Lau Hoi Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 134-157). / Abstracts in English and Chinese. / PUBLICATIONS BASED ON WORK IN THIS THESIS --- p.I / ABSTRACT --- p.II / ACKNOWLEDGEMENTS --- p.VI / TABLE OF CONTENTS --- p.VIII / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Emesis --- p.3 / Chapter 1.2.1 --- Introduction --- p.3 / Chapter 1.2.2 --- Retching & Vomiting --- p.3 / Chapter 1.2.3 --- Nausea --- p.4 / Chapter 1.2.4 --- Motor Components of Emetic Reflex --- p.5 / Chapter 1.2.4.1 --- Pre-ejection Phase --- p.5 / Chapter 1.2.4.2 --- Ejection Phase --- p.5 / Chapter 1.2.4.3 --- Post-ejection Phase --- p.6 / Chapter 1.2.5 --- Components of Emetic Reflex --- p.6 / Chapter 1.2.5.1 --- Area Postrema (AP) --- p.6 / Chapter 1.2.5.2 --- Nucleus Tractus Solitarius (NTS) --- p.7 / Chapter 1.2.5.3 --- Vomiting Centre --- p.8 / Chapter 1.2.5.4 --- Vestibular System --- p.10 / Chapter 1.2.5.5 --- Abdominal Visceral Afferents --- p.10 / Chapter 1.2.5.6 --- Forebrain --- p.11 / Chapter 1.2.6 --- Neurotransmitters & Receptors --- p.12 / Chapter 1.2.7 --- Anti-emetics --- p.13 / Chapter 1.3 --- Models of Nausea --- p.16 / Chapter 1.3.1 --- Introduction --- p.16 / Chapter 1.3.2 --- Conditioned Taste Aversion --- p.18 / Chapter 1.3.3 --- Pica Behaviour --- p.20 / Chapter 1.3.4 --- Studies of the Involvement of Vasopressin --- p.21 / Chapter 1.3.5 --- Tachygastria --- p.24 / Chapter 1.3.6 --- Locomotor Activity --- p.26 / Chapter 1.4 --- Markers of Neuronal Activity --- p.27 / Chapter 1.4.1 --- General Comments --- p.27 / Chapter 1.4.2 --- c-fos Expression as a Marker of Neuronal Activity --- p.28 / Chapter 1.4.2.1 --- What is c-fos? --- p.28 / Chapter 1.4.2.2 --- Regulation of c-fos Expression --- p.30 / Chapter 1.4.2.2.1 --- Calcium Response Element --- p.31 / Chapter 1.4.2.2.2 --- Serum Response Element --- p.32 / Chapter 1.4.2.3 --- Types of Receptors Involved in c-fos Expression --- p.32 / Chapter 1.4.2.4 --- Feasibility of Using c-fos Expression as Marker of Cellular Activity --- p.36 / Chapter 1.4.2.5 --- Identification of Emetic Pathway by c-fos Immunohistochemistry --- p.36 / Chapter 1.5 --- Aims & Objectives --- p.37 / Chapter CHAPTER 2 --- METHODS --- p.42 / Chapter 2.1 --- Animals --- p.42 / Chapter 2.1.1 --- Ferrets --- p.42 / Chapter 2.1.2 --- Suncus murinus --- p.42 / Chapter 2.2 --- Measurement of Animal Behaviour --- p.43 / Chapter 2.2.1 --- Experiment Design --- p.43 / Chapter 2.2.2 --- Recording of Animal Behaviour --- p.43 / Chapter 2.2.3 --- Calibration of Equipment Used to Record Spontaneous Locomotor Activity --- p.44 / Chapter 2.2.4 --- Behaviour Recorded by the Observer --- p.45 / Chapter 2.3 --- Administration of Drugs --- p.46 / Chapter 2.3.1 --- Ferrets --- p.46 / Chapter 2.3.1.1 --- General Comments --- p.46 / Chapter 2.3.1.2 --- Drug Antagonism Studies --- p.47 / Chapter 2.3.2 --- Suncus murinus --- p.47 / Chapter 2.3.2.1 --- General Comments --- p.47 / Chapter 2.3.2.2 --- Dose-Response Studies --- p.48 / Chapter 2.3.2.3 --- Drug Antagonism Studies --- p.48 / Chapter 2.4 --- c-fos Expression Studies in Ferret Brainstems --- p.50 / Chapter 2.4.1 --- Animals and Anaesthesia --- p.50 / Chapter 2.4.2 --- Perfusion and fixation --- p.50 / Chapter 2.4.3 --- Dehydration of brains --- p.51 / Chapter 2.4.4 --- Embedding of tissue --- p.52 / Chapter 2.4.5 --- Sectioning --- p.52 / Chapter 2.4.6 --- Staining --- p.52 / Chapter 2.4.7 --- Antibodies used --- p.55 / Chapter 2.4.8 --- Positive Control Slides --- p.55 / Chapter 2.5 --- Experimental Design and Statistics --- p.56 / Chapter 2.5.1 --- Randomization of Treatments --- p.56 / Chapter 2.5.2 --- Statistics --- p.57 / Chapter 