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C:\Documents and Settings\lproctor\LocaI Settings\Temporary Internet Files\OLK8\Copyright - thesis (2).doc M UCL REGULATION OF HAEM OXYGENASE-1 BY NITROSATIVE STRESS IN CARDIAC CELLS PATRICK NAUGHTON THESIS SUBMITTED FOR THE DEGREE OF DOCTORATE OF PHILOSOPHY IN THE UNIVERSITY OF LONDON 2005 FROM THE VASCULAR BIOLOGY UNIT, DEPARTMENT OF SURGICAL RESEARCH, NORTHWICK PARK INSTITUTE FOR MEDICAL RESEARCH, HARROW, MIDDLESEX, HA1 3UJ. UMI Number: U592173 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. Dissertation Publishing UMI U592173 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 “The body possesses the high art of wrecking and also of restoring. ” Paracelsus (1493-1541) ACKNOWLEDGEMENTS I would like to acknowledge a number of people who have made this Thesis possible. Firstly, my supervisor and Head of the Vascular Biology Unit, Dr. Roberto Motterlini, and Prof. Colin J. Green Director of the Northwick Park Institute for Medical Research, for allowing me to undertake this research work and providing me with continuous support and encouragement throughout the project. I would also like to thank current members of the Department of Surgical Research at the Northwick Park Institute for Medical Research, namely: Dr. James Clark for his technical assistance in the laboratory and advice and help in writing this Thesis; Aaron Southgate for his assistance with the formatting; Dr. Roberta Foresti for her helpful advice and instruction with the cell culture and biochemical procedures; and Hadil Abuarqob, Sandip K. Bains, Jehad Hammad, Martha Hoque, Montaser Musameh, Yasin Owais, Ashraf Sandouka and Philip Sawle, for their assistance and camaraderie in and out of the laboratory. ABSTRACT The reactive nitrogen species (RNS) nitric oxide (NO), nitroxyl anion (N0‘) and \ nitrosonium cation (NO+), modulate a myriad of biological processes. The microsomal haem oxygenases (HO-1, HO-2 and HO-3) oxidatively catabolise haem to bilirubin, carbon monoxide (CO) and ferrous iron (Fe2+). Sensitivity of the inducible isoform (HO-1) to a variety of inducers has identified HO-1 as an effective endogenous cytoprotectant against oxidative stress. Although nitrosative stimuli can enhance HO-1 expression, little is known about the biochemistry and mechanisms of this response. This Thesis examines a number of aspects related to HO-1 and nitrosative stimuli in cardiac cells, including: 1. induction by NO'; 2. the biochemistry of NO'/NO-mediated induction of HO-1; 3. identification of a possible mechanism for the activation of HO-1 by NO congeners; 4. the antinitrosative potential of bilirubin; and 5. the potential of glyceryl trinitrate (GTN), a clinically used NO donor, to activate the haem oxygenase pathway. These different aspects of HO-1 were addressed using biochemical, molecular biology and cell culture techniques. The results indicate that NO', in analogy with other RNS, is a potent inducer of haem oxygenase activity and HO-1 mRNA and protein expression. A proposed mechanism for this response is modulation of thiol groups within redox- sensitive transcription factors. An antinitrosative and HO-1 inducing capacity was identified for bilirubin and GTN, respectively. Collectively, these findings suggest that the haem oxygenase pathway can act both as a sensor to, and target of, redox based mechanisms involving RNS, and extend our knowledge on the biological function of HO-1 in response to nitrosative stress. LIST OF ABBREVIATIONS pl microlitre gM micromolar AS Angeli’s salt ' BR bilirubin BVR biliverdin reductase + O CO calcium CaM calmodulin 2-(4-carboxyphenyl)-4I4,5,5-tetramethylimidazoline-1-oxyl C-PTIO 3-oxide (potassium salt) CO carbon monoxide cGMP cyclic guanidine monophosphate DeaNO diethylamine NONOate DetaNO diethylenetriamine NONOate DMEM Dulbecco’s modified eagle’s medium DPBS Dulbecco’s phosphate buffered saline Fe3+ ferric iron Fe2+ ferrous iron GTN glyceryl trinitrate g gram(s) h hour(s) HO haem oxygenase HO-1 ,-2 and -3 haem oxygenase-1, -2 and -3 I litre(s) min minute(s) ml mililitre mM milimolar MW molecular weight n number of replicates per group NADH reduced nicotinamide adenine dinucleotide NADP oxidised-nicotinamide adenine dinucleotide phosphate NADPH reduced nicotinamide adenine dinucleotide phosphate NO nitric oxide NOS nitric oxide synthase N03‘ nitrate N02‘ nitrite NO+ nitrosonium cation NO' nitroxyl anion OD optical density P probability RNS reactive nitrogen species ROS reactive oxygen species Sec seconds S.E.M. standard error of the mean sGC soluble guanylate cyclase vs. versus xfif times gravity iii TABLE OF CONTENTS 1 INTRODUCTION ..........................................................................1 \ 1.1 Historical perspective on nitric oxide ...............................................1 1.2 Nomenclature and biochemistry of nitric oxide ..............................3 1.3 Nitroxyl anion: chemistry, sources and physiological aspects.. 6 1.4 Nitric oxide biosynthesis .................................................................. 9 1.5 The nitric oxide synthases ............................................................. 10 1.5.1 Neuronal nitric oxide synthase ....................................................... 11 1.5.2 Inducible nitric oxide synthase ....................................................... 11 1.5.3 Endothelial nitric oxide synthase ...................................................12 1.5.4 Mitochondrial nitric oxide synthase ................................................ 13 1.6 Nitric oxide synthases: structure and mechanism of action ...... 13 1.6.1 Regulation of nitric oxide production ..............................................20 1.7 Cyclic guanosine monophosphate and guanylate cyclases .......24 1.7.1 Activation of soluble guanylate cyclase .........................................29 1.7.2 Modulation of soluble guanylate cyclase .......................................31 1.8 Physiological and pathophysiological aspects of nitric oxide.. 33 1.8.1 Nitric oxide in the nervous system .................................................34 1.8.2 Nitric oxide in the vascular system ................................................36 1.8.3 Nitric oxide synthases and nitric oxide in the heart ....................... 43 1.8.4 Nitric oxide in the immune system .................................................52 1.8.5 Nitric oxide in the reproductive system ..........................................57 1.9 Oxidative and nitrosative stress: sources, biochemistry and relevance to cellular function and injury ...................................... 60 1.10 Nitric oxide donors ...........................................................................65 1.10.1 Diazeniumdiolates ........................................................................ 66 1.10.2 Chemistry and sources of sodium trioxodinitrate (Angeli’s salt)... 68 iv 1.11 The structure of haem ....................................................................72 1.11.1 The haem biosynthesis pathway .................................................74 1.11.2 Regulation of haem biosynthesis ................................................. 76 1.12 The haem oxygenase pathway......................................................77