Neste estudo efetuou-se uma análise comparativa da resposta inflamatória e estresse oxidativo produzido pela administração intratraqueal (i.tr.) das PED e/ou 1,2-NQ em vias aéreas de ratos. O efeito destes poluentes sobre a funcionabilidade dos macrófagos foi também avaliado. A injeção i.tr. das PED, numa dose incapaz de causar edema, promoveu efeito aditivo no edema e influxo de leucócitos induzido pela 1,2-NQ nas vias aéreas. O edema foi inibido por antagonistas de taquicininas ou capsaicina, mas o influxo de leucócitos não. O tratamento com estes poluentes aumentou a expressão gênica dos receptores TRPV1, taquicininas e TNFR1, as quais foram reduzidas, mas a iNOS aumentada, em animais depletados de neuropeptídeos. Concentrações elevadas de citocinas pró-inflamatórias foram encontradas no brônquio de ratos saudáveis e, mais intensamente, naqueles tratados com capsaicina. Níveis basais de 3-NT, IL-10 ou IFN-?? no brônquio não foram alterados pelos poluentes. A fagocitose de partículas de zimosan foi aumentada após a exposição de animais saudáveis e depletados de neuropeptídeos aos poluentes. A inflamação nas vias aéreas de ratos expostos ao MP do diesel é influenciada por concentrações de 1,2-NQ no ambiente, via mecanismo neurogênico. / Pharmacological approaches on the healthy side effects evoked by the interaction between environmental pollutants are poorly studied. Here we tested the hypothesis that the environmental chemical 1,2-naphthoquinone (1,2-NQ) is implicated in the exacerbation of airways diseases induced by exposure to diesel exhaust particles (DEP), and that involves a neurogenic-mediated mechanism. Intra-tracheal (i.tr.) injection of DEP (1 and 5 mg/kg) or 1,2-NQ (35 and 100 nmol/kg) caused oedema in rat airways. DEP (at a dose unable to produce oedema) increased the 1,2-NQ-induced responses in the rat airways in a additive manner. This effect was reduced by L-732,138, an NK1 receptor antagonist, and in a lesser extent by the NK2 receptor SR48968. Capsaicin treatment also markedly reduced pollutants-induced oedema. Exposure to pollutants increased the TRPV1, NK1 and NK2 receptors gene expression in bronchus, an effect nearly abolished by capsaicin treatment. No evidence of increased 3-NT in main bronchus was found. Our results are consistent with the hypothesis that DEP-induced airways oedema is highly influenced by increased ambient levels of 1,2-NQ, and takes place by neurogenic-mediated mechanisms involving up-regulation of TRPV1 and tachykinin receptors.

Os receptores canabinoides do tipo 1 (CB1) e vaniloides de potencial transitório 1 (TRPV1) presentes no córtex pré-frontal medial ventral (CPFMv) modulam de maneira oposta a resposta emocional condicionada (REC) no modelo do medo condicionado contextual (MCC). Enquanto a ativação de receptores CB1 reduz as respostas comportamental e cardiovascular da REC, a ativação de TRPV1 aumenta tais parâmetros. Além destes receptores, receptores de glutamato do tipo NMDA e o sistema nitrérgico no CPFMv estão envolvidos na modulação da REC. Possivelmente, tanto a resposta modulada pelo receptor CB1 quanto pelo TRPV1 estão ligadas à modulação da liberação de glutamato e produção de óxido nítrico (NO). Outro neurotransmissor que também possui papel importante na REC é a acetilcolina (ACh) e que provavelmente atua via NO e eCBs. O favorecimento desta neurotransmissão no CPFMv aumenta a REC por meio da ativação de receptores muscarínicos M3. É descrito que a ativação de receptores muscarínicos induz a produção de NO, o qual pode aumentar a liberação de glutamato e, assim, aumentar a REC. Além disso, a ativação de receptores muscarínicos também podem induzir a produção de endocanabinoiodes (eCBs), como a anandamida (AEA), neuromoduladores que podem influenciar a liberação de glutamato, via CB1 ou TRPV1 e, consequentemente, podem afetar a REC. Portanto, o objetivo do trabalho foi avaliar se um antagonista CB1 (NIDA41020) e um agonista TRPV1 (capsaicina) atuam através da via NMDA/NO e se o aumento dos níveis de ACh modula a neurotransmissão gluatamatérgicapor meio de eCBs e NO. Ratos wistars com cânulas direcionadas para o CPFMv foram submetidos ao protocolo de medo condicionado ao contexto. No dia seguinte, cateter de polietileno foi implantado na artéria femoral para posterior registro cardiovascular. 