin (Sim and Chai, 1996) Binding studies also revealed that the angiotensin receptor exists in two guanine nucleotide-differentiated subtypes in the same pulmonary. .. angiotensin II-induced NO release and increase in constitutive nitric oxide synthase (cNOS) activity, respectively, via the AT4 receptor in porcine pulmonary arterial endothelial cells since divalinal-angiotensin IV blocked both angiotensin II- and angiotensin IV-induced NO release and increases in cNOS activity while AT1 and AT2 blockade failed to influence these responses Interestingly, AT1 and AT4 receptors. .. from the heart to the lung Secondly, we demonstrate the contribution of bioactive angiotensin fragments, e.g., angiotensin III and angiotensin IV, to the fineregulation of pulmonary vascular control 1 In the isolated tissue study, differential responses of the endothelium-denuded sections of the rabbit pulmonary trunk (the vessel arising from the right ventricle of the heart prior to its bifurcation into... angiotensin responses in the pulmonary trunk, which receives blood directly from the right ventricle, may play a role in the protection of the delicate pulmonary capillaries from excessive systolic pressure 5 Understanding these protective mechanisms may have important therapeutic implications in the treatment of pulmonary hypertension 6 CHAPTER 1 GENERAL INTRODUCTION 1.1 Introduction The renin angiotensin... metabolized into active angiotensin peptides by enzymes acting at the two ends of the peptide (Figure 1.2) Its precursor, angiotensin I, can be similarly degraded Four angiotensin fragments are of biological interest: angiotensin III, which is obtained by deletion of the N-terminal aspartic acid from angiotensin II; angiotensin IV, which is obtained by deletion of the N-terminal arginine from angiotensin III;... Distribution in vasculature AT1 receptors are predominant in the control of angiotensin II-induced vascular functions (Sadoshima, 1998; de Gasparo et al., 2000) In the vasculature, AT1 receptors are present at high levels in smooth muscle cells and relatively low levels in the adventitia and are undetectable in the endothelium (Zhuo et al., 1998; Allen et al., 2000) Conversely, AT2 receptors predominate in the. .. converting enzyme in the lungs The existence of a local tissue RAS is evidenced by the localization of many of its components in tissues All components of the RAS, except renin, have been demonstrated to be produced in the vasculature The locally produced angiotensin II exerts paracrine and autocrine effects in the vicinity of its site of formation 1.2.2 Bioactive angiotensin fragments Angiotensin II is metabolized . from the rabbit pulmonary trunk and artery by receptor binding and Northern blot analysis. Finally, we evaluated the signal transduction pathways coupled to these receptors by measuring the. involved in mediating this response. Amastatin, a selective aminopeptidase A and N inhibitor, blocked the angiotensin III-induced vasoconstrictor and vasodilator response in the pulmonary artery. signaling in tissues of the rabbit pulmonary vasculature. Angiotensin II significantly increased the production of both prostaglandin E 2 and PGI 2 in the rabbit pulmonary trunk and artery