Dissertations / Theses on the topic 'Tachykinin'

The tachykinins are highly active mediators of neuronal action in the CNS. In order to understand their action and provide templates for drug development, analogues based on the active sequence of Substance P have recently been synthesised. These analogues often have conformational constraints incorporated into their sequence. For instance, the linear agonists [L and D-Pro9]SP6-11 (l) & (2) and [SucAsp6, NMePhe8]SP6-11 (3) have receptor subtype selectivities for NK1, NK2 and NK3 respectively. The novel cyclic peptides [GlnTrpPheGlyLeuMet] (4) and [GlnTrpPheGly[ANC-2]MetLeu] (5), and their linear counterpart AcLeuMetGlnTrpPheGlyNH2 (6) have high antagonist activity for the NK2 receptor. The NK2 receptor is the preferred subtype for endogenous agonist neurokinin A (7). Knowing their conformational properties and rationalising these against their activities, may provide the basis for understanding the process of molecular recognition and lead to directed drug design. The conformational properties of these peptides in DMSO and methanol were investigated using 1 and 2D NMR techniques. Particularly important was the application of the recently developed ROESY and TOCSY experiments, which greatly simplify the assignment and conformational analysis of these compounds. Distinct conformations were found for the linear analogues (1), (2) and (3). This was shown to be due to cis/trans isomerism about the N-alkylated peptide bond. However, despite the known turn preferences for the constraints incorporated into these peptides, no global conformational characteristics were identified. All the linear peptides were found to exist in a chiefly extended state in DMSO. Peptides (1) and (2) occupied 'random coil' conformational space in methanol. The cyclic peptides (4) and (5) exhibited specific conformational preferences. Using restraints provided by their NMR parameters, molecular modelling showed a ss II turn in the LeuMet region for both of these peptides. The similarity in conformation of these cyclics may explain their analogous pharmacology. The synthesis of novel Substance P analogues with a beta-lactam constraint in the (RS)Phe[ANC-2]Gly position is presented, together with the attempted cyclisation of the resulting linear peptides.

L’objectif de cette thèse est d’étudier le rôle de neuropeptides cérébraux, telle que la Neurokinin B codée par le gène tac3, dans le contrôle de la reproduction d’une espèce en danger, l’anguille Européenne, Anguilla anguilla. La maturation sexuelle de l’anguille est bloquée à un stade prépubertaire avant la migration océanique. Etant donnée sa position phylogénétique basale parmi les téléostéens, l’anguille est un modèle pertinent pour étudier l’évolution moléculaire et fonctionnelle de neuropeptides d’intérêt. Deux gènes paralogues tachykinine 3 (tac3) ont été identifiés dans le génome de l’anguille, chacun codant pour deux peptides. Ces gènes paralogues résultent de la duplication complète du génome spécifique aux téléostéens, comme le montrent les analyses phylogénétiques et synténiques. Les analyses de qPCR montrent que les deux gènes sont exprimés dans le cerveau. Les quatre peptides d’anguille ont été synthétisés et testés sur des cultures primaires de cellules hypophysaires d’anguille. Les quatre peptides inhibent l’expression de l’hormone lutéinisante et d’un récepteur à la gonadolibérine, révélant un double rôle inhibiteur dans le contrôle de la reproduction
The aim of this PhD is to investigate the role of brain neuropeptides, such as neurokinin B, encoded by tac3 gene, in the control of reproduction of an endangered species, the European eel, Anguilla anguilla. The sexual maturation of the eel is blocked at a prepubertal stage before the oceanic migration. Due to its basal phylogenetic position among teleosts, the eel is also a relevant model for studying molecular and functional evolution of key neuropeptides. Two tachykinin 3 (tac3) paralogous genes were identified in the eel genome, each encoding two peptides. These paralogs result from the teleost-specific whole genome duplication, as shown by phylogeny and synteny analyses. Both genes are expressed in the brain as shown by qPCR. The four eel peptides were synthesized and tested on primary cultures of eel pituitary cells. The four peptides inhibited the expression of luteinizing hormone and gonadotropin-releasing hormone receptor, revealing a dual inhibitory role in the control of reproduction

The purpose of this series of investigations was to elucidate the endogenous control mechanisms active at primary afferent terminals of nociceptive origin in the dorsal laminae of the spinal cord grey matter. Such studies may enable the researcher to make certain conclusions about endogenous mechanisms of pain control in vivo. Furthermore, these studies may help to identify novel areas for the development of analgesic drugs or protocols of therapeutic value in both human and veterinary pain management. Experiments were centred on one particular family of neuropeptides, the tachykinin peptides, and their release in response to peripheral noxious stimulation as determined by the antibody microprobe technique. A review of the anatomy and physiology of tachykinins in the spinal cord is presented at the start of this thesis. The antibody microprobe technique itself is also fully described. Various means of moduling tachykinin release pharmacologically were tested and are presented in this thesis. The results presented here relate to studies on 1) morphine, 2) noradrenaline and the imidazoline derivative drug, medetomidine, 3) neuropeptide Y. A short review on the pharmacological actions of each of these drugs is included. All the results presented are derived from experiments on barbiturate anaesthetised, spinalised cats.