24h após, as drogas foram administradas no CPFMv e o tempo de congelamento e a resposta autonômica foram avaliados durante a reexposição ao contexto. Tanto o NIDA quanto a capsaicina aumentaram a expressão da REC, independentemente de a administração ser na porção PL ou IL. A resposta do antagonismo de CB1 parece depender da ativação de TRPV1 e a resposta do antagonismo TRPV1 depende da ativação de CB1. O aumento da REC induzida por antagonista CB1 ou agonista TRPV1 foi prevenida com a administração prévia de antagonista NMDA ou inibidor da enzima nNOS. A administração de um sequestrador de NO ou de um inibidor da enzima guanilato ciclase solúvel (GCs) preveniu apenas a resposta do antagonismo CB1. O aumento da REC evocado pelo agonista TRPV1 foi prevenido com a microinjeção de antioxidante/sequestrador de radicais livres. Desta maneira, os resultados demonstram que no CPFMv o receptor CB1 modula a expressão da REC através da via NMDA/NO/GCs e o receptor TRPV1 através da via NMDA/NO/Estresse nitrosativo. Além disso, a administração de um inibidor da enzima acetilcolinesterase (AChE) aumentou a REC, sendo este efeito prevenido com a administração prévia de antagonista NMDA, inibidor da nNOS, sequestrador de NO, inibidor da GCs e antagonista de receptores TRPV1. O aumento da REC evocado pelo antagonista CB1 e agonista TRPV1 não foi prevenido pela administração local prévia de antagonista de receptores M3. Este resultado indica que a resposta promovida pela ACh modula a neurotransmissão glutamatérgica possivelmente através da produção de NO e ativação de TRPV1pela AEA e que os eCBs não modulam a transmissão colinérgica no CPFMv. Portanto, podemos sugerir que a re-exposição ao contexto aversivo aumenta os níveis de ACh no CPFMv e, assim, ativa receptores M3 que, por sua vez, induzem a produção de eCBs, possivelmente AEA, e NO. O NO atuaria pré- sinapticamente aumentando a liberação de glutamato, e a AEA ativaria receptores TRPV1 pós-sinápticos que ativaria mecanismos de estresse nitrosativo decorrentes da produção do NO. / CB1 and TRPV1 receptors present in the ventromedial prefrontal cortex (vmPFC) have been related in the modulation of defensive behavior, as fear conditioning response. In contextual fear conditioning, CB1 and TRPV1 antagonism increase and decrease, respectively, the behavior and autonomic response during the reexposure to aversive context. CB1 and TRPV1 activation lead to decrease and increase of glutamate release, respectively. Glutamate is an important neurotransmitter in vmPFC involve in cardiovascular and behavioral response. NMDA activation can promote nitric oxide (NO) production, and this mediator could regulate the pre-synaptic and post-synaptic signaling. Another important neurotransmission related to REC and eCBs/NO is Acetylcholine (ACh). AChE inhibitor in vmPFC increase conditioned response expression through M3 receptor activation. Muscarinic activation leads to NO production and this event can increase the glutamate release. Moreover, muscarinic activation also can induce endocannabinoid (eCBs) production and modulation of glutamatergic neurotransmission by CB1 and TRPV1 receptors. Thus, NO and eCBs production by muscarinic activation probably affect conditioned response through glutamate release. Our aim in this study was to investigate if CB1 antagonism and TRPV1 agonism promote an increase in conditioned response by NMDA/NO pathway. In addition, AChE inhibitor inject in vmPFC modulate glutamatergic neurotransmission by NO and eCBs. Male wistars rats with guide cannulas invmPFC were submitted to contextual fear conditioning. 