THE EFFECT OF OMEGA-3 FATTY ACIDS ON AIRWAY INFLAMMATION, HYPERPNEA-INDUCED BRONCHOCONSTRICTION, AND AIRWAY SMOOTH MUSCLE CONTRACTILITY IN ASTHMA Sally K. Head Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Cellular and Integrative Physiology, Indiana University September 2011 ii Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. ____________________________________ Timothy D. Mickleborough, Ph.D., Chair ____________________________________ Susan J. Gunst, Ph.D. ____________________________________ Maureen A. Harrington, Ph.D. Doctoral Committee ____________________________________ Michael S. Sturek, Ph.D. May 31, 2011 ____________________________________ Robert S. Tepper, M.D., Ph.D. ____________________________________ Johnathan D. Tune, Ph.D. iii ACKNOWLEDGEMENTS This thesis is the product of the cooperation of multiple Indiana University departments and campuses. The author would like to thank the Medical Scientist Training Program and Department of Cellular and Integrative Physiology on the Indianapolis campus as well as the Department of Kinesiology on the Bloomington campus for their dedication to this research and the author’s development as a doctoral candidate. In particular, the author is grateful to her advisor, Dr. Timothy D. Mickleborough, and the members of her research committee, Drs. Susan J. Gunst, Maureen A. Harrington, Michael S. Sturek, Robert S. Tepper, and Johnathan D. Tune, for their guidance. Additionally, the author wishes to thank the subjects who participated in the studies conducted in Bloomington. This work was supported by the Department of Cellular and Integrative Physiology, the Department of Kinesiology, the AAU/Bell- Updyke-Willet Research Fund, the National Institute of General Medical Sciences, GM077229-02, and the National Institutes of Health, HL29289 and HL074099. iv ABSTRACT Sally K. Head THE EFFECT OF OMEGA-3 FATTY ACIDS ON AIRWAY INFLAMMATION, HYPERPNEA-INDUCED BRONCHOCONSTRICTION, AND AIRWAY SMOOTH MUSCLE CONTRACTILITY IN ASTHMA Asthma, a chronic inflammatory disease of the airways, affects nearly 25 million Americans. The vast majority of these patients suffer from exercise-induced bronchoconstriction (EIB), a complication of asthma. Although traditionally treated pharmacologically, nutritional strategies provide a promising alternative for managing EIB as the prevalence of asthma may be due in part to changes in diet. Our objective was to determine the effects of novel nutritional strategies on hyperpnea-induced bronchoconstriction (HIB) in asthmatic individuals. HIB uses rapid breathing to identify EIB in a research or clinical setting. Fish oil, a combination of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docsahexaenoic acid (DHA), has been shown to be effective in suppressing EIB. However, its use in combination with other nutritional supplements, the optimal fish oil formula, and its effect on smooth muscle contractility have not been fully explored. An in vivo study (study 1) was conducted in individuals with both asthma and HIB to determine whether a combination of fish oil and vitamin C was more effective than either one alone in alleviating HIB. Pulmonary function was significantly improved with both fish oil and the combination treatment but not with vitamin C alone. In study 2, individuals with both asthma and HIB were supplemented with DHA alone since the optimal formula for fish oil has yet to be ascertained; previous in vitro studies have suggested DHA may be the more potent omega-3 fatty acid in fish oil. However, no significant changes in pulmonary function or airway inflammation were seen with DHA supplementation. v For study 3, canine airway smooth muscle tissue was treated with fish oil to determine the in vitro effect of fish oil on smooth muscle contractility. Acute treatment with fish oil relaxed smooth muscle strips that had been contracted with acetylcholine or 5-hydroxytryptamine. These minor relaxations in smooth muscle tension with fish oil may represent significant changes at the level of the smaller airways. These