En aquesta tesi doctoral, s’han aplicat metodologies biofísiques per estudiar i caracteritzar l'estructura dels pèptids substància P (SP), neuroquinina A (NKA) i scyliorinina I (ScyI), que pertanyen a la família de les taquiquinines (TK), així com la seva manera d'interacció amb membranes model. Els pèptids TKS són agonistes dels receptors de neuroquinina acoblats a la proteïna G. Els TKS estan involucrats en diversos processos fisiològics i malalties com el càncer, malalties neurodegeneratives i respostes inflamatòries, i això els converteix en un objecte de gran interès per a l'estudi estructural en la recerca d'agents terapèuticament rellevants. Els tres pèptids SP, NKA i ScyI mostren una seqüència comuna en el domini C-terminal i es diferencien en el seu domini N-terminal. Per tant, sorgeixen preguntes: Què determina el seu diferent potencial d’activació? Quina és la conformació activa dels pèptids quan interactuen amb el receptor de NK per transferir la senyal? Quina és la seva manera d'interacció amb la superfície de la membrana? La superfície de la membrana, afecta la conformació del pèptid? Tenen els pèptids capacitat d’associar-se i si és així, quin tipus d'estructures formen i en quines condicions? A la primera part dels resultats i discussió, s’utilitzen diferents mètodes d'espectroscòpia de fluorescència, per estudiar les interaccions dels sistemes de membranes amb TKS mimètics (micel·les SDS i vesícules DMPG / DMPC). Les cadenes laterals Trp i Phe CN es van utilitzar com fluoròfors intrínsecs, mentre la fluoresceïna fosfatidiletanolamina (FPE) i els lípids bromats es van utilitzar com sondes externes. Ja que SP i NKA no posseeixen el residu Trp, es va substituir el residu Phe en la posició 6 i 8 amb Trp, respectivament per als pèptids SP i NKA. L’espectroscòpia CD va confirmar la similitud de l'estructura general de SP i NKA amb els seus anàlegs. A més, mitjançant l'ús d'espectroscòpia de fluorescència de Trp (principalment λmax), es demostra que, en solució i en estat sub-micel·lar de SDS, la cadena lateral de Trp en SPW i NKAW està en un entorn hidròfob, mentre que apareix desplaçat a ambient hidrofílic després de la formació de micel·les. D'altra banda, l'espectroscòpia de CD mostra la transició de l’estructura PPII dominant en solució, a estructura α helicoïdal (en cas de SP i ScyI) o a la barreja de conformació desordenada i d’hèlix α (en el cas de NKA) una vegada es formen micel · les. Aplicant una metodologia de fluorescència diferent, ha sigut possible separar el procés d'unió del pèptid a la superfície de la membrana, del procés d'inserció del pèptid / plegament al nucli hidrofòbic. SP, NKA i ScyI interaccionen amb DMPC i amb els liposomes amb càrrega negativa DMPG. No obstant això, les afinitats d'unió dels pèptids als liposomes de DMPC estan en el rang de 20-40 vegades menor, en comparació amb les afinitats a DMPG. A més, les afinitats d'unió de SP, NKA i ScyI correlacionen amb les seves càrregues netes, quan interactuen amb DMPG, però no amb DMPC. A la segona part dels resultats i discussions, els factors que regeixen les estructures secundàries de les taquiquinines en estat monomèric (com el medi, la càrrega neta del pèptid i la càrrega superficial de la membrana) s’estudien per espectroscòpia CD. A més, es va aplicar la dispersió de raigs X d'angle petit per determinar l'estructura secundària del NKA en solució. En solució aquosa, l'estructura dominant de totes les taquiquininas és PPII. Per espectroscòpia d'infraroig, l’estructura flexible desordenada es va detectar per les taquiquininas en una concentració de 1,5 mM en solució. A més, en aquestes condicions, les estructures girs β i estructures esteses de làmina β es van detectar, respectivament, per SP i ScyI. En TFE, l'estructura dominant de les taquiqinines és helicoïdal, indicant la propensió helicoïdal intrínsec dels pèptids. Igual que en solució, les taquiquinines mostren estructura PPII en presència de vesícules d’ions híbrids. En els liposomes carregats negativament, SP i ScyI posseeixen estructura α mentre NKA mostra una barreja d’estructura desordenada i conformacions en hèlix α. Els canvis conformacionals de les taquiquinines en augmentar la fracció de DMPG de la vesícula composta de DMPC / DMPG demostren clarament que l’estructura α dels pèptids depèn fortament de la quantitat relativa de DMPG aniònic en les vesícules. Això reflecteix la importància de les interaccions electrostàtiques dels pèptids amb els caps de la membrana. A la part III dels resultats i discussió, s'estudia l'estat d'agregació de les taquiquinines. Es mostra que les taquiquinines són capaces de formar estructures fibril·lars. En solució, les taquiquinines a una concentració de 3 mM formen fibril·les amb diferent morfologia. En SP, es veuen llargues fibril·les retorçades i filaments individuals rectes, mentre que en ScyI i NKA només s’observen fibril·les rectes. Les taquiquinines en una concentració per sobre de 1,5 mm formen fibril·les immediatament en presència de vesícules de càrrega negativa, mentre que no es van detectar en les fibril·les de DMPC. Aquest fet indica la importància de les càrregues negatives en el procés de fibril·lizació. L’espectroscòpia FTIR mostra un augment significatiu de l'estructura de làmina β per les taquiquinines a una concentració de 3 mM i en presència de les vesícules de DMPG, que s'atribueix a la formació de fibril·les. A més, en aquesta condició, FTIR mostra estructura helicoïdal en tots els TKS i algunes conformacions de gir β per SP i NKA. Sobre la base de TEM i espectroscòpia de CD, es mostra que la fibril·lizació de SP (100 mM) es produeix durant la transició d'estructura PPII a fulla β després de la incubació en concentracions de SDS prop de la CMC. En contrast, SPW no mostra fibril·lizació en les mateixes condicions. Sobre la base d'assaig THT, es van detectar fibril·les amiloides per NKA però de moment no tenim cap evidència sobre la formació amiloide en SP i ScyI. Els resultats indiquen que la formació d'amiloide en NKA disminueix a pH alcalí. En contrast, NKAW és capaç de formar fibril·les amiloides a pH àcid i alcalí però no a pH neutre. Analitzant l'activitat metabòlica de la línia cel·lular PC12 mitjançant la prova de TMM, es demostra que NKA a una concentració de més de 25 µM pot induir toxicitat, mentre que no es va observar cap disminució significativa de l'activitat metabòlica en presència de fins a 250 µM de SP o de ScyI
In this doctoral thesis, biophysical methodologies were applied to study and characterize the structures of substance P (SP), neurokinin A (NKA) and scyliorhinin I (ScyI) peptides, which belong to the tachykinin (TK) family, and their mode of interaction with model membranes. The TKs peptides are agonists of Neurokinin G-protein coupling receptors. TKs are involved in several physiological processes and diseases such as neurodegenerative disorders, cancer and inflammatory responses, what make them an object of high interest for structural study in search of relevant therapeutically agents. The three peptides SP, NKA and ScyI are homologous sharing common C domain –terminal and differ in their N terminal domain. Therefore, questions arise: What determine their different activity potential? What is the active conformation of the peptides when they interact with the NK receptor to transfer the signal? What is their mode of interaction with the membrane surface? Does the membrane surface affect the peptide conformation? Do the peptides self associate and if they have these ability, what kind of structure they form and at which conditions? In part I of the Results and Discussion, by using different fluorescence spectroscopy approaches, the mode of the interactions of TKs with membrane mimetic systems (SDS micelles and DMPG/DMPC vesicles) were studied. The Trp and Phe-CN amino acids were used as intrinsic fluorophores, while fluorescein phosphatidylethanolamine (FPE) and brominated lipids were used as external probes. Since SP and NKA lack the intrinsic Trp residue, we substituted Phe residue in position 6 and 8 with Trp, respectively for SP and NKA peptides. CD spectroscopy confirmed the similarity of the overall structure of SP and NKA with their analogues. Furthermore, by using Trp fluorescence spectroscopy (mainly λmax), we understood that in solution and in sub-micellar state of SDS, the Trp side chain of SPW and NKAW are in a hydrophobic environment, while they appear displaced to hydrophilic environment upon formation of micelles. On the other hand, CD spectroscopy show the transition of the dominant PPII helical structure in solution to α helical structure (in case of SP and ScyI) or to the mixture of unordered and α helical conformation (in the case of NKA) upon formation of micelles. Applying different fluorescence methodology it was possible to separate the process of peptide binding to a membrane surface from the process of peptide insertion/folding into the hydrophobic core. SP, NKA and ScyI bind to both zwitterionic DMPC and negatively charged DMPG liposomes. However, binding affinities of the peptides to DMPC liposomes are in the range 20-40 times lower, compared to the affinities to DMPG. Moreover the binding affinities of SP, NKA and ScyI correlate with their net charges, when they interact with DMPG, but not with DMPC. In part II of the Results and Discussions, the factors governing the secondary structures of the tachykinins in monomeric state (such as environment, peptide net charge and membrane surface charge) are studied by CD spectroscopy. Moreover, small angle X-ray scattering was applied to determine the NKA secondary structure in solution. In aqueous solution, the dominant structure of all tachykinins is PPII. By FTIR spectroscopy, flexible unordered structure was detected for tachykinins in a concentration of 1.5 mM in solution. Moreover, in these condition, β turn and extended β sheet structures were detected, respectively for SP and ScyI. In the TFE the dominant structure of tachykinins is alpha helical which indicates the intrinsic helical propensity of peptides. Like in solution, tachykinins have PPII structure in the presence of the zwitterionic vesicles. In negatively charged liposomes, SP and ScyI are in α helical structure while NKA shows a mixture of the unordered and α helical conformations. Conformational changes of tachykinins upon increasing of the DMPG fraction of the vesicle composed of DMPC/DMPG demonstrate clearly that the α helical fold of peptides strongly depends on the relative amount of anionic DMPG in the vesicles and reflecting the importance of the electrostatic interactions of peptides with headgroup of the membrane. In part III of the Results and Discussion, the aggregation state of the tachykinins is studied. We understood that tachykinins are able to form fibrillar structures. In solution, 3 mM of tachykinins formed fibrils with different morphology. In SP long twisted fibrils and straight single filaments were seen while in ScyI and NKA fibrils are only single-straight. Tachykinins in a concentration of above 1.5 mM formed fibrils immediately in the presence of negatively charge vesicles, while no fibrils were detected in DMPC for any tachykinins. This fact indicates the importance of the negatively surface charges on fibrillization. FTIR spectroscopy shows a significant increase of the β sheet structure for tachykinins in a concentration of 3 mM and in the presence of the DMPG vesicles which is attributed to the fibrils formation. Moreover, in this condition, FTIR shows helical structure in all TKs and some β turn conformations for SP and NKA. Based on TEM and CD spectroscopy, we understood that fibrillization of SP (100 µM) occurs upon transition of PPII structure of peptide to β sheet after incubation in SDS concentrations close to CMC. In contrast, SPW was not able to make fibrils in the same condition. Based on ThT assay, amiloid fibrils were detected for NKA but at the moment we do not have any evidence about the amiloid formation of SP and ScyI. Moreover we found that amyloid formation of NKA decreases at alkaline pH. In contrast, NKAW is able to form amyloid fibrils at acidic and alkaline pH but not at the neutral pH. Analyzing of the PC12 cell line metabolic activity by TMM test indicates that NKA in a concentration of more than 25 µM can induce toxicity, while no significant decrease of metabolic activity was seen in the presence of up to 250 µM SP or ScyI.