1 day after conditioning, a polyethylene catheter was implanted in the femoral artery for cardiovascular recording. Following 24h, drugs were administrated in vmPFC and freezing behavior and autonomic response was recorded during context reexposure. CB1 antagonism and TRPV1 agonism increased the expression of conditioned emotional response and the response was not different when injected in PL or IL subareas. The response of CB1 antagonism depends on TRPV1 activation and response of TRPV1 antagonism depends on CB1 activation, demonstrating the relation of these receptors. The effect induced by CB1 antagonism and TRPV1 agonism were prevented by an NMDA antagonism and preferential neuronal NO synthase inhibitor. In case of CB1 antagonism, NO scavenger and a soluble guanylate cyclase inhibitor (sGC) also prevented this response, but not response induced by TRPV1 agonism. Effect of TRPV1 agonism was prevented by administration of antioxidant/free radical scavenger. In addition, inhibition of AChE in vmPFC increased the conditioned response and this effect was prevented by NMDA antagonist, nNOS inhibitor, NO scavenger, sGC inhibitor and TRPV1 antagonist. CB1 antagonist and TRPV1 agonist increased conditioned response and M3 antagonist was not able to prevent this effect. Our results demonstrated that the response promoted by ACh modulate glutamatergic neurotransmission through NO and TRPV1 activation (by AEA). Moreover, endocannabinoid system did not affect cholinergic neurotransmission. Therefore, we suggest that reexposure to aversive context increase ACh concentration in vmPFC and thus induce activation of the M3 receptor. M3 receptor promote NO and eCBs production. NO act in pre-synaptic terminalenhancing glutamate release and AEA activate the TRPV1 receptor in the postsynaptic terminal that act by nitrosative stress in NO pathway.

Le canal cationique TRPV1 (Transient Receptor Potential Vanilloid 1) est activé par la capsaïcine, une acidose, de fortes températures ainsi que par les anesthésiques volatils (AV) dans les neurones sensoriels. Dans le muscle squelettique, TRPV1 est impliqué dans le métabolisme énergétique et l'exercice d'endurance. Grâce à des techniques d'immunomarquage et d'imagerie calcique, la première partie de la thèse vise à caractériser TRPV1 en tant que canal de fuite fonctionnel du réticulum sarcoplasmique (RS) dans les cellules musculaires squelettiques isolées de FDB (Flexor Digitorum Brevis) de souris. Par la suite, nous nous sommes intéressés à son rôle physiopathologique dans le muscle strié squelettique. Ainsi, dans une seconde partie nous supposons une implication de TRPV1 dans les crises d'hyperthermie maligne (HM) chez l'homme. Cette pathologie musculaire correspond à une crise de métabolisme exacerbé du muscle strié squelettique menant à une brusque montée en température chez le patient (>42°C) endormi au moyen d'AV. Dans cette deuxième étude nous démontrons, à travers une approche combinant imagerie calcique et outils pharmacologiques spécifiques du canal, que TRPV1 est activé lors de l'exposition des cellules musculaires à l'isoflurane. TRPV1 est donc une cible des AV dans la cellule musculaire. Puis, des variants de TRPV1 (T612M et N394del) de patients susceptibles à l'HM ont été découvertes. Nous avons pu montrer, suite à la transfection in vivo de ces variants dans des souris déficientes en TRPV1 et grâce à la mesure de flux calciques intracellulaires, que les variants humains de TRPV1 rendent ces canaux plus sensibles aux anesthésiques volatils que le canal TRPV1 humain sauvage. La troisième partie de la thèse a pour but de déterminer le rôle de TRPV1 dans le muscle squelettique en conditions physiologiques par des études fonctionnelles (fonction locomotrice, consommation d'oxygène) sur animal entier. Les résultats préliminaires de cette étude tendent à montrer que l'entraînement physique est moins efficace sur la fonction musculaire des souris déficientes en TRPV1. En conclusion, l'ensemble de ces résultats révèlent pour la première fois que TRPV1 est un canal calcique de fuite fonctionnel du RS pouvant faire le lien entre le déclenchement de l'HM au cours des anesthésies et la