studies have confirmed that fish oil represents a viable treatment modality for asthmatic individuals with EIB and suggest that fish oil may influence airway smooth muscle contractility. Timothy D. Mickleborough, Ph.D., Chair vi TABLE OF CONTENTS LIST OF TABLES ix LIST OF FIGURES xi CHAPTER 1: INTRODUCTION 1 Asthma 1 Exercise-Induced Bronchoconstriction 5 Bronchoprovocation Tests to Diagnose Exercise-Induced Bronchoconstriction 5 Pharmacotherapy for Exercise-Induced Bronchoconstriction 8 Diet and Asthma 10 Omega-3 Fatty Acids and Smooth Muscle Contractility 18 Summary and Proposed Experimental Aims 21 CHAPTER 2: THE EFFECT OF FISH OIL, VITAMIN C, AND THEIR COMBINATION ON HYPEPNEA-INDUCED BRONCHOCONSTRICTION IN ADULTS WITH ASTHMA 26 Abstract 26 Introduction 27 Methods 30 Results 37 Discussion 79 Acknowledgements 86 Funding 86 CHAPTER 3: THE EFFECT OF THE OMEGA-3 POLYUNSATURATED FATTY ACID DOCISAHEXAENOIC ACID (DHA) ON HYPERPNEA-INDUCED BRONCHOCONSTRICTION IN ADULTS WITH ASTHMA 87 Abstract 87 Introduction 88 vii Methods 90 Results 97 Discussion 119 Acknowledgements 121 Funding 122 CHAPTER 4: THE ASSOCIATION BETWEEN FISH OIL TREATMENT OF ISOLATED CANINE TRACHEAL SMOOTH MUSCLE TISSUE AND THEIR CONTRACTILITY 123 Abstract 123 Introduction 124 Methods 126 Results 131 Discussion 165 Acknowledgements 173 Funding 173 CHAPTER 5: DISCUSSION 174 Summary of Findings 174 Clinical Implications 177 Future Directions and Proposed Studies 180 Concluding Remarks 182 APPENDIX A: INSTITUTIONAL REVIEW BOARD DOCUMENTS FOR CHAPTER 2 184 APPENDIX B: INSTITUTIONAL REVIEW BOARD DOCUMENTS FOR CHAPTER 3 204 APPENDIX C: RAW DATA FOR CHAPTER 2 228 APPENDIX D: RAW DATA FOR CHAPTER 3 262 viii APPENDIX E: RAW DATA FOR CHAPTER 4 285 REFERENCES 310 CURRICULUM VITAE ix LIST OF TABLES Table 2-1. Baseline characteristics of the subjects at their first (pre- supplementation) laboratory visit 38 Table 2-2. Baseline characteristics of the “responders” at their first (pre- supplementation) laboratory visit 38 Table 2-3. Resting pulmonary function of the subjects in the Fish Oil Group 39 Table 2-4. Resting pulmonary function of the subjects in the Vitamin C Group 39 Table 2-5. Summary of the treatment effects in the Fish Oil Group at each laboratory visit 40 Table 2-6. Summary of the treatment effects in the Vitamin C Group at each laboratory visit 41 Table 2-7. Summary of the treatment effects for all subjects at the pre- supplement and combination treatment tests. 42 Table 2-8. Average intake amounts of selected nutrients for the Fish Oil Group and the Vitamin C Group 78 Table 3-1. Baseline characteristics of the subjects at their first (pre-supplementation) laboratory visit 99 Table 3-2. Resting pulmonary function 99 Table 3-3. Summary of the treatment effects 100 Table 3-4. Average intake amounts of selected nutrients 118 Table 4-1. Percent composition of arachidonic acid, eicosapentaentoic acid (EPA), and docosahexaenoic acid (DHA) in tissues incubated in control, vehicle, soybean oil, or fish oil media for 4 hours 135 Table 4-2. Percent composition of arachidonic acid, eicosapentaentoic acid (EPA), and docosahexaenoic acid (DHA) in tissues incubated in control, vehicle, soybean oil, or fish oil media for 15 hours 135 x Table 4-3. Comparison of the arachidonic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) fatty acid composition reported in the current study and the literature 167 [...]... hyperresponsiveness, it is still not clear whether the epithelial abnormalities are a cause or an effect of asthma (48) Airway Inflammation The other key feature of asthma is airway inflammation, which can occur acutely or chronically, and is the target for many other asthma medications In fact, guidelines for managing asthma tend to concentrate on treating airway inflammation (18) Acute inflammation in asthma. .. significantly relax the canine tracheal smooth muscle response to 10-7 M 5-hydroxytryptamine 165 Figure 5-34 Proposed mechanism of how omega-3 fatty acids reduce airway inflammation and constriction in hyperpnea-induced bronchoconstriction 