Eight cyclic analogues of Substance P were made in order to investigate the conformation of the C-terminal end of the peptide. These analogues were designed to test three literature models describing the active conformation of substance P. Although the potencies of the analogues were low (in the micromolar range), our results support Cotrait's and Hospital's model (1986). Several substance P antagonists were synthesized. These compounds did not demonstrate agonistic activity nor anatagonistic activity. The tryptophan side chain is contributing to the antagonistic activity of these analogues, and not just the chirality of the α-carbon. Highly potent and selective photoaffinity ligands of H-Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE) and D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH₂ (CTP) were synthesized. These compounds will be useful in the isolation of δ and μ opioid receptors. Several new amino acids designed and synthesized to contain both the natural amino acid side chain and a thiol group which can be used to make disulfide constraints. The racemic amino acids made were as follows: (1) 2-amino-4-methyl-2- [(p-methylbenzyl)thiomethyl] pentanoic acid; (2) 2-amino-2- [(p-methylbenzyl)thiomethyl] -3-phenylpropanoic acid; (3) 2-amino-e- [(p-methylbenzyl)thio] pentanoic acid; and (4) 2-amino-3- [(p-methylbenzyl)-thio] -3-phenyl-pentanoic acid. These amino acids will be useful in the conformational restriction of peptides. To investigate the δ-opioid receptor conformation proposed for DPDPE by Hruby et al. (1988) and the μ-opioid receptor conformation proposed for Tyr-c [Abu₂,Gly,Phe,Leu] by Mierke et al. (1988), constrained phenylalanine amino acids were incorporated into H-Try-D-Pen-Gly-Phe-D-Pen-OH (DPDPE) in the four position. Our results indicate that these models are correct. And in an investigation into the physical-chemical properties of the delta opioid receptor, our results suggest that the δ receptor topochemical site for the Phe⁴ residue contains a partial positive charge on its surface and has specific steric requirements.