présence des RyR1 mutés dans le muscle squelettique / TRPV1 (Transient Receptor Potential Vanilloid 1) cation channel is activated by capsaicine, acidosis, high temperature and by volatile anaesthetics (VA) in sensory neurons. In skeletal muscle, TRPV1 appears to be implied in exercice endurance and energy metabolism. The present work aims first to characterize the functionality of this channel using immnostaining and calcium imaging. We report that TRPV1 is functionally expressed in isolated mouse skeletal muscle cells of FDB (Flexor Digitorum Brevis). These experiments point out that TRPV1 acts as a SR calcium leak channel. In contrast to earlier reports, our analysis shows that TRPV1 is only located to the sarcoplasmic reticulum (SR) membrane. Subsequently, we have studied its physiological role in skeletal muscle. Thus, in a second part, we suppose that TRPV1 could be involved in malignant hyperthermia (MH) crisis in human. MH is a muscular pathology linked to an abrupt increase in body temperature (> 42°C) in patients. MH crisis is a severe and feared complication of anesthesia. Nevertheless, any studies have demonstrated that RyR1 mutants are activated by VA. If the triggering agents of MH are known, their targets remain to be determined. By combining calcium imaging and pharmacological agents, our data first demonstrate that TRPV1 is activated by isoflurane in skeletal muscle cells. TRPV1 is so a target of volatile anaesthetics in skeletal muscle. Afterwards, TRPV1 mutants (T612M and N394del), obtained from susceptibles MH patients, were discovered. In the second part of the work, using in vivo transfection of TRPV1 mutants in TRPV1-/- mice and intracellular calcium measurements we have been able to demonstrate that human TRPV1 mutants are more sensitive to VA than human wild type TRPV1. The last part of the work investigates the physiological role of TRPV1 in skeletal muscle, using a functional exploration (locomotor function, oxygen consumption) in TRPV1-/- mice. Preliminary data point out that training seems to be less effective on skeletal muscle function of TRPV1-/- mice. To conclude, these results indicate for the first time that TRPV1 is a functional SR calcium leak channel and that TRPV1 may be the missing link between MH induction and RyR1 mutants in skeletal muscle during anesthesia

A técnica de laserterapia é um método não invasivo utilizado por diversas áreas da saúde, e tem se mostrado eficaz no tratamento da diminuição da sensibilidade a dor. O presente estudo visa elucidar os efeitos desta terapia nas alterações moleculares induzida pela constrição crônica (CCI) do nervo isquiático de ratos. Esta técnica é aplicada em dias intercalados e tem a duração de 10 sessões. Ao finalizarmos o tratamento com laserterapia os animais foram eutanasiados e os gânglios das raízes posteriores (DRG L4L6) e o nervo isquiático foram retirados para posterior análise. Os DRGs foram processados para detecção da Substância P (SP) e do receptor de potencial transitório vanilóide tipo I (TRPV1). Ainda submetemos o nervo isquiático para a detecção do fator de crescimento neural (NGF) e proteína zero (P0). Nossos resultados demonstraram uma reversão da hipernocicepção dos animais tratados com LASER. Ainda, observamos um aumento da expressão de SP e TRPV1 nos animais com lesão e uma diminuição após o tratamento com LASER. Com relação aos ensaios referente ao nervo isquiático, podemos observar que houve um aumento da densidade óptica para o NGF e para a P0 após tratamento. Esses resultados podem ser observados de modo qualitativo por meio da técnica de imunohistoquímica de fluorescência. Nossos resultados em conjunto, demonstram a eficácia da laserterapia para reversão da hipernocicepção dos animais com lesão. Ainda, esta técnica é capaz de modular liberação de mediadores que participação do processo álgico, além de induzir a regeneração do nervo isquiático. / The laser therapy technique is a noninvasive method used by several health areas, and has been shown to be effective in treat