2.5.2.1 --- Ferrets --- p.57 / Chapter 2.5.2.2 --- Suncus murinus --- p.59 / Chapter 2.6 --- Drugs and Chemicals Used --- p.60 / Chapter 2.6.1 --- Drugs Used --- p.60 / Chapter 2.6.2 --- Chemicals Used --- p.62 / Chapter CHAPTER 3 --- RESULTS --- p.63 / Chapter 3.1 --- Ferret --- p.63 / Chapter 3.1.1 --- "The Effect of Ondansetron and CP-99,994 on Emesis and Locomotor Activity Changes Induced by Cisplatin in the Ferret" --- p.63 / Chapter 3.1.2 --- The Effect of Domperidone on Emesis and Locomotor Activity Changes Induced by Apomorphine in the Ferret --- p.69 / Chapter 3.1.3 --- "The Effect of CP-99,994 on Emesis and Locomotor Activity Changes Induced by Apomorphine in the Ferret" --- p.74 / Chapter 3.1.4 --- c-fos Expression Studies in Ferret Brainstems --- p.79 / Chapter 3.1.4.1 --- Cisplatin-treated Ferrets --- p.79 / Chapter 3.1.4.2 --- Positive Control Slides --- p.84 / Chapter 3.2 --- Suncus murinus --- p.88 / Chapter 3.2.1 --- The Emetic Potential of Nicotine and its Effects on the Spontaneous Locomotor Activity of Suncus murinus --- p.88 / Chapter 3.2.2 --- "The Effect of CP-99,994 on Emesis and Locomotor Activity Changes Induced by Nicotine in Suncus murinus" --- p.92 / Chapter 3.2.3 --- The Emetic Potential of Copper Sulphate and its Effects on the Spontaneous Locomotor Activity of Suncus murinus --- p.95 / Chapter 3.2.4 --- "The Effect of CP-99,994 on Emesis and Locomotor Activity Changes Induced by Copper Sulphate in Suncus murinus" --- p.98 / Chapter 3.2.5 --- The Emetic Potential of Cisplatin and its Effects on the Spontaneous Locomotor Activity of Suncus murinus --- p.101 / Chapter 3.2.6 --- The Effect of Ondansetron on Emesis and Locomotor Activity Changes Induced by Cisplatin in Suncus murinus --- p.104 / Chapter 3.2.7 --- "The Effect of CP-99,994 on Emesis and Locomotor Activity Changes Induced by Cisplatin in Suncus murinus" --- p.107 / Chapter 3.2.8 --- "The Effects of Ondansetron and CP-99,994 on Locomotor Activity in Suncus murinus" --- p.110 / Chapter CHAPTER 4 --- DISCUSSION --- p.113 / Chapter CHAPTER 5 --- GENERAL SUMMARY --- p.130 / REFERENCES --- p.134

Wong Hei Lui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 192-226). / Abstracts in English and Chinese. / Publications Based On The Work In This Thesis --- p.i / Abstract --- p.ii / Acknowledgements --- p.vii / Abbreviations --- p.viii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Normal joint --- p.1 / Chapter 1.11 --- Biology of joint --- p.1 / Chapter 1.12 --- Structure of synovial joint --- p.1 / Chapter 1.13 --- Components of the mature synovial joint --- p.3 / Chapter 1.131 --- Articular cartilage --- p.3 / Chapter 1.1311 --- Water --- p.4 / Chapter 1.1312 --- Cartilage matrix --- p.4 / Chapter 1.1313 --- Chondrocyte --- p.5 / Chapter 1.132 --- Synovium --- p.5 / Chapter 1.1321 --- Synovium vasculature --- p.6 / Chapter 1.1322 --- Synovial blood flow --- p.7 / Chapter 1.133 --- Synovial fluid --- p.8 / Chapter 1.134 --- Bone --- p.9 / Chapter 1.2 --- Pathological processes of arthritis --- p.11 / Chapter 1.21 --- Activation of immune cells in arthritis --- p.11 / Chapter 1.22 --- Synovial proliferation --- p.13 / Chapter 1.221 --- Synovial lining cell activation --- p.13 / Chapter 1.222 --- Pannus invasion --- p.14 / Chapter 1.23 --- Cartilage and bone degradation --- p.14 / Chapter 1.231 --- Depletion of proteoglycan (GAG) --- p.15 / Chapter 1.232 --- Collagen denature --- p.15 / Chapter 1.3 --- Tachykinins (TKs) --- p.17 / Chapter 1.31 --- History --- p.17 / Chapter 1.32 --- "Synthesis, storage and release of TKs" --- p.17 / Chapter 1.33 --- Tachykinin receptors --- p.18 / Chapter 1.331 --- Characterization