Doctor of Philosophy (PhD)
Little is known about the role that tachykinins, such as substance P and its receptor, the neurokinin-1 receptor, play in the generation of sympathetic nerve activity and the integration within the ventrolateral medulla (VLM) of many vital autonomic reflexes such as the baroreflex, chemoreflex, somato-sympathetic reflex, and the regulation of cerebral blood flow. The studies described in this thesis investigate these autonomic functions and the role of tachykinins through physiological (response to hypercapnoea, chapter 3), anatomical (neurokinin-1 receptor immunohistochemistry, chapter 4) and microinjection (neurokinin-1 receptor activation and blockade, chapters 5 and 6) experiments. In the first series of experiments (chapter 3) the effects of chemoreceptor activation with hyperoxic hypercapnoea (5%, 10% or 15% CO2 in O2) on splanchnic sympathetic nerve activity and sympathetic reflexes such as the baroreflex and somato-sympathetic reflex were examined in anaesthetized rats. Hypercapnoea resulted in sympatho-excitation in all groups and a small increase in arterial blood pressure in the 10 % CO2 group. Phrenic nerve amplitude and phrenic frequency were also increased, with the frequency adapting back to baseline during the CO2 exposure. Hypercapnoea selectively attenuated (5% CO2) or abolished (10% and 15% CO2) the somato-sympathetic reflex while leaving the baroreflex unaffected. This selective inhibition of the somato-sympathetic reflex while leaving the baroreflex unaffected was also seen following neurokinin-1 receptor activation in the rostral ventrolateral medulla (RVLM) (see below). Microinjection of substance P analogues into the RVLM results in a pressor response, however the anatomical basis for this response is unknown. In the second series of experiments (chapter 4), the distribution of the neurokinin-1 receptor in the RVLM was investigated in relation to catecholaminergic (putative sympatho-excitatory “C1”) and bulbospinal neurons. The neurokinin-1 receptor was demonstrated on a small percentage (5.3%) of C1 neurons, and a small percentage (4.7%) of RVLM C1 neurons also receive close appositions from neurokinin-1 receptor immunoreactive terminals. This provides a mechanism for the pressor response seen with RVLM microinjection of substance P analogues. Neurokinin-1 receptor immunoreactivity was also seen a region overlapping the preBötzinger complex (the putative respiratory rhythm generation region), however at this level a large percentage of these neurons are bulbospinal, contradicting previous work suggesting that the neurokinin-1 receptor is an exclusive anatomical marker for the propriobulbar rhythm generating neurons of the preBötzinger complex. The third series of experiments (chapter 5) investigated the effects of neurokinin-1 receptor activation and blockade in the RVLM on splanchnic sympathetic nerve activity, arterial blood pressure, and autonomic reflexes such as the baroreflex, somato-sympathetic reflex, and sympathetic chemoreflex. Activation of RVLM neurokinin-1 receptors resulted in sympatho-excitation, a pressor response, and abolition of phrenic nerve activity, all of which were blocked by RVLM pre-treatment with a neurokinin-1 receptor antagonist. As seen with hypercapnoea, RVLM neurokinin-1 receptor activation significantly attenuated the somato-sympathetic reflex but did not affect the sympathetic baroreflex. Further, blockade of RVLM neurokinin-1 receptors significantly attenuated the sympathetic chemoreflex, suggesting a role for RVLM substance P release in this pathway. The fourth series of experiments (chapter 6) investigated the role of neurokinin-1 receptors in the RVLM, caudal ventrolateral medulla (CVLM), and nucleus tractus solitarius (NTS) on regional cerebral blood flow (rCBF) and tail blood flow (TBF). Activation of RVLM neurokinin-1 receptors increased rCBF associated with a decrease in cerebral vascular resistance (CVR). Activation of CVLM neurokinin-1 receptors decreased rCBF, however no change in CVR was seen. In the NTS, activation of neurokinin-1 receptors resulted in a biphasic response in both arterial blood pressure and rCBF, but no significant change in CVR. These findings suggest that in the RVLM substance P and the neurokinin-1 receptor play a role in the regulation of cerebral blood flow, and that changes in rCBF evoked in the CVLM and NTS are most likely secondary to changes in arterial blood pressure. Substance P and neurokinin-1 receptors in the RVLM, CVLM and NTS do not appear to play a role in the brainstem regulation of tail blood flow. In the final chapter (chapter 7), a model is proposed for the role of tachykinins in the brainstem integration of the sympathetic baroreflex, sympathetic chemoreflex, cerebral vascular tone, and the sympatho-excitation seen following hypercapnoea. A further model for the somato-sympathetic reflex is proposed, providing a mechanism for the selective inhibition of this reflex seen with hypercapnoea (chapter 3) and RVLM neurokinin-1 receptor activation (chapter 5). In summary, the ventral medulla is essential for the generation of basal sympathetic tone and the integration of many vital autonomic reflexes such as the baroreflex, chemoreflex, somato-sympathetic reflex, and the regulation of cerebral blood flow. The tachykinin substance P, and its receptor, the neurokinin-1 receptor, have a role to play in many of these vital autonomic functions. This role is predominantly neuromodulatory.

Excessive secretion of the peptide, neurokinin B (NKB) into the circulation during the third trimester of pregnancy has been reported in women with the gestational disease, pre-eclampsia (PE). Therefore, the objective of this work was to develop a clinic friendly two-site enzyme-linked immunosorbent assay (ELISA) to detect levels of NKB directly from plasma samples of pregnant women and to determine the mechanism's of precursor processing. !t was hoped to confIrm the link between elevated levels of this peptide and PE. Initial assays of pregnancy plasmas using the two-site NKB ELISA were inconclusive in determining a link, as were those conducted using an ELISA developed to the adjacent C-terminal portion of the precursor peptide. Consequently, extraction and purification ofNKB from the human placenta was undertaken, with separation on the basis of size and hydrophobicity. A commercial one-site competitive NKB radioimmunoassay was used at each stage to identify immunoreactive fractions and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS) ,was performed on the purified samples. These experiments indicated that NKB extracted from the placenta had a higher molecular weight than NKB reported from the brain. The mass difference indicated one of two potential posttranslational modifications in the placenta of either a di-methylation or phosphocholine (PC) modification. These hypotheses were investigated through the development of specific two-site ELISAs. Putative processing mechanlsms were also explored through synthetic peptide digests with the enzyme carboxypeptidase B. Analysis of the digests via thin layer chromatography and MS proved that the dimethylation hampered exopeptidase processing. Quantitative PCR for the closely-related membrane-bound enzyme carboxypeptidase M, showed highest expression in the placenta. Synthetic NKB peptides carrying a dirnethylation however, had no biological activity either in in vitro ligand binding studies at the NK3 recepto~ or in in vivo models involving both rats and mice. Larger scale placental purification disproved the dimethylation theory and provided evidence for the PC modification thus far unprecedented in mammals, where pilot assays detected signals in both placental extracts and plasma samples. Collectively, these results provide evidence for the existence ofa unique placental NKB peptide, the structure of which has yet to be fully elucidated, as does the exact nature of the involvement ofthis peptide in PE.