pain sensitivity. The present study aims to elucidate the effects of this therapy on molecular changes induced by chronic constriction (CCI) in rats. This technique was applied every other day, during 10 sessions. At the end of treatment, the animals were euthanized and the posterior root ganglia (DRG L4-L6) and sciatic nerve were removed for further analysis. The DRG\'s were processed for the detection of Substance P (SP) and transient potential receptor type I (TRPV1). In addition, we submit the sciatic nerve for the detection of neural growth factor (NGF) and zero protein (P0). Our results demonstrated a reversal of hypernociception in animals treated with LASER. Furthermore, we observed an increase in SP and TRPV1 expression in animals with lesion and a decrease after LASER treatment. With respect to the tests referring to the sciatic nerve, we could observe that there was an increase in the optical density for NGF and P0 after treatment. These results can be observed qualitatively by fluorescence immunohistochemistry. Our results together demonstrate the efficacy of laser therapy for the reversal of hypernociception of animals with lesions. Furthermore, this technique is able to modulate the release of mediators that participate in the algic process; in addition, this technique is also able to induce regeneration of sciatic nerve in rats.

Les cellules beta pancréatiques sécrètent l’insuline lors d’une augmentation post-prandiale du glucose dans le sang. Ce processus essentiel est contrôlé par des facteurs physiologiques, nutritionnels et pathologiques. D’autres sources d’énergie, comme les acides aminés (leucine et glutamine) ou les acides gras potentialisent la sécrétion d’insuline. Une sécrétion d’insuline insuffisante au besoin du corps déclanche le diabète. Le rôle que joue l’augmentation du calcium intracellulaire et les canaux K+/ATP dans la sécrétion d’insuline est bien connu. Bien que le mécanisme exact de la potentialisation de la sécrétion d’insuline par les lipides est inconnu, le cycle Glycérolipides/Acides gras (GL/FFA) et son segment lipolytique ont été reconnu comme un composant essentiel de la potentialisation lipidique de la sécrétion d’insuline. Le diacylglycérol, provenant de la lipolyse, a été proposé comme un signal lipidique important d’amplification. Cependant, l’hydrolyse des triglycérides et des diacylglycérides a été démontrée essentielle pour la sécrétion d’insuline stimulée par le glucose, en suggérant un rôle du monoacylglycérol (MAG) dans ce processus. Dans cette étude, on démontre que la réduction de la sécrétion d’insuline stimulée par le glucose, lors d’une inhibition de la lipolyse, est restaurée par l’addition de MAG. Dans les cellules beta pancréatiques, le niveau de MAG augmente en présence des concentrations élevées du glucose, et également lorsqu’on inhibe l’enzyme MAG hydrolase abhydrolase-6 (ABHD6) avec l’inhibiteur spécifique WWL70. L’analyse lipidomique a démontré qu’après la stimulation des cellules beta pancréatiques avec le glucose et aussi avec le WWL70, l’espèce la plus accumulée de MAG était le 1-stearoylglycérol (1-SG). L’addition de 1-SG, de 1-palmitoylglycérol (1-PG) ou de WWL70 augmente la sécrétion d’insuline stimulée par le glucose, et cette augmentation est indépendante de la génération de acides gras à partir de MAG. Cela suggère que le MAG est un signal lipidique pour la potentialisation de la sécrétion d’insuline stimulée par le glucose. De plus, la surexpression du gène d’ABHD6 dans les cellules INS832/13 cause une réduction de la sécrétion d’insuline, due probablement à la diminution des niveaux intracellulaire de MAG. Avec le but de comprendre le mécanisme moléculaire impliqué dans la potentialisation de la sécrétion d’insuline par le MAG, on a bloqué l’action du récepteur vanilloid-1 (TRPV1) liant le MAG par l’agent pharmacologiste, AMG9810. Le traitement des cellules beta pancréatique par AMG9810 entraîne une diminution de la potentialisation de la sécrétion de l’insuline induite par le MAG. Il est a noter