Tyrosine Kinase Pathways, Smooth Muscle Function and Inos Induction
Total Page:16
File Type:pdf, Size:1020Kb
University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies Legacy Theses 1997 Tyrosine kinase pathways, smooth muscle function and iNOS induction Zheng, Xilong Zheng, X. (1997). Tyrosine kinase pathways, smooth muscle function and iNOS induction (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/17957 http://hdl.handle.net/1880/26938 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca THE UNIVERSITY OF CALGARY TYROSINE KINASE PATHWAYS, SMOOTEI MUSCLE FUNCTION AND iNOS INDUCTION by Xiiong Zheng A DISSERTATION SUBMKTED TO TEE FACULTY OF GRADUATE STUDiES IN PARTIAL FULE*ILMENT OF THE REQUIREMENTS FOR TEE DEGREE OF DOCTOR OF PEILOSOPHY DEPARTMENT OF MEDICAL SCIENCE CALGARY, ALBERTA JUNE, 1997 0Xüong Zheng 1997 IYPIIOMI uurary OIUIIUU I~UUI IOUUI I~JU 1*1 oiCanada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Weilington Street 395, nie Wellington Ottawa ON KlA ON4 Ottawa ON K1A ON4 Canada Canada The author has granted a non- L'auteur a accordé une Licence non exclusive licence ailowing the exclusive permettant a la National Library of Canada to Bibliothèque nationafe du Canada de reproduce, loan, distriiute or sell reproduire, prêter, distniuer ou copies of this thesis in microfonn, vendre des copies de cette thése sous paper or electronic formats. la forme de rnicrofiche/fh, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni Ia thèse ni des extraits substantiels may be printed or othewise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation- ABSTRACT The role(s) of tyrosine kinase (TYK) pathways in both a conirade response and the induction of induciile nitric oxide synthase (iNOS)have been studied in vasniIar and gastric smooth muscle (SM) tissue. In guinea pig (GP) gastric Iongituduial muscle (LM), contractions caused by epidermal growth factor-urogastrone (EGF), ethanol and a thrombin receptor activating peptide (PAR@) were found to share signalhg pathway enzymes, including TM(, diacylglycerol lipase and cyclooxygenase. The LM responses to EGF and PAR,AP also involved MAP kinase-kinase (MEX), phosphatidyhositol3-kinase and protein kinase C, whereas the ethanol response did not. Ethanol PAR,AP and EGF- induced responses were al1 dependent on extracellular Ca2'; the EGF and PAR,AP responses involved a voltage-operated Ca2+charnel whereas the ethanol response involved a receptor-operated channel. Neither the ethano1 nor the PAR,AP contractile response were due to transactivation of the EGF receptor. In the organ bath, WOS was induced spontaneously in rat aorta (RA) and rat gastric circular muscle (RGCM)tissue, but not in RGLM.in the RA, but not in the CM, both a tyrosine kinase inhibitor (AG213) and a tyrosine phosphatase inhibitor (vanadate) blocked fùnctional iNOS induction. Whereas vanadate blocked both iNOS mRNA induction and the induction of BOS enzyme activity, AG2 13 blocked only the appearance of enzyme activity, as assesseci by an L-arginine relaxation response. In the RA but not in RGCM, iNOS induction was dependent on NF-kB activity; in the RGCM but not in the RA tissue, iNOS induction depended on the synthesis of a cycloheximide-sensitive factor. In the RGCM tissue, tNOS induction occurred in the macrophage-related cens, but not, as in the RA tissue, in the iii smooth muscle element. It was concluded that, depending on the smooth muscle phenotype, TYKs and tyrosine phosphatases may play a role in smooth muscle bction, both in tems of regulating the aaite contractile response and in tems of modulating enzyme induction as typified by NOS. There are no words in the world that can be found for me to express my appreciation to my supervisor, Dr. Morley D. Hollenberg, to whom 1 am deeply indebted. Without Dr. Hollenberg's generous support and bis excellent supervision, the work presented in this thesis wuld not have been accomplished. 1would iike to thank Drs. David L. Severson and Chris R. Triggle, my supervisory cornmittee members, for their wonderfiil suggestions and encouragement during my study. Dr. Keith A Sharkey is highly appreciated for his help with the immunohistochemical studies in this thesis. 