Unilateral exercise can produce certain contralateral strength effects. Deleterious events can be cross-transferred as well, as illustrated by a strict symmetry in some chronic inflammatory diseases. To date, knowledge on the effects of marked overuse of skeletal muscles is limited, and there is largely no information if unilateral overuse affects the contralateral muscles. In view of this, the present study was undertaken to test the hypothesis that unilateral muscle overuse causes alterations in tissue structure and the tachykinin system, with a focus on substance P (SP), not only in the exercised muscles, but also in the contralateral muscles. SP is a well-known neuromodulator that is known to be proinflammatory. An experimental rabbit model with unilateral muscle overuse of the soleus and gastrocnemius muscles caused by exercise via electrical muscle stimulation (E/EMS) was used. In total, 40 rabbits were randomly divided into seven groups of which two groups served as controls. The rabbits were anaesthetized and then set on a “kicking machine” to perform exercise via EMS for 2h every second day. Experimental periods for groups 1-3 were 1, 3 and 6w, respectively, whereas groups 4-6 were exercised for 1w but also subjected to injections in the peritendinous tissue with SP, NaCL, Captopril (C), an ACE inhibitor, and DL-Thiorphan (Th) which inhibits the activity of neural endopeptidase. One group was not subjected to the experiment at all. The day after the last session of E/EMS, the soleus muscle and the gastrocnemius muscle from both legs were collected for analysis. Alterations in muscle structure and the tachykinin system were analyzed with enzyme and immunohistochemical techniques, in situ hybridization and EIA methods. After 1w of E/EMS, focal areas of the exercised muscles contained a mild infiltration of inflammatory cells (myositis) and small morphological changes. After 3 and 6w of E/EMS, distinct myositis and muscle changes were bilaterally present in focal areas of both muscles. The structural changes, which mainly were observed in myositis areas, consisted of increased fiber size variability, split fibers, internal myonuclei, necrotic fibers, fibrosis, fat infiltration, and small fibers containing developmental MyHCs. Bilateral morphological changes, such as loss of axons, were also observed in nerves. In addition, expressions of tachykinin and the SP-preferred receptor, the neurokinin-1 (NK-1R), were bilaterally upregulated in nerve structures and blood vessel walls.  Infiltrating white blood cells exhibited tachykinin–like and NK-1R immunoreactivity. NK-1R immunoreactions were also found in necrotic and regenerating muscle fibers. The concentration of tachykinin (SP) was significantly increased in both soleus and gastrocnemius muscles after E/EMS. There was a significant correlation between the two sides in concentration of tachykinin and in the intensity of tachykinin-like immunoreaction in blood vessel walls. The muscle fiber size and capillary supply of fibers were bilaterally decreased after 3w of EMS. The myositis areas contained an increased number of vessels with a larger size than capillaries, while areas with increased amount of connective tissue contained a very low number of capillaries. A bilateral fiber type shift against a lower proportion of slow MyHCI fibers and higher proportion of fast MyHCII fibers was observed in both muscles. The local injections of C+Th and SP+C+Th led to marked structural changes in the muscle tissue and marked increased NK-1R and tachykinin-like immunoreactivity in the myositis areas and increased tachykinin concentration in the tissue. In conclusion, the repetitive unilateral muscle overuse caused by E/EMS led overtime to muscle injury and myositis. The affected areas contained both degenerative and regenerative alterations in the muscle tissue and nerves, and an upregulation of the tachykinin system. Most interestingly, the changes not only occurred in the exercised side, but also in the homologous contralateral muscles. The tachykinin system appears to be an important factor in the processes of crossover effects.

La surrénale de la grenouille Rana ridibunda est richement innervée par un réseau de fibres contenant deux tachykinines, i. E. La ranakinine et la [Leu3, Ile7]neurokinine A. Ces deux tachykinines stimulent la sécrétion des corticostéroïdes in vitro. L'objectif du présent travail était de déterminer le mode d'action de la ranakinine sur la surrénale de grenouille et d'analyser les mécanismes de transduction associés à son effet corticotrope. Les observations en microscopie optique et électronique ont montré que les fibres A tachykinines sont étroitement associées aux seules cellules chromaffines. Par ailleurs, la ranakinine stimule la sécrétion de stéroïdes par des tranches de surrénale mais non par des cellules dissociées. Le marquage cytoautoradiographique des cellules surrénaliennes en culture primaire indique que les sites de liaison aux tachykinines sont exclusivement localisés sur les cellules chromaffines. Enfin, la mesure des concentrations cytosoliques de calcium [Ca2+]i par la technique de microfluorimétrie a montré que les tachykinines sont dépourvues d'effet sur les cellules stéroïdogènes alors qu'elles provoquent une augmentation massive, transitoire et dose-dépendante de la [Ca2+]i dans les cellules chromaffines. L'ensemble de ces résultats suggère que, chez les amphibiens, les tachykinines exercent une stimulation indirecte de la stéroïdogénèse surrénalienne par activation des cellules chromaffines. L'effet des tachykinines met en jeu des récepteurs spécifiques, couplés positivement à une phospholipase C, dont les caractéristiques pharmacologiques sont intermédiaires entre celles des récepteurs NK-1 et NK-2 des mammifères. L'activation de ces récepteurs se traduit par une production d'IP3 qui provoque une mobilisation de calcium a partir des pools intracellulaires. Le calcium ainsi libéré va vraisemblablement favoriser l'exocytose des granules chromaffines contenant divers facteurs capables de stimuler l'activité des cellules corticosurrénaliennes adjacentes.

Thesis (Ph. D.)--University of Sydney, 2007.
Title from title screen (viewed 1 November 2007). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Discipline of Physiology, Faculty of Medicine. Degree awarded 2007 ; thesis submitted 2006. Bibliography: leaves 239-284. Also issued in print.

Tachykinins are a family of neuropeptides characterised by the conserved C-terminal motif FXGLM-NH2, where X represents a hydrophobic amino acid. Substance P (SP) a member of the tachykinin family has recently been shown to stimulate platelet aggregation and a SP-like immunoreactivity has been demonstrated in platelets and shown to be released upon platelet activation, suggesting that SP may act as a secondary platelet agonist. In recent years a gene encoding new members of the tachykinin family has been identified named TTAC4, which unlike the classical tachykinins is predominantly expressed in the periphery, with high expression in the megakaryocytic cell line HEL. The predicted products of the human TAC4 gene, endokinins A and B share high homology with SP and display similar binding characteristics as SP for the neurokinin-1 (NKl) receptor, which is present on the platelet surface. The high sequence homology between endokinins A and B and SP renders them indistinguishable using SP-imunoassays raising the possibility that platelets may be a source of endokinins. The purpose of this study was to assess the roles of peripheral tachykinins in regulating platelet function.