que le MAG pourrait activer TRPV1 par une liaison physique dans la membrane cellulaire interne; ce qui entraînerai l’entrée du calcium dans la cellule, et ensuite la stimulation de l’exocytose des granules à insuline. En soutien de cette hypothèse, on a trouvé une diminution du calcium intracellulaire lorsqu’on traite au AMG9810 des cellules beta pancréatique de rat (provenant des îlots dispersés) stimulées au glucose et au WWL70. L’ensemble des résultats suggère que le MAG est un médiateur de la potentialisation lipidique de la sécrétion d’insuline stimulée par le glucose. Vu que l’inhibition pharmacologique d’ABHD6 augmente la sécrétion d’insuline, on pourra conclure que cette enzyme représente une cible thérapeutique potentielle dans le développement des médicaments anti-diabétiques, visant une augmentation de la sécrétion d’insuline. / Insulin secretion by the pancreatic b-cell in response to post-prandial increase in blood glucose levels is an essential physiological process that is governed by cellular, nutritional and pathological factors. Other fuels including amino acids like leucine and glutamine and also fatty acids contribute to further augment insulin secretion. Failure to secrete adequate amount of insulin according to the changing demands of the body by b-cell is a key determinant of diabetes. The role played by the elevated Ca2+ influx and K+-ATP channels in insulin secretion is well known. Even though the precise mechanism of the lipid amplification of insulin secretion and the involved molecular signals are not clear, Glycerolipid/Free fatty acid (GL/FFA) cycle and its lipolytic segment have been recognized as essential components in the lipid amplification pathway of insulin secretion. Diacylglycerol produced by lipolysis was proposed as an important lipid amplification signal. However, hydrolysis of triglycerides and also of diacylglycerols is shown to be essential for glucose stimulated insulin secretion (GSIS), indicating a possible role for monoacylglycerol (MAG) in this process. In the present study we demonstrate that the obliterated GSIS due to lipolysis inhibition in b-cells can be restored by providing exogenous MAG. In the b-cells MAG levels increase significantly in the presence of high glucose concentration and specific inhibition of the major MAG hydrolase, abhydrolase-6 (ABHD6), in b-cells and islets with WWL70 leads to accumulation of MAG with concomitant increase in insulin secretion. Lipidomics analysis indicated that the major MAG species that is elevated by high glucose as well as WWL70 addition is 1-stearoylglycerol (1-SG). Exogenously added 1-SG and also 1-palmitoylglycerol (1-PG) strongly enhanced GSIS and this augmentation is not dependent on the generation of FFA by these MAGs. This indicates that MAG is a potential candidate for being the lipid signal for GSIS amplification. Further evidence for this was provided by the observation that overexpression of the MAG hydrolase ABHD6 in INS832/13 cells, resulted in decreased insulin secretion, probably owing to the lowered MAG level inside the b-cells. Pharmacological studies using AMG9810, a specific antagonist of transient receptor potential vanilloid-1 (TRPV1) receptor that binds MAG, revealed that a blockade of TRPV1 strongly attenuated the MAG-augmented insulin secretion. Since MAG is a potential activator of TRPV1, it is likely that MAG binds on the inner surface of the cell membrane to TRPV1, which in turn triggers rapid influx of Ca2+ thereby promoting insulin granule exocytosis. Thus, AMG9810 was found to lower Ca2+ influx into dispersed rat islet cells that was induced by high glucose and also WWL70. These results collectively suggest that MAG is the potential mediator of the lipid amplification of glucose-stimulated insulin secretion. Our results also indicate that pharmacological intervening at the ABHD6 hydrolysis step enhances insulin secretion; this enzyme protein can be a promising thrapeutic target for the development of anti-diabetic drugs that promote insulin secretion.