of NK1 receptor --- p.19 / Chapter 1.332 --- Characterization of NK2 receptor --- p.19 / Chapter 1.333 --- Characterization of NK3 receptor --- p.20 / Chapter 1.34 --- Effector systems of TKs --- p.21 / Chapter 1.35 --- Termination of TK signals --- p.21 / Chapter 1.351 --- Enzymatic breakdown --- p.21 / Chapter 1.352 --- Receptor desensitization --- p.22 / Chapter 1.353 --- Receptor endocytosis --- p.22 / Chapter 1.36 --- TK receptor antagonists --- p.23 / Chapter 1.361 --- Selective NK1 receptor antagonists --- p.23 / Chapter 1.362 --- Selective NK2 receptor antagonists --- p.24 / Chapter 1.363 --- Selective NK3 receptor antagonists --- p.25 / Chapter 1.4 --- Roles of tachykinins in arthritis --- p.28 / Chapter 1.41 --- Correlation between tachykinins and joint inflammation --- p.28 / Chapter 1.42 --- Roles of tachykinins in immune cell activation --- p.30 / Chapter 1.43 --- Roles of tachykinins in synovial proliferation --- p.31 / Chapter 1.44 --- Roles of tachykinins in cartilage degradation --- p.32 / Chapter 1.5 --- Animal model of arthritis --- p.33 / Chapter 1.51 --- Instability model --- p.33 / Chapter 1.52 --- Immobilization model --- p.34 / Chapter 1.53 --- Noxious agent-induced model --- p.34 / Chapter 1.531 --- Collagen-induced erosive arthritis --- p.34 / Chapter 1.532 --- Cartilage oligometric matrix protein-induced arthritis --- p.35 / Chapter 1.533 --- Oil-induced arthritis --- p.35 / Chapter 1.534 --- Streptococcal cell wall-induced arthritis --- p.35 / Chapter 1.535 --- Adjuvant-induced arthritis --- p.36 / Chapter 1.536 --- Pristane-induced arthritis --- p.36 / Chapter 1.6 --- Current anti-arthritic therapies --- p.39 / Chapter 1.61 --- Non steroid anti-inflammatory drugs --- p.39 / Chapter 1.62 --- Glucocorticoid --- p.44 / Chapter 1.63 --- Second-line treatment --- p.46 / Chapter 1.631 --- Sulfasalazine --- p.46 / Chapter 1.632 --- Gold salts --- p.47 / Chapter 1 633 --- D-penicillamine --- p.48 / Chapter 1.634 --- Antimalarial --- p.49 / Chapter 1 .635 --- Methotrexate --- p.51 / Chapter 1.64 --- New trends for treatment of arthritis --- p.53 / Chapter 1.641 --- Anti-cytokine therapy --- p.53 / Chapter 1.642 --- Anti-angiogenesis therapy --- p.54 / Chapter 1.7 --- Aims of study --- p.57 / Chapter Chapter 2 --- Material and drugs --- p.62 / Chapter Chapter 3 --- Methodology --- p.62 / Chapter 3.1 --- Animals used and anaesthetization --- p.62 / Chapter 3.2 --- Measurement of plasma protein extravasation --- p.63 / Chapter 3.3 --- Measurement of knee joint sizes --- p.64 / Chapter 3.4 --- Measurement of knee joint blood flow --- p.65 / Chapter 3.5 --- Measurement of histological changes --- p.65 / Chapter 3.51 --- Dissection and fixation --- p.65 / Chapter 3.52 --- Decalcification --- p.66 / Chapter 3.53 --- Processing --- p.66 / Chapter 3.54 --- Embedding --- p.67 / Chapter 3.55 --- Sectioning --- p.67 / Chapter 3.56 --- Staining --- p.69 / Chapter 3.6 --- Data analysis --- p.69 / Chapter 3.61 --- Scoring systems --- p.72 / Chapter Chapter 4 --- A model of monoarthritis in rats --- p.72 / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.2 --- Method --- p.73 / Chapter 4.3 --- Results --- p.73 / Chapter 4.31 --- Lewis rats --- p.73 / Chapter 4.32 --- Sprague-Dawley (SD) rats --- p.74 / Chapter 4.33 --- Comparison of FCA-induced changes in Lewis and SD rats --- p.74 / Chapter 4.34 --- Histological studies on arthritic SD rats --- p.75 / Chapter 4.4 --- Discussion --- p.93 / Chapter 4.5 --- Conclusions --- p.95 / Chapter Chapter 5 --- Effect of Substance P on adjuvant-induced arthritis --- p.96 / Chapter 5.1 --- Introduction --- p.96 / Chapter 5.2 --- Method --- p.98 / Chapter 5.3 --- Results --- p.99 / Chapter 5.31 --- Evans blue extravasation --- p.99 / Chapter 5.32 --- Joint size --- p.100 / Chapter 5.33 --- Knee joint blood flow --- p.101 / Chapter 5.34 --- Histology results --- p.102 / Chapter 5.341 --- Infiltration of immune cells in synovial tissue --- p.102 / Chapter 5.342 --- Synovial tissue proliferation --- p.102 / Chapter 5.343 --- Cartilage degradation --- p.103 / Chapter 5.344 --- Bone degradation --- p.103 / Chapter 5.4 --- Discussion --- p.120 / Chapter 5.5 --- Conclusions --- p.125 / Chapter Chapter 6 --- Effects of tachykinin receptor antagonists on FCA-induced arthritis / Chapter 6.1 --- Introduction --- p.126 / Chapter 6.2 --- Method --- p.128 / Chapter 6. 21 --- Intravenous NK1 receptor antagonists on FCA-induced arthritis --- p.128 / Chapter 6. 22 --- Intraperitoneal TK receptor antagonists on FCA-induced arthritis --- p.128 / Chapter 6.3 --- Results --- p.129 / Chapter 6.31 --- Intravenous NK1 227}0اreceptor antagonists on FCA-induced arthritis Evans blue extravasation and joint swelling --- p.129 / Chapter 6.32 --- Intraperitoneal tachykinin receptor antagonists on FCA- induced arthritis Evans blue extravasation and joint swelling --- p.129 / Chapter 6.33 --- Intraperitoneal tachykinin receptor antagonists on FCA- induced immune cell accumulation --- p.130 / Chapter 6.34 --- Intraperitoneal tachykinin receptor antagonists on FCA- induced synovial tissue proliferation --- p.131 / Chapter 6.35 --- Intraperitoneal tachykinin receptor antagonists on FCA- induced cartilage degration and bone erosion --- p.131 / Chapter 6.4 --- Discussion --- p.159 / Chapter 6.5 --- Conclusions --- p.162 / Chapter Chapter 7 --- Individual and combined effects of dexamethasone and TK receptor antagonists on FCA-induced arthritis --- p.163 / Chapter 7.1 --- Introduction --- p.163 / Chapter 7.2 --- Method --- p.166 / Chapter 7.3 --- Results --- p.167 / Chapter 7.31 --- Evans blue extravasation --- p.167 / Chapter 7.32 --- Knee joint size --- p.167 / Chapter 7.33 --- Body weight --- p.168 / Chapter 7.34 --- Cellular infiltration --- p.168 / Chapter 7.35 --- Synovial tissue proliferation --- p.168 / Chapter 7.36 --- Cartilage degradation --- p.169 / Chapter 7.4 --- Discussion --- p.184 / Chapter 7.5 --- Conclusions --- p.187 / Chapter Chapter 8 --- General discussions and conclusions --- p.188 / References --- p.192

Les peptides appartenant à la famille des tachykinines telle que la substance P (SP) sont des acteurs essentiels contribuant à l’hyperalgésie primaire et secondaire. La SP libérée par les neurones afférents primaires, ne provoque pas à elle seule des décharges nociceptives, mais elle potentialise l’effet de divers neurotransmetteurs tel que le glutamate. Pour ces différentes raisons, de nombreuses recherches ont été effectuées avec des antagonistes des récepteurs neurokinines et en particulier des récepteurs NK1. Cependant, malgré des études pré-cliniques prometteuses, les antagonistes du récepteur NK1 n’ont pas montré d’effet significatif chez l’Homme. La biosynthèse des neuropeptides actifs passe par la maturation protéolytique des pro-neuropeptides. La compréhension des mécanismes de la maturation enzymatique des précurseurs des tachykinines, ainsi que l’étude de la stabilité métabolique de la substance P (SP) permettraient d’élucider des stratégies de traitement innovateur en favorisant l’inhibition du processus de maturation ou la production de fragments peptidiques moins actifs ou inactifs. Le premier objectif de cette étude était d’élucider le rôle de la Proproteine convertase 1 (PC1) et de la Proproteine convertase 2 (PC2) dans la maturation de la protachykinine