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Distribution of Neurokinin 2 and 3 Receptor Mrna in the Normal Distribution of Neurokinin 2 and 3 Receptor mRNA in the Normal Equine Gastrointestinal Tract and Effect of Inflammation on Expression of Neurokinin 1, 2 and 3 Receptor mRNA in the Jejunum by Dr. Christina Martin A Thesis presented to The University of Guelph In partial fulfillment of requirements for the degree of Masters of Science in Clinical Studies Guelph, Ontario, Canada Dr. Christina Martin, April, 2014 ABSTRACT DISTRIBUTION OF NEUROKININ 2 AND 3 RECEPTOR mRNA IN THE NORMAL EQUINE GASTROINTESTINAL TRACT AND EFFECT OF INFLAMMATION ON EXPRESSION OF NEUROKININ 1, 2 AND 3 RECEPTOR mRNA IN THE JEJUNUM Dr. Christina E. W. Martin Advisor: University of Guelph, 2014 Dr. Judith B. Koenig This study is an investigation of the distribution of neurokinin receptors in the equine gastrointestinal tract. The objectives of this research were to determine the relative distribution of neurokinin 2 (NK2) and 3 (NK3) receptor mRNA in the normal equine gastrointestinal tract, and also to determine changes in neurokinin 1 (NK1), NK2 and NK3 receptor mRNA expression after ischemia/reperfusion injury or intraluminal distension in the jejunum. Samples from 9 regions in the gastrointestinal tract (duodenum, jejunum, ileum, caecum, right ventral colon, left ventral colon, pelvic flexure, right dorsal colon and left dorsal colon) were harvested from 5 mature healthy horses, euthanized for reasons unrelated to the gastrointestinal tract, for the study of NK2 and NK3 mRNA distribution in the normal intestinal tract. To evaluate the effect of inflammation on NK1, NK2 and NK3 receptor mRNA distribution, samples were taken from 6 horses whose jejunum underwent one of three treatments: control (sham-operated), intraluminal distension (ILD) or ischemic strangulation obstruction and subsequent reperfusion injury (ISO). NK2 and NK3 receptor primers were ii designed and real-time PCR was used to quantify NK1 (primers previously described), NK2 and NK3 receptor mRNA expression in the treatment groups described above. In healthy horses, NK2 mRNA expression in the small intestine was highest in the duodenum and lowest in the ileum. NK2 mRNA expression in the large intestine was highest in the caecum. NK3 mRNA expression was more variable between individuals than NK2 expression overall. No significant difference was found between concentrations of NK1 or NK3 receptor mRNA between control, ILD or ISO treatments. A trend was noted for NK1 mRNA to be lower in ILD treatments than control. For NK2 receptor mRNA, ILD and ISO values were significantly lower than that of control. Tachykinin agonists and antagonists have shown therapeutic value in intestinal inflammation and motility disorders in laboratory animals and humans. Neurokinin receptor mRNA is present in the equine intestinal tract. Relative levels appear to be altered by inflammation, although the clinical significance of this finding needs to be further evaluated. The current study suggests that tachykinin therapy may have a potential utility in the medical treatment of equine post-operative ileus and equine colic, however further investigation into the physiology of neurokinin receptors in the horse is warranted. iii iv ACKNOWLEDGEMENTS I wish to thank my advisor, Dr. Judith B. Koenig for her guidance and support throughout this process. Drs. Ioana Sonea and Jon LaMarre have also provided invaluable advice and encouragement. I am grateful to Dr. Nicole Solinger for her patient teaching of laboratory techniques and continued dedication to the project. I also wish to thank Sukhminder Sawhney for her work on RNA isolation and help with analysis, as well as Tim Gingerich for insight in interpretation of results. iv v DECLARATION OF WORK PERFORMED I declare that all the work reported in this thesis was performed by Dr. Christina E. W. Martin. v vi TABLE OF CONTENTS page Abstract ii Acknowledgements iv Declaration of work performed v Table of contents vi 1.0 Introduction 1 1.1 Hypothesis and Objectives 2 1.2 Literature review 3 1.2.1 Tachykinins 3 1.2.2. The Role of Tachykinins in Gastrointestinal Inflammation 6 1.2.2.1. Models of Inflammation and the Benefit of Antagonists 7 a. Trinitrobenzene Sulfonic Acid Model 7 b. Acetic Acid and Dextran Sodium Sulfate Models 8 c. Toxin A Model 9 d. Cryptosporidium Parvum Model 11 1.2.2.2. Measuring Substance P Levels in Human Tissues 12 1.2.2.3. Water flux – Determination of the Role of SP in Hypersecretion 13 a. Non-absorbable Markers in Induced Hypersecretion 13 b. Ussing Chambers – Electrophysiologic Studies 14 1.2.2.4. Tachykinins and Food Allergy 15 vi vii 1.2.2.5. Summary 15 1.2.3. Inflammatory Bowel Diseases in the Horse 16 1.2.3.1. Granulomatous Enteritis 16 1.2.3.2. Lymphocytic-plasmacytic Enterocolitis 17 1.2.3.3. Multisystemic Eosinophilic Epitheliotropic Disease/ Idiopathic Eosinophilic Enterocolitis 17 1.2.4. Ischemia/Reperfusion Injury and Intraluminal Distension of the Equine Small Intestine 18 1.2.4.1. Ischemia/reperfusion 18 1.2.4.2. Distension 20 1.2.4.3. Summary 21 1.2.5. Normal Motility in the Equine Gastrointestinal Tract 22 1.2.5.1. The MMC 23 1.2.5.2. Interstitial Cells of Cajal 23 1.2.6. The Role of Tachykinins in Gastrointestinal Motility 24 1.2.6.1. Isometric Studies 25 1.2.6.2. Inhibitory Effects 27 1.2.6.3. Motility and Pathology 28 a. Post-operative Ileus 28 b. Stress 29 c. Inflammation 29 1.2.6.4. In Vivo Studies 30 vii viii 1.2.6.5. Summary 32 1.2.7. Ileus: Pathophysiology and Relevance 33 1.2.7.1. Risk Factors 33 1.2.7.2. Etiology 34 1.2.7.3. Recognition and Treatment 36 1.2.7.4. Summary 37 1.2.8. Tachykinins Research and The Equine Gastrointestinal Tract 38 1.2.8.1. Immunoreactivity of Substance P in the Equine Gastrointestinal Tract 38 1.2.8.2. Studies for Determination of NK Receptor Distribution in Equine Gastrointestinal Tract 39 1.2.8.3. Pathological Receptor Changes 40 1.2.8.4. Isometric Studies on Isolated Muscle Preparations 41 1.2.8.5. In Vivo Studies of Response to Substance P 42 1.2.8.6. Summary 42 1.3 References 44 2.0 Distribution of Neurokinin Receptors 2 and 3 in the Normal Equine Gastrointestinal Tract and Effect of Inflammation on Distribution of Neurokinin receptors 1, 2 and 3 in the Jejunum 63 2.1 Abstract 64 viii ix 2.2 Introduction 65 2.3 Materials and Methods 68 2.4 Results 73 2.5 Discussion 76 2.6 Figures 81 2.6 References 85 3.0 Conclusions and Future Studies 92 3.1 References 95 4.1 Appendix 100 ix INTRODUCTION The distribution of neurokinin 2 (NK2) and neurokinin 3 (NK3) receptors in the equine gastrointestinal tract is unknown. Tachykinins, the natural ligands for neurokinin receptors, have been implicated in gastrointestinal inflammation and motility (61; 62; 63). Gastrointestinal disorders are serious and common conditions in the horse (71; 138) Tachykinin agonists and antagonists have been studied in both human and animal models for the potential treatment of inflammatory and motility disorders of the gut. In other species, NK2 and NK3 receptors are present in intestinal tissues (61). Tachykinins have been shown to affect intestinal motility, and provide protective effects in the case of intestinal inflammation (61; 62; 63). Characterizing the distribution of neurokinin receptors in equine tissues, both in normal conditions and in the case of ischemia and distension, would be useful information for the determination of the potential therapeutic uses of tachykinin agonists and antagonists in the horse. 1 HYPOTHESIS AND OBJECTIVES This study was designed to test the hypothesis that NK2 and NK3 receptors are present in the equine intestinal tract, and that NK1, NK2 and NK3 receptors may be useful targets during equine colic, with the following objectives: 1) To determine the relative expression of NK2 and NK3 receptor mRNA in normal equine tissue throughout 9 regions of the intestinal tract. 2) To determine the relative expression of NK1, NK2 and NK3 receptor mRNA in the equine jejunum under normal (sham operated), ischemic strangulation obstruction, and intraluminal distension conditions. 2 1.2.1 Tachykinins Tachykinins are a family of neuropeptides with the conserved carboxy (C)-terminal sequence: Phe-x-Gly-Leu-Met-NH2, where ‘x’ signifies either a hydrophobic or aromatic group (22; 131). The mammalian tachykinins include Substance P (SP), Neurokinin A (NKA) and Neurokinin B (NKB). The first tachykinin to be discovered was SP. It was extracted from intestinal and cerebral equine tissues and then tested on longitudinal smooth muscle samples from rabbit intestine (40). These samples were pre-treated with atropine, which decreases gastrointestinal secretion and motility, and it was found that their peripheral vasodilation and contractility subsequently increased after treatment with SP (40). Eleven amino acids form SP (114), whereas NKA and NKB are decapeptides (24). Peptide Amino Acid Sequence Substance P Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met NH2 NKA His-Lys-The-Asp-Ser-Phe-Val-Gly-Leu-Met NH2 NKB Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met NH2 Table 1: Amino acid sequences for SP, NKA and NKB (24). The preprotachykinin A gene (PPT-A) on chromosome 7 (22) encodes for SP and NKA. It is then differentially spliced to produce either SP or NKA. Preprotachykinin B (PPT-B) on chromosome 12 encodes for NKB (131). Tachykinins act centrally or peripherally via NK receptors NK1, NK2 or NK3 (13). The natural ligand for SP is considered to be NK1, NKA the natural ligand for NK2 and NKB for NK3 (13). However, all three peptides are effective agonists for each receptor, according to the following rankings: NK1 receptor: SP>NKA>NKB; NK2 receptor: NKA>NKB>SP; NK3 3 receptor: NKB>NKA>SP (116).
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