1 would also Iike to thank Dr. Sultan Ahmad for his heIp with the molecular biological studies and Ms. Winnie Ho for her technical help with irnmunoùistochemical studies. Dr. Mike Walsh and Dr. Roger Loutzenhiser who were examiners in my candidacy examination are also highly appreciated. The input of Drs. Steven L. Pelech and Keith A Sharkey as the extemal examiners of this thesis is also gratefdiy acknowIedged. Partial sdary support that provided for the completion of this thesis work was provided by Wiam H. Davies Medical Research Scholarship. DEDICATION TO MY MOTHER TO MY WIFE, YU AND MY DAUGHTER JIE TABLE OF CONTENTS APPROVALPAGE ................................................... u.. DEDICATION ...................................................... vi TABLEOFCONTENTS .............................................vi LISTOFTABLES ............................................ xiv LISTOFFIGURES .................................................. xv LISTOFABBREVIATIONS ......................................... xxi CHAPTER ONE: INTRODUCTION ..................................... 1 1.1.0veMew .................................................. 1 1.2. Mechanisrns of smooth muscle contraction and relaxation .............. 5 1 -2.1. Caz' dependent contractile machinery ...................... 5 1.2.2. Regdation of intraceMar fiee Ca2' ....................... 6 1.2.3. Myosin regdation ..................................... 9 1.2.4. Thin filament regdation of smooth muscle ................. 12 1.2.5. Protein kinaseC ..................................... 14 1.3. The role of tyrosine kinase in signal transduction .................... 15 1.3.1.Introduction ........................................ 15 1.3 .2. Tyrosine kinase receptors .............................. 16 1-3 -3 . Activation of c-Src ................................... 18 1-3 .4 . The mitogen activated protein (MAP) kinase cascade ......... 19 1 -3.5 . Phosphatidylinositol3- (PI 3-j kinase ..................... 22 1 .3.6.G protein coupled receptors: Introduction .................. 23 1.3.7. Gpy and the MAP kinase cascade ........................ 27 1 -3.7.1. Gpy-mediated Ras dependent MAP kinase pathway ... 28 1.3.7.2. Tyrosine kinase pathways and MAPK ..............29 1 -3.7.3 . Tyrosine kinases as candidates in the activation of MAPK by G protein coupled receptor .................... 30 1-3 -7 .4 . ûther signaling moIdes ....................... 35 1.3.8. G protein Gq and the activation of MAP kinase ............. 37 1.3.9. Tyrosine kinase pathways and smooth muscle fbnction ........ 38 1.4. Signal transduction pathways and the induction of nitric oxide synthase (mas) ................................................. 41 1 .4.1. Nïm~oxide synthesis ................................. 41 viii 1.4.2. Sigaalling byN0 .................................... 42 1 .4.3. NOS isoforms and distribution .......................... 45 1.4.4. Structure of NOS .................................... 50 1.4.5. Signal transduction and ïNOS induction ...................51 1.4.5.2. NOS gene ..................................51 1.4.5.2.ïNQS mRNA stabüity .......................... 52 1 .4.5 .3 . Functional regulation of 240s ................... 52 1.4.5.4. Signalling for iNOS gene transcription .............53 1.4.5.5. Tyrosine kinase pathway and BOS induction ........ 56 1.5 . PreIiminary observations and rationale for the work described in the thesis ...................................................... 58 1.5.1.Background ........................................ 58 1.5.2. Preliary expenmeuts ............................... 60 1.5.3. Main goak of the thesis ............................... 61 CHAPTER TWO: MATERIALS AND METHODS ........................ 63 2.1. Materials and reagents ........................................ 63 2.2. Bioassay procedures ......................................... 64 2.2.1. Gastric smooth muscle preparation ....................... 64 2.2.2. Rat aorta preparation ................................. 67 2.3. Western blot analysis ......................................... 68 2.4. Preparation of tissue RN& reverse transcriptase-polymerase chah reaction analysis and nuckotide quencing ............................ 69 2.5. L-arginïne-mediateci relaxation assay ............................. 70 2.6. Immunohistochemistry ....................................... 71 CHAPTER THREE: SIGNALLING PATBWAYS FOR THE CONTRACTLE ACTION OF ETHANOL, EGF AND THROMBIN RECEPTOR ACTIVATINGPEPTIDE ........................................ 72 3.1.Introduction ............................................... 72 3.2. Ethanol-induced contractile response ............................. 75 3.2.1. Effts of tyrosine kinase inhibitors, indomethacin and inhibitors of nerve-released