en utilisant des fractions S9 de la moelle épinière des souris du type sauvage (WT), PC1-/+ et PC2-/+. La caractérisation et la quantification des neuropeptides ont été réalisées à l’aide de la chromatographie liquide à haute performance et de la spectrométrie de masse. Les résultats montrent que PC1 et PC2 interviennent dans la maturation de la protachykinine et ces deux enzymes sont essentielles pour la biosynthèse de la Tachykinine58-71, le précurseur de la SP. Une réduction de plus de 50% de la vitesse de formation dans les fractions S9 de la moelle épinière de souris mutantes PC1 et PC2 a été observée. Les résultats obtenus révèlent que PC1 et PC2 sont impliquées dans la protéolyse de la protachykinine et suggèrent un rôle important de ces enzymes dans la maturation de la protachykinine-1. La protéolyse régule probablement les concentrations extracellulaires de la SP, mais peu d'études ont été menées sur le métabolisme des tachykinines. Dans ce présent travail, nous démontrons que la protéolyse contrôle le niveau de la SP dans la moelle épinière menant à la formation de fragments C-terminaux actifs. La stabilité métabolique de la β-tachykinine58-71 et de la SP était très courte, avec une demi-vie de 5.7 et 3.5 min, respectivement. Plusieurs fragments C-terminaux ont été identifiés, y compris la SP3-11, la SP5-11 et la SP8-11, qui conservent leurs affinités vis-à-vis des récepteurs neurokinines. La stabilité métabolique des fragments C-terminaux était significativement supérieure à celle de la β-Tachykinine58-71 et de la SP. Deux inhibiteurs de Prolyl endopeptidase spécifiques ont été utilisés et ont montré une réduction significative de la vitesse de formation de SP3-11 et de SP5-11. Ainsi, nous avons démontré que le Prolyl endopeptidase est impliqué dans le traitement N-terminal de la SP dans la moelle épinière et dans la formation de la SP3-11 et la SP5-11. Étant donné que la régulation des niveaux endogènes de peptides opioïdes (DynA, Leu-Enk, Met-Enk) et des tachykinines (Tach58-71, SP) dépend fondamentalement de l'activité de PC1 et de celle de PC2, l'analyse des tachykinines et des neuropeptides opioïdes ont été réalisées. Les résultats obtenus révèlent une diminution significative des neuropeptides pro nociceptifs la Tach58-71 (p <0,05), de la SP (p <0,01) et du NKA (P <0,001)), et des neuropeptides opioïdes DynA (p <0,01), de Leu-Enk (p <0,001), de Met-Enk (p <0,001), dans la moelle épinière de souris PC1 - / + et PC2 - / +. Par conséquent, la modulation de l'activité des PCs a un impact important sur les peptides pro-nociceptifs, mais également sur le système opioïde endogène et par conséquent elle affectera significativement les voies modulatrices de la douleur. Ces résultats suggèrent également que la réduction significative des concentrations de peptides pro-nociceptifs peut altérer la réponse du système opioïde endogène. Les analyses des concentrations des peptides opioïdes chez les souris Tac1-/- ont montré spécifiquement que les concentrations en Endomorphine-2 (EM2), en Leu-Enk et en Dyn A sont significativement inférieures que celles obtenues dans la moelle épinière chez les souris WT. Par conséquent, l’absence de la SP a un impact sur les mécanismes endogènes de modulation de la douleur. Mots clés : Tachykinines, substance P, proprotéines convertases, protéolyse, peptides opioïdes, moelle épinière, douleur, chromatographie liquide à haute performance, spectrométrie de masse. / SP is a major proteolytic product of the protachykinin-1 primarily synthesized in neurons and plays a central role in nociceptive transmission. The SP does not acte alone to cause nociceptive discharges, but