176 xviii CHAPTER 1 INTRODUCTION Asthma Epidemiology of Asthma Asthma is a chronic inflammatory disease of the airways characterized by recurrent wheezing, breathlessness,... Mechanism of smooth muscle contraction Following cell membrane depolarization, the calcium (Ca2+) concentration increases Myosin light chain kinase (MLCK), whose activation depends on calcium, phosphorylates myosin This allows myosin to bind with actin to produce smooth muscle contraction Myosin light chain phosphatase (MLCP) dephosphorylates myosin to cause relaxation Airway smooth muscle contractility. .. dependent on the overlying epithelium Epithelial functions include creating a barrier between the airways and the external environment as well as secreting many factors (48) Epithelial secretions include arachidonic acid metabolites involved in airway smooth muscle tone, mucus secretion, and inflammation (48) Nitric oxide, growth factors involved in 2 respiratory tissue repair, and proinflammatory cytokines... As airway narrowing and hyperresponsiveness are key features of asthma, airway smooth muscle contraction is an important mechanism Consequently, medications that relax airway smooth muscle and thus dilate the airways are among the most widely prescribed treatments for asthma These include long- and short-acting β2agonists, such as salmeterol and albuterol, respectively 1 Smooth muscle contraction (figure... medications to optimally control their symptoms Combination therapies targeting the acute and chronic symptoms of asthma are increasingly prescribed since monotherapy is often inadequate (31) Appropriate asthma treatment and management is thus an important issue due to the substantial burden asthma has placed on American society in terms of lost productivity and healthcare costs Airway Smooth Muscle Contractility. .. to the airways (18) Chronic inflammation in asthma is characterized by activated T-cells, eosinophils, mast cells, macrophages, epithelial cells, fibroblasts, and bronchial smooth muscle cells in the airways (18) The eosinophils in particular secrete proinflammatory mediators, cytotoxic mediators, and cytokines which cause many of the features of asthma, including mucus secretion, smooth muscle contraction,... pathogenesis of EIB, the hyperosmolarity theory and the airway re-warming theory According to the hyperosmolarity theory, the airway surface liquid becomes hypertonic due to water loss during exercise; the ensuing hyperosmolar environment in the airway cells results in the release of proinflammatory mediators that cause bronchoconstriction (90) Alternatively, the less widely accepted airway re-warming theory... asthmatics leading to less bronchoconstriction 17 Figure 1-6 Site of action for fish oil supplementation The omega-3 polyunsaturated fatty acids in fish oil produce prostaglandins and leukotrienes that are less proinflammatory than their omega-6 fatty acid counterparts Fish oil supplementation thus increases this pathway to reduce bronchoconstriction Omega-3 Fatty Acids and Smooth Muscle Contractility Conflict... tightness, and coughing (87) The hallmark features of asthma are airway inflammation, airway hyperresponsiveness, and airway narrowing (90) In 2009, 24.6 million Americans reported having asthma with 60% of those 5-17 years of age missing at least one day of school and 34% of those over 18 years of age missing at least one day of work due to asthma symptoms; this translated to 10.5 million missed school . AAU/Bell- Updyke-Willet Research Fund, the National Institute of General Medical Sciences, GM077229-02, and the National Institutes of Health, HL29289 and HL074099. iv ABSTRACT Sally K. Head THE. author would like to thank the Medical Scientist Training Program and Department of Cellular and Integrative Physiology on the Indianapolis campus as well as the Department of Kinesiology on. wishes to thank the subjects who participated in the studies conducted in Bloomington. This work was supported by the Department of Cellular and Integrative Physiology, the Department of Kinesiology,