Transgenic and knockout mouse models of disease and gene function have revolutionised the field of biomedical research and the targeted mutations of genes expressed in the brain are revealing the mechanisms underlying normal behaviour and behavioural abnormalities which has led to the development of behavioural neuroscience. However, it is also clear that the phenomena know as "redundancy" can limit the effectiveness of traditional knockout models by the effects of compensatory mechanisms. This thesis utilises transgenic and knockout mouse models to explore the role of the tachykinins in the pathogenesis of anxiety, depression and epilepsy focusing on the TACl gene products and their functionality via the TACRl (NKl) receptor. The generation of a novel double knockout line via the cross breeding of single knockout models relevant for the tachykinin signalling pathways circumnavigates the problems associated with redundancy and also reveals that this phenomena is contributing to the data generated using the single knockouts. The use of these animals in a variety of experimental models to compare their function with the original "parental" knockouts and their corresponding wildtype controls reveals novel models for the action of the tachykinins in epilepsy and suggests a correlation with serotonin levels with an ultimate effect on the behavioural responses observed.

Parmi les nombreux peptides identifies dans les voies aeriennes, les tachykinines, notamment la substance p et la neurokinine a, suscitent beaucoup d'interet en raison de leurs effets pulmonaires bronchoconstricteurs et pro-inflammatoires et de leur possible implication dans la maladie asthmatique. Dans ce travail, nous avons recherche le role des tachykinines dans l'hyperreactivite et les phenomenes inflammatoires des voies aeriennes observees dans trois modeles experimentaux chez le cobaye: un modele d'exposition a un agent anti-parasitaire, l'isethionate de pentamidine, un modele d'exposition a un agent infectieux, l'endotoxine, et un modele d'exposition a un agent toxique, l'yperite (moutarde au soufre). Nous avons montre que les tachykinines etaient impliques dans la bronchoconstriction et l'augmentation de permeabilite vasculaire tracheo-bronchique provoquees par l'administration intra-tracheale d'isethionate de pentamidine chez le cobaye. Nous avons egalement montre une implication des tachykinines dans l'hyperreactivite bronchique et l'afflux de cellules inflammatoires provoquees par l'administration intra-tracheale d'endotoxine. Dans le modele d'intoxication par l'yperite, nous avons mis en evidence une hyperreactivite des voies aeriennes a la substance p 14 jours apres l'administration du toxique, liee a la diminution de l'activite de l'endopeptidase neutre de l'epithelium tracheal. Dans ce dernier modele, les glucocorticoides inhibent l'hyperreactivite a la substance p, en restaurant l'activite de l'endopeptidase neutre epitheliale. Ces resultats montrent que l'hyperreactivite et les phenomenes inflammatoires des voies aeriennes provoquees par les tachykinines peuvent etre la consequence soit d'une liberation accrue de tachykinines, comme le montrent nos deux premiers modeles experimentaux, soit d'une diminution de leur degradation, du fait d'une reduction de l'activite de l'endopeptidase neutre, comme dans le modele d'intoxication par l'yperite

Les récepteurs couplés aux protéines G (RCPGs) représentent la plus grande famille de récepteurs cellulaires de surface. Les RCPGs sont constitués par une seule chaîne poly-peptidique comportant sept régions hydrophobes transmembranaires, associée à un groupe hétéro-trimérique de protéines intracellulaires : les protéines G. Ces protéines sont constituées d'une sous-unité Ga liée au GDP et de deux sous-unités Gbg indissociables. Lorsqu'ils sont activés par leur ligand, les RCPG catalysent l'échange du GDP par du GTP. Les protéines Ga d'une part, et Gbg d'autre part, deviennent alors capables de moduler l'activité de différents effecteurs intracellulaires : enzymes, canaux, échangeurs ioniquesLa signalisation des RCPGs, médiée par leurs agonistes, requière donc un changement conformationnel dans la protéine récepteur impliquant au moins deux sites de liaison topographiquement distincts, l'un pour l'agoniste, l'autre pour la protéine G. Ces récepteurs sont ainsi, par nature, des protéines allostériques (du grec 'autre site'). Il est maintenant connu que les RCPGs peuvent aussi former des complexes ternaires avec d'autres ligands ou des protéines 'accessoires' et présenter ainsi des modifications dans les propriétés de liaison et/ou de signalisation en rapport avec le complexe binaire agoniste-récepteur. Les sites des ligands allostériques des RCPGs représentent des cibles thérapeutiques de choix. En effet, les modulateurs allostériques possèdent des avantages théoriques par rapport aux ligands du site dit primaire (ligand endogène) tels que un effet plafonnable ou une meilleure sélectivité potentielle des sous-types de RCPGs (Christopoulos and Kenakin, 2002). De par la nature non compétitive du phénomène allostérique, la détection et la quantification de tels effets demande la combinaison d'approches expérimentales multiples telles que des études de liaison du ligand à l'équilibre, de cinétiques de liaison et de réponses fonctionnelles. Ce travail de thèse présente principalement la recherche et la caractérisation de tels modulateurs sur le récepteur des tachykinines de type 2 (NK2) vis-à-vis de son agoniste principal, la neurokinine A (NKA). La neurokinine NKA est un neuropeptide appartenant à la famille des tachykinines incluant également la substance P et la neurokinine B. Les tachykinines sont libérées dans de nombreuses régions du système nerveux central et périphérique et agissent sur des récepteurs couplés aux protéines G, les récepteurs aux tachykinines NK1, NK2 et NK3. La stimulation de ces récepteurs par des agonistes sélectifs mène à de multiples évènements intracellulaires tels que l'élévation de la concentration de calcium intracellulaire via l'activation de la phospholipase C, l'accumulation d'AMPc, l'activation de la cascade des MAP kinases, la stimulation du métabolisme d'acide arachidonique ou la stimulation des phospholipases (Quartara et al. , 2001). D'autre part, il a été montré in vitro que le récepteur NK1R pouvait se coupler non seulement à Gaq/11 mais aussi à GaS et Gao (Roush and Kwatra, 1998). Le récepteur NK2, qui présente la meilleure affinité pour la NKA, est fortement exprimé dans les muscles lisses respiratoires et les appareils gastro-intestinal et génital. Son expression dans le système nerveux central est restée longtemps matière à débat mais de récentes études ont démontré que l'ARNm du NK2R avait une expression détectable dans diverses régions du cerveau humain, incluant le noyau caudé, le putamen, l'hippocampe, la substance noire et le cortex cérébral. La distribution de l'ARNm du récepteur NK2R dans le cortex révèle un expression majoritairement frontale et temporale comparée aux régions occipitales et pariétales (Bensaid et al. , 2001). Les antagonistes des neurokinines ont été le sujet de nombreuses investigations au niveau fondamental et industriel depuis l'association de leurs effets centraux et périphériques sur le traitement de nombreuses pathologies telles que l'asthme, les douleurs chroniques comme la fibromyalgie ou l'arthrite rumathoide, l'émésie et certains désordres psychiatriques (Lecci et al. , 2000; Quartara and Maggi, 1998). Le criblage de molécules issues de la chimiothèque de la Faculté de Pharmacie de Strasbourg a été réalisé sur cellules vivantes par la technique de FRET en cinétiques de liaison de l'agoniste NKA lié au fluorophore bodipy sur le récepteur NK2 fusionné en N-terminal à la protéine fluorescente GFP (Vollmer et al. , 1999). A l'image de ce qui a été décrit pour les récepteurs muscariniques (Tucek and Proska, 1995) et sérotoninergiques (Massot et al. , 1996), nous avons identifié des molécules dont l'effet principal n'est pas d'entrer en compétition avec l'agoniste naturel du récepteur NK2R des tachykinines, mais d'accélérer sa vitesse de dissociation. [. . . ]