it potentiates the effect of various neurotransmitters such as glutamate. For these various reasons, much research has been carried out with antagonists of neurokinin receptors and in particular NK1 receptors. However, despite promising pre-clinical studies, NK1 receptor antagonists have not shown significant effect in Humans. The proteolysis control of endogenous protachykinins has a profound impact on pain perception. Proprotein convertases (PCs) are extensively expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue but the role of PCs remains unclear. The first objective of this study was to decipher the role of PC1 and PC2 in the proteolysis of protachykinins using cellular fractions of spinal cords from wild type (WT), PC1 -/+ and PC2 -/+ mices and mass spectrometry. The results clearly demonstrate that both PC1 and PC2 mediate the formation of SP and β-Tachykinin58-71, an important SP precursor, with over 50 % reduction of the rate of formation in mutant PC1 and PC2 mouse S9 spinal cord fractions. The results obtained revealed that PC1 and PC2 are involved in the C-terminal processing of protachykinin peptides and suggest a major role in the maturation of the protachykinin-1 protein. The proteolysis is suspected to regulate extracellular SP concentrations but few studies were conducted on the metabolism of proneuropeptides and neuropeptides. In the present study, we provide evidence that proteolysis controls SP levels in the spinal cord leading to the formation of active C-terminal fragments. The metabolic stability of β-Tachykinin58-71 and SP were very short resulting in half-life of 5.7 and 3.5 min, respectively. Several C-terminal fragments were identified, including SP3-11, SP5-11 and SP8-11, which conserve affinity for the neurokinin receptors. Interestingly, the metabolic stability of C-terminal fragments were significantly superior. Two specific Prolyl endopeptidase inhibitors were used and showed a significant reduction in the rate of formation of SP3-11 and SP5-11 providing strong evidence that Prolyl endopeptidase is involved into N-terminal processing of SP in the spinal cord. The role of proprotein convertases (PCs) in the proteolysis of proneuropeptides was previously established but few studies have shown the direct impact of PCs on the regulation of specific tachykinin and opioid peptides in the central nervous system. This study has determined the relative concentration of targeted neuropeptides in the spinal cord of WT, PC1- / + and PC2- /+ mice to establish the impact of a restricted PCs activity on the regulation of specific neuropeptides. The results revealed a significant decrease of Dyn A (p < 0.01), Leu-Enk (p < 0.001), Met-Enk (p < 0.001), Tach58-71 (p < 0.05), SP (p < 0.01) and NKA (p < 0.001) spinal cord concentrations in both, PC1 -/+ and PC2 -/+ mice. Therefore, the modulation of PCs activity has an important impact on specific pronociceptive peptides (SP and NKA), but the results also showed that endogenous opioid system is hindered and consequently it will affect significantly the pain modulatory pathways. Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in Tac1-/- spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. Our results reveal that Endomorphin-2 (EM2), Leu-Enk and Dyn A were down regulated in Tac1-/- spinal cord tissues that strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These results may have insightful impact on future analgesic drug developments and therapeutic strategies. Key words: Tachykinins, substance P, proprotein convertases, proteolysis, opioid peptides, spinal cord, pain, high performance liquid chromatography, mass spectrometry.