N b'tr .q)_ GENE EXPRESSION OF NERVE GROWTH FACTOR IN THE DEVELOPING SPONTANEOUSLY HYPERTENSIVE RAT A Thesis submitted to the University of Adelaide in fulfrlment of the requirements for the degree of Doctor of Philosophy ln The Department of Clinical and Experimental Pharmacology, University of Adelaide by Patrick Helmer James Falckh B.Sc.(Hons.) February 1992 LIST OF PUBLICATTONS IN SUPPORT OF THTS THESIS Falckh PHJ, Irvine R, Rush R, Bridges D and Head RJ, 1989, Gene Expression of Nerve Growth Factor in the Mouse Salivary Gland, Clin. Exp. Pharmacol. Physiol., (Suppl. 16); Falckh PHJ, Rush R, Bridges D and Head RJ, 1990, Expression of Nerve Growth Factor (NGÐ and NGF-mRNA, Blood Vessels, YoL27(l):36: Falckh PHJ, Harkin LA and Head RJ, Expression of NGF in the Hea¡t and Kidney in the Developing Spontaneously Hypertensive Rat, Clin. Exp. Pharmacol Physiol., (in press); Falckh PHJ, Harkin LA and Head RJ, Resistance Vessel Gene Expression of Nerve Growth Factor is Elevated in Young Sponøneously Hypertensive Rats, Hypertension, (submitted 199 1) ll ACKNOWLEDGMENTS There are so many individuals to whom I would like to express my sincere gratitu"de for their help and support during my PhD. candidature that the list is far too long to include here, however, there are several who I feel I must mention. Professor Richard John Head, not only provided me the opportunity to conduct my studies at the CSIRO, Division of Human Nutrition, but arranged a scholarship with CSIRO, and provided the impetus of the project. Thanlcs to Mark Mano for hís friendship, discussions, support and technical adt,i¡B over the last 5 years and to many of the staff at CSIRO who provided technícal help and friendship especíally Roger King, Colin Chandler, Yvette Schaeffer and lan Pannent. David Courøge for his uncanny ability to "pull-a-rat-ont-of-a-hat", his wasted weekends and forbearance of the extensive breeding program I required over the years. Kath llles and Pat Nayda for their advíce, friendshíp and the annoyance I must have caused wíth interuuptions to have my thesis, and drafts, printed. Diana Bridges for her technical expertise in conducting the NGF determinations and Professor Robert (Bob) Rush for allowing the use of his laboratory for this work. My most sincerest thanlcs to Dr Mavis Abbey, CSIRO, Divisíon of Hwnan Nutrition, for her expert discussiotts and assistance in understanding molecular biology, her willingness to guide a confused mínd and the "lost deys" of reading my thesis. To many members of the Department for theír friendship, stimulating discussions and elbow-bending especially Rod lrvine, Gordon Crabb and Julie Jonsson. To Sotiria Bexis who has kept me sane (?) throughout my postgrad years with her unwavering friendship and loyalty,for the hours and days I "bent her ears",for the discussions we have shared and to whom the completion of this thesis is attributed for never letting me quit; I owe my greatest thanks and appreciatíon. Finally to my wift, Heather, and her family for their encouragement and support during the dfficult months of writing the thesis; especially for my bad moods. ThIs thesis is dedicated to my mother, who unfortunately did not see me complete my Universiry studies, but whom I love and miss greatly. lu ABSTRACT Hypemoradrenergic innervation of the vasculature is a recognised as a key characteristic in the spontaneously hypertensive rat (SHR), a genetic animal model of hypertension. Nerve growth factor (NGF), a protein essential for the development and maintenance of sympathetic innervation, has been shown to be elevated in the young SHR when compared to the normotensive conEol, the Wistar-Kyoto (WKY) rat. NGF concentrations have been shown to correlate with gene expression for the protein and with the degree of sympathetic innervation in many species. The NGFmRNA levels in cardiovascular tissue of neonatal, and developing, SHR and WKY rats were investigated to determine if there is a link between the gene expression of NGF and the pattern of abnormal sympathetic innervation in this model. The reliability of the cDNA probe used for the determination of NGFmRNA concentrations was initially verified by investigating NGFmRNA concentrations in the submaxillary salivary gland of the mouse after pharmacological manipulation. Mesenteric and caudal arteries of the SHR were found to contain significantly elevated levels of NGFmRNA (> 5 fold) consistent with the hypernoradrenergic state reported for these blood vessels. The kidney of the SHR also displayed an elevaæd NGFmRNA production, consistent with the elevated innervation reported for this tissue. Cardiac and aortic tissues, which do not exhibit hypernoradrenergic innervation in the SHR, displayed low levels of NGFmRNA which were generally similar to