Trois tachykinines (la substance p, la neurokinine a et la neurokinine b) et trois recepteurs specifiques de ces peptides (nk-1, nk-2 et nk-3) ont ete caracterises chez les mammiferes. Sp arg-pro-lys-pro-gln-gln-phe-phe-gly-leu-met-nh#2 nk-1. Nka his-lys-thr-asp-ser-phe-val-gly-leu-met-nh#2 nk-2. Nkb asp-met-his-asp-phe-phe-val-gly-leu-met-nh#2 nk-3. Pour preciser les roles physiologiques associes a ces trois tachykinines, des agonistes selectifs de chacun des recepteurs sont necessaires. A partir d'un modele de conformation bvioaffine d'agonistes nk-1, deduit de l'analyse structurale de la substance p dans le methanol, des analogues conformationnellement contraints de la substance p ont ete synthetises. Une etude systematique de la conformation bioaffine de la partie c-terminale, a l'aide d'acides amines n-methyles et de proline a conduit a des agonistes selectifs du recepteur nk-1 (ex. : pro#9 sp). Les liaisons peptidiques de la region c-terminale ont ensuite ete remplacees par des chaines hydrocarbonees pour preciser l'enveloppe sterique des interactions substrat-recepteur. Suivant la meme methodologie, un agoniste hydrosoluble du recepteur nk-3 a ete propose: pro#7 nkb. D'autres resultats de correlation structure-affinite ont conduit a la synthese d'un analogue cyclique: cys#2#,#5 nkb. L'analyse conformationnelle par rmn de ces deux peptides a permis d'elaborer un modele de conformation bioaffine pour les agonistes nk-3. La synthese d'analogues de la neurokinine a, conformationnellement contraints par introduction d'acides amines n-methyles est finalement decrite

Numerous studies in experimental animals have emphasized the importance of tachykinin peptides in hypothalamic function. There is however, little information on the location of these peptides in the human brain. In the first study, in situ hybridization was used to map the distribution of neurons expressing the substance P (SP) or neurokinin B (NKB) gene transcripts. The distribution of neurons containing tachykinin mRNAs was found to be distinct and complementary: SP was the predominant tachykinin in the striatum, posterior hypothalamus and intermediate, ventromedial and mammillary nuclei; there were more NKB mRNA containing neurons than SP neurons in the magnocellular basal forebrain, the bed nucleus, and the preoptic/anterior hypothalamic regions. Comparable numbers of neurons expressing both mRNAs were identified in the infundibular nucleus and amygdala. Because numerous neurons containing NKB mRNAs were identified in the nucleus basalis of Meynert, it was next determined if NKB mRNA and choline acetyltransferase (ChAT) mRNAs are colocalized in this region. It was found that approximately 30% of the cholinergic neurons in the nucleus basalis also expressed NKB gene transcripts. This is the first identification of peptide colocalization in a significant population of magnocellular cholinergic neurons in the human basal forebrain. The nucleus basalis of Meynert plays an important role in higher brain functions in humans. There is considerable evidence suggesting that SP and gonadotropin releasing hormone (GnRH) neurons are anatomically and functionally connected in the human brain. In this study, double in situ hybridization with 35S-labeled SP receptor (SPR) and digoxigenin-labeled GnRH riboprobes was used to determine if GnRH neurons contain SPR mRNAs. The radiolabeled GnRH riboprobe hybridized with scattered neurons in the preoptic-septal regions and medial basal hypothalamus. A digoxigenin-labeled GnRH probe labeled cells in the medial basal hypothalamus, the primate control center for reproduction. SPR mRNA was identified in numerous magnocellular basal forebrain neurons, however, GnRH neurons containing SPR mRNAs were not identified. Although the possibility that SPR mRNA may be present in these cells but below the level of detection remains, present data suggests that a link between SP and GnRH neurons does not exist in the human brain.

L'yperite fait partie des armes chimiques dont le risque d'utilisation est le plus important en raison de la simplicite de sa synthese et de son emploi. La peau, l'il et l'appareil respiratoire constituent les principales cibles de cet agent alkylant. Bien que les lesions respiratoires soient la principale cause de mortalite et a l'origine de la morbidite la plus grave, leurs effets ont ete peu etudies. Dans ce travail, nous avons mis au point un modele animal pour 1) decrire les lesions respiratoires induites par l'yperite et leur reparation, 2) pour etudier les consequences fonctionnelles d'une telle intoxication et 3) pour rechercher un role eventuel des tachykinines dans les atteintes fonctionnelles observees. Nous avons egalement etudie la possibilite de limiter les sequelles fonctionnelles par un traitement par les glucocorticoides. Nous avons montre que, chez le cobaye ayant recu une injection intra-tracheale d'yperite, les principales lesions siegent dans l'epithelium des voies aeriennes centrales. La regeneration epitheliale, obtenue principalement a partir des cellules basales, conduit a un epithelium desorganise dont la hauteur et la densite cellulaire sont diminuees. Ces lesions epitheliales persistantes s'accompagnent d'une hyperreactivite des voies aeriennes et du muscle lisse tracheal a la substance p, liees a une diminution de l'activite de l'endopeptidase neutre de l'epithelium des voies aeriennes. L'administration de glucocorticoides durant la phase de regeneration epitheliale supprime l'hyperreactivite du muscle lisse tracheal a la substance p, en restaurant l'activite de l'endopeptidase neutre de l'epithelium tracheal. Ce traitement augmente la hauteur et la densite cellulaire de l'epithelium tracheal en favorisant la proliferation cellulaire, mise en evidence par immunohistochimie

