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Local Control of Human Umbilical Vessel Tone LOCAL CONTROL OF HUMAN UMBILICAL VESSEL TONE. Thesis submitted to the University of London for the degree of Doctor of Philosophy in the Faculty of Science. by Anita-Jane Sexton B.Sc. (Hons.) Department of Anatomy and Developmental Biology University College London. ProQuest Number: 10017796 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 complete 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 10017796 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT This thesis examines the influence of the vascular endothelium on the local control of human feto-placental vascular tone and how these influences may vary with gestation. Electron microscopic studies have revealed that human umbilical vessels are devoid of nerves throughout gestation. Structural changes in the arterial and venous wall could be detected throughout gestation. The presence of a subpopulation of endothelial cells containing nitric oxide synthase (NOS)-like immunoreactivity was identified only in late pregnancy. The umbilical artery and vein, from both early and late pregnancy, did not relax to acetylcholine (ACh), adenosine 5-triphosphate (ATP), substance P (SP) and calcium ionophore A23187, known to be endothelium-dependent vasodilators, but did relax to sodium nitroprusside (SNP). Using the method of in vitro pharmacology, it was shown that 5- hydroxytryptamine (5-HT), histamine and endothclin-1 (ET-1 ) arc all vasoconstrictors in early and late pregnancy, acting independently of the endothelium in late pregnancy. 5-HT was shown to mediate constriction of the human umbilical artery and vein by an action on 5-HT^ and 5-HTo receptors. This contraction was found to be significantly more potent in early pregnancy. Evidence for endothelial cells as a source of 5-HT, histamine and ET-1 was found only in late pregnancy and is presented with electron immunocytochemical localization of these substances in the endothelial cells of the human umbilical artery and vein. A physiological role for these substances is suggested since exogenous addition of these substances had an effect on isolated vessel preparations. ATP and a,y5-methylene ATP (a,y5-MeATP) induced vasconstriction in the human umbilical artery from early and late pregnancy, acting independently of the endothelium in late pregnancy. 2-Methylthio ATP (2-MeSATP) and ATP did not initiate vasodilatation of the umbilical artery or vein from either early or late pregnancy. Receptor binding studies and autoradiography have identified the presence of P 2X"purinoceptors on the smooth muscle of umbilical arteries and veins from early and late pregnancy. The results reported here contribute to the understanding of the local control mechanisms regulating feto-placental vascular tone. Of particular interest is the fact that umbilical endothelial cells do not directly modify blood flow throughout gestation, but substances shown to be localised in umbilical endothelial cells may be released to act in an autocrine/paracrine manner. ACKNOWLEDGEMENTS I would like to thank my supervisor, Professor Geoffrey Bumstock, for his advice and encouragement during this studentship and in the preparation of this thesis. I wish to thank all those who have collaborated in this work; in particular, the excellent nursing staff of the Labour Suite, University College London, for never tiring of my persistence; Dr. L. Wong from the MRC Tissue Bank, Hammersmith Hospital for help in obtaining cords; Dr. John Kingdom for help in obtaining cords, as well as advice and friendship; Doreen Bailey and Dr. P. Bodin for their continual help and sharing of experimental tissue; Mark Turmaine, for carrying out the electron microscopy in Chapter 3; Dr. A. Loesch who performed the electron immunocytochemistry in Chapters 4 and 5; Dr. X. Bo and Dr. M. Zhao for the autoradiography in Chapter 6 ; Jane Pendjiky and Chris Syms for the excellent photographic assistance; Dr. D. Christie for all her editorial assistance; 1 would also like to thank my past and present colleagues, for help, friendship and encouragement over the last four years. 1 would like to thank my parents and grandparents for their endless support during my years as a student, and the friends who have shared the anxieties of the last four years with me. Finally, a special thank-you goes to Dr Gill Knight, for her never ending patience, time and friendship. This work was supported by the Anatomical Society of Great Britain and Ireland. CONTENTS PAGE Abstract ............................................................................................................... 2 Acknowledgements ................................................................................................. 3 Contents .............................................................................................................. 5 List of Tables and Figures ......................................................................................... 8 Publications Arising from this Thesis .................................................................... 9 List of Abbreviations .............................................................................................. 11 Preface ................................................................................................................. 13 Chapter 1 HISTORICAL INTRODUCTION 1.1 THE FETO-PLACENTAL CIRCULATION ........................... 16 1.1.1 The discovery of the placental circulation .................................. 16 1.1.2 Anatomy of the feto-placental circulation ................................ 17 1.2 LOCAL CONTROL OF FETO-PLACENTAL VASCULAR TONE ................................................................. 24 1.2.1 Prostanoids ...................................................................................... 26 1.2.2 Endothelium-derived relaxing factor ....................................... 28 1.2.3 Endothelin ..................................................................... 30 1.2.4 Atrial natriuretic peptide .............................................................. 31 1.2.5 Renin-angiotensin system ........................................................... 32 1.2.6 ............................................................................................. 1.2.7 Arachidonic acid ....................................................................... 33 1.2.8 Purines ...................................................................................... 34 1.3 ENDOTHELIUM AS A SOURCE OF VASOACTIVE SUBSTANCES ....................................................................... 35 1.4 CLINICAL IMPLICATIONS ................................................. 36 Chapter 2 METHODS AND MATERIALS 2.1 COLLECTION OF TISSUE ................................................... 39 2.2 ELECTRON MICROSCOPY ................................................. 39 2.3 IN VITRO PHARMACOLOGY OF ISOLATED BLOOD VESSELS ............................................................... 40 2.3.1 Preparation of tissue ................................................................. 40 2.3.2 Application of agonists ............................................................ 40 2.3.3 Application of antagonists ...................................................... 41 2.3.4 Analysis of results .................................................................... 42 2.4 HISTOCHEMISTRY ............................................................... 42 2.4.1 Endothelial cell studies ............................................................ 42 2.4.2 Electron immunocytochemistry .............................................. 43 2.5 RADIOLIGAND BINDING STUDIES ................................ 45 2.6 AUTORADIOGRAPHY ......................................................... 45 2.6.1 Drugs ........................................................................................... 46 2.6.2 Statistical analysis ..................................................................... 46 2.7 SOURCES OF DRUGS, PEPTIDES, ANTIBODIES AND CHEMICALS ............................................................. 47 EXPERIMENTAL RESULTS Chapter 3 A Ultrastructural Study of the Developing Human Umbilical Vessels ..................................................... 49 Chapter 4 Electron Immunolabelling of Vasoactive Substances in Human Umbilical Endothelial Cells and Their Actions From Early and Late Pregnancy ......................... 75 Chapter 5 Nitric Oxide and Human Umbilical Vessels: Pharmacological and Immunohistochemical Studies ................................................................................... 100 Chapter 6 Autoradiographic and Pharmacological Evidence for the Presence of a P 2x~P*Jrinoceptor in Human Umbilical Vessels from Early and Late Pregnancy .............................................................................. 133 GENERAL DISCUSSION ................................................................................ 148 REFERENCES ..................................................................................................... 160 LIST OF TABLES AND FIGURES.
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