levels seen in cardiac and aortic tissues from V/KY rats. In the hypernoradrenergically lv innervared tissues the NGFmRNA levels were elevated as early as 2 days of age and the elevated level sustained for 6 weeks. The laner period corresponds to the normal time course for sympathetic innervation of the vasculature. It is proposed that the elevated degree of innervation seen in resistance vessels and organs (for example in the kidney) is due, in part, to a sustained elevation of NGFmRNA and that the elevated NGFmRNA is present at bifih. The findings also provide a rational basis for the elevated noradrenaline (NA) content of vessels in the SHR, the larger releasable pool of NA in the SHR and the elevated levels of NGF peptide in vessels from the SHR. The results provide sufficient stimulus for examination of a role of NGF in the initiation of hypertension in the SHR. v ABBREVIATIONS USED IN THIS THESIS o wall sÍess 32P 32Phosphorus 6-OHDA 6-hydroxydopamine Ad adrenalin Azso absorbance at 750nm ABS Australian Bureau of Statistics BC back cross BC2 second generation back cross bp base pairs BP blood pressure BSA bovine serum albumin cDNA complemenøry DNA cm centimeüe CNS cennal nervous system cpm counts per minute CsCl cesium chloride oc degrees celsius DPH dopamine p-hydroxylase dATP 2'-deoxyadenosine triphosphate DBP diastolic blood pressure dCTP 2' -deoxy cytidine triph o sph ate DEPC diethyl pyrocarbonate vi dGTP 2'-deoxyguanosine triphosphate DNA deoxyribonucleic acid dNTP deoxyribonucleotide triphosphates DOCA deoxycorticosterone acetate DR Dahl salt-resistant strain DS Dahl salt-sensitive strain dTTP 2'-deoxythymidine triphosphate ECso concentration required to produce 50 percent of the maximum response ECD elecEochemical detection EDTA ethylenediameteEa acetic acid EtBr ethidium bromide Fl first generation progeny F2 second generation progeny g grams I gravity unis GH Genetically hypertensive strain H/BW heart to body weight ratio HPLC high performance liquid chomatography hrs hours kb kilo base pairs LH Lyon hypertensive strain LL Lyon low blood pressure strain LN Lyon normotensive strain M moles per litre mg milligrams vll MHS Milan hypertensive strain min mlnute rnl millilitre Inm millimetres mM millimoles per liue mmHg millimetres of mercury MNS Milan normotensive strain MOPS 3-[N-morpholino] propane sulfonic acid NA noradrenaline NaOH sodium hydroxide ng nanogT¿un NGF nerve growth factor NGFrnRNA NGF messenger RNA NGF-R-mRNA NGF receptor m€ssenger RNA NGF-Rt NGF receptor NHF National Heart Foundation nm nanometre P Eansmural pressure PCA perchloric acid pmol picomoles r radius RNA ribonucleic acid rpm revolutions per minute SBH Sabra hypertensive strain SBN Sabra normotensive strain SBP systolic blood pressure viii SDW sterile distilled water sec seconds SEM standa¡d error of the mean SHR Spontaneously Hyperten sive Rat SHRSP SHR-stroke prone strain SMC smooth muscle cells SNA sympathetic nervous activity SNS sympathetic nervous system Soln 4 homogenising solution Soln D denaturing solution SSC standa¡d sodium/citrate buffer TAE tris-acetateÆDTA buffer l¡g mrcrogram pl microlitre ¡rM micromoles per litre V volts VSM vascular smooth muscle w wall thickness WKY Wistar Kyoto Rat x multiply lx CONTENTS DECLARATION LIST OF PUBLICATIONS IN SUPPORT u ACKNOWLEDGMENTS iï ABSTRACT iv ABBREVIATIONS vi CONTENTS x CHAPTER I : GENERAL INTRODUCTION I I.l Hypertension I I.1.1 Definition 1 1.1.2 Aetiology of Essential Hypertension 4 I.1.3 Genetic Hypertension 5 I.1.4 Sympathetíc Nervous System 6 I.I.5 Structural Changes 1 3 1.2 Animal Models of Hypertension l5 I.2.1 Spontaneously Hypertensive Rat 16 l.2.l.l Hypernoradrenergic Innervation 19 1.2.1.2 Structural Changes 23 I.3 Nerve Growth Factor 25 1.4 Introduction Summary 35 I.5 Aims 36 x CHAPTER II : GENERAL METHODS 37 II.] Glassware and Solutions 37 ll^z Gel Electrophoresis 37 Il.2.l Equipment 38 1L2.2 RNA Electrophoresis 38 11.2.2.1 Buffers 39 11.2.2.2 Gel Preparation 39 11.2.2.3 Sample Preparations q 11.2.2.4 Visualísation û II.2.3 DNAElectrophoresis 43 11.2.3.I Buffers 43 11.2.3.2 Gel Preparatíon 43 11.2.3.3 Sample Prepøratiort M 11.2.3.4 Visualisation M II.3 NorthernTransfers 45 II.4 Slot Blots 45 ll.4.l Sample Preparation 46 I1.4,2 Sample Application 46 II.5 The cDNA Probe 47 il.5.1 NGFrr¡RNA Probe Production 4t II.6 Radiolabelling of the cDNA Probe 52 II.6.1 NickTranslation 52 lL7 Hybridisation of Nitrocellulose Filters 56 lI.7.l Prehybridisation 56 1I.7.2 Hybridisation 57 1I.7.3 Stríngency Washes 59 II.8 Autoradiography 59 II.9 DNA Determinations 60 u.r0 Protein Determinations 60 xl CHAPTER III : ISOLATION OF RNA USING GUANIDINIUM- PHENOL.CHLOROFORM METHODOLOGIES 62 IfI.l Introduction 62 III.2 Isolation of RNA 63 III.2.1 The Chomczynski-Sacchi Method 63 lll.2.l.l