Les résultats présentés dans ce mémoire portent sur la substance P (SP) et d'autres peptides appellés tachykinines. Pendant longtemps, la SP était la seule tachykinine connue chez les mammifères; récemment, deux nouvelles tachykinines ont été découvertes, soit la neurokinine A (substance K, SK) et la neurokinine B (neuromédine K, NK).Dans un premier temps, nous avons cherché des tissus sensibles aux tachykinines pour développer des préparations pharmacologiques adéquates à l'étude des effets biologiques (généralement des effets contractiles) et des récepteurs des tachykinines. Parmi plusieurs tissus, nous avons choisis le duodénum de rat et les vessies de cobaye, de hamster et de chien à cause de leur sensibilité très élevée aux tachykinines. Dans une deuxième série d'expériences, les deux nouvelles tachykinines (SK et NK) ont été testées sur des préparations pharmacologiques déjà connues, à savoir l'iléon et la trachée de cobaye et l'artère carotide de chien. Sur toutes ces préparations (7 au total) nous avons mesuré les effets myotropes des trois tachykinines déjà mentionnées et d'autres peptides d'origines amphibienne, à savoir la physalaemine (PHYS), l'élédoisine (ELED) et la kassinine (KASS).L'étude des récepteurs des tachykinines a été pratiquée en utilisant trois critères: la mesure de l'ordre de puissance des agonistes, la mesure des affinités d'antagonistes spécifiques et compétitifs et des essais de désensibilisation. L'application du premier critère a fourni les indications suivantes: a) sur l'artère carotide de chien et la vessie isolée de cobaye la substance P est la plus puissante, alors que la SK et la NK sont les agonistes les plus faibles; b) sur le duodénum de rat et la trachée de cobaye, la SK est l'agoniste le plus puissant et la SP le plus faible; c) sur les vessies de chien et de hamster, la KASS et la NK sont plus puissantes que les autres tachykinines. La mesure des affinités des antagonistes (deuxième critère de classification des récepteurs) a fourni des données disparates et d'interprétation difficile, à cause de la faible affinité et du manque de spécificité des antagonistes. Cependant, les affinités de certains antagonistes étaient plus fortes contre l'une ou l'autre des tachykinines étudiées (SP et SK) ou dans l'une ou l'autre des préparations. Dans l'ensemble, les données obtenues avec les antagonistes tendent à confirmer l'existence de trois groupes de préparations et possiblement de trois récepteurs. nfin, des expériences de désensibilisations croisées nous ont permis de constater que les effets de la SP sont généralement éliminés dans les tissus désensibilisés à la SK, alors que cette dernière maintient une certaine activité sur des préparations désensibilisées avec la SP. Ceci suggère l'existence d'au moins deux types de récepteurs pour les tachykinines dans un même tissu.

The bladder mucosa plays an essential role in bladder function. Several receptors are expressed on mucosal cells and the mucosal release of mediators is considered to contribute to bladder afferent signalling. However, mucosal motor activity has not been previously examined. The aim of this thesis was to examine the role of vanilloid TRPV1 and tachykinin NK2 receptors in the bladder mucosa. In addition, the expression and function of novel acid sensing ion channels (ASICs) was explored. Pig and rat models were employed to examine potential species specific differences. In organ bath experiments, the tachykinin, neurokinin A (NKA), was demonstrated to be a potent contractile agent of the pig mucosa. Mucosal responses to NKA were higher than those in intact strips and were mediated by the NK2 receptor, as evidenced by antagonist studies. Immunostaining revealed smooth muscle actin expression on suburothelial myofibroblasts, suggesting their potential role in mucosal contractile activity. To examine the endogenous sources of NKA, contraction and ATP release in response to TRPV1 agonists. capsaicin and acid, were assessed in pig and rat mucosa. In pig mucosal strips, capsaicin did not evoke contraction or ATP release. However, acid (in the physiological range of normal urine) resulted in contraction and a 25-fold increase in ATP release compared with basal. In contrast, in rat mucosal strips, both capsaicin and acid were potent stimuli for ATP release. Acid-evoked ATP release was shown to be mediated via both the TRPV1 and ASIC receptors. ASIC receptor mRNA expression was demonstrated in the rat bladder mucosa and detrusor. Results from this thesis demonstrate novel characteristics of the bladder mucosa: its ability to contract, distinct from the detrusor muscle. The contraction of the mucosa via NK2 receptors has provided a new (so far, overlooked) target for drugs in the treatment of bladder diseases. In addition, acid has been shown to be an important stimulus in modulating bladder function. With the focus of current bladder research shifting towards the sensory pathways, acid may represent an important endogenous initiator of bladder sensation.

Les objectifs de cette thèse ont été d'étudier le rôle des tachykinines dans la transmission nerveuse au niveau des voies aériennes. Nous avons ainsi : 1) examiné le rôle des tachykinines dans la potentialisation par l'endotheline-1 de la contraction des bronches humaines isolées induite par la stimulation électrique des nerfs cholinergiques ; et 2) étudié chez le lapin, dans un modèle de reflux gastrooesophagien, le rôle des tachykinines dans la bronchoconstriction et l'extravasation plasmatique au niveau des voies aériennes induites par l'instillation d'HCI dans l'oesaphage. L'endotheline-1 accroît les contractions des bronches humaines isolées induites par la stimulation électrique de champ. .
1- The aim of this thesis is studying role of tachykinins in the airway neurotransmission. 2- This study demonstrated that, at least, in part, the ET-1 potentiation of cholinergic nervemediated contraction is mediated by tachykinin release, suggesting that, in addition to nerves, several type of cells, such as airway smooth muscle cell, may participate to neuropeptide production. 3- Intraoesphgeal (i. Oe. ) instillation of HC1 in anaesthetized rabbit induce a significant bronchoconstriction. . .