Neste estudo efetuou-se uma análise comparativa da resposta inflamatória e estresse oxidativo produzido pela administração intratraqueal (i.tr.) das PED e/ou 1,2-NQ em vias aéreas de ratos. O efeito destes poluentes sobre a funcionabilidade dos macrófagos foi também avaliado. A injeção i.tr. das PED, numa dose incapaz de causar edema, promoveu efeito aditivo no edema e influxo de leucócitos induzido pela 1,2-NQ nas vias aéreas. O edema foi inibido por antagonistas de taquicininas ou capsaicina, mas o influxo de leucócitos não. O tratamento com estes poluentes aumentou a expressão gênica dos receptores TRPV1, taquicininas e TNFR1, as quais foram reduzidas, mas a iNOS aumentada, em animais depletados de neuropeptídeos. Concentrações elevadas de citocinas pró-inflamatórias foram encontradas no brônquio de ratos saudáveis e, mais intensamente, naqueles tratados com capsaicina. Níveis basais de 3-NT, IL-10 ou IFN-?? no brônquio não foram alterados pelos poluentes. A fagocitose de partículas de zimosan foi aumentada após a exposição de animais saudáveis e depletados de neuropeptídeos aos poluentes. A inflamação nas vias aéreas de ratos expostos ao MP do diesel é influenciada por concentrações de 1,2-NQ no ambiente, via mecanismo neurogênico. / Pharmacological approaches on the healthy side effects evoked by the interaction between environmental pollutants are poorly studied. Here we tested the hypothesis that the environmental chemical 1,2-naphthoquinone (1,2-NQ) is implicated in the exacerbation of airways diseases induced by exposure to diesel exhaust particles (DEP), and that involves a neurogenic-mediated mechanism. Intra-tracheal (i.tr.) injection of DEP (1 and 5 mg/kg) or 1,2-NQ (35 and 100 nmol/kg) caused oedema in rat airways. DEP (at a dose unable to produce oedema) increased the 1,2-NQ-induced responses in the rat airways in a additive manner. This effect was reduced by L-732,138, an NK1 receptor antagonist, and in a lesser extent by the NK2 receptor SR48968. Capsaicin treatment also markedly reduced pollutants-induced oedema. Exposure to pollutants increased the TRPV1, NK1 and NK2 receptors gene expression in bronchus, an effect nearly abolished by capsaicin treatment. No evidence of increased 3-NT in main bronchus was found. Our results are consistent with the hypothesis that DEP-induced airways oedema is highly influenced by increased ambient levels of 1,2-NQ, and takes place by neurogenic-mediated mechanisms involving up-regulation of TRPV1 and tachykinin receptors.

L’inflammation neurogène cutanée (INC) est une inflammation de la peau induite par l’activation des fibres nerveuses intra-épidermiques qui secrètent des neuropeptides tels que la substance P (SP). L’INC est impliquée dans des dermatoses inflammatoires prurigineuses comme le psoriasis, la dermatite atopique (DA) et le syndrome de Netherton (SN). Un nouveau concept émerge, suggérant que les kératinocytes sont également des acteurs majeurs de l’INC. Le récepteur activé par des protéases de type 2 (PAR-2) est fortement incriminé dans l’INC associée à ces dermatoses, ce qui permet de comprendre les voies du prurit non-histaminergique. Les enjeux thérapeutiques sont de taille puisqu’il n’existe actuellement aucun traitement efficace permettant la prise en charge spécifique du prurit histamino-indépendant au cours des dermatoses prurigineuses associées à l’INC.Bien que le rôle de PAR-2 dans la sécrétion de neuropeptides à partir des neurones sensoriels soit clairement établi, son implication dans la modulation de gènes pouvant contribuer à l’entretien ou l’amplification de l’INC reste méconnue. Le rôle inflammatoire de PAR-2 a également été démontré sur des kératinocytes cultivés en monocouche via la sécrétion de cytokines par des mécanismes dépendants du Ca2+. La surexpression de PAR-2 et la perte d’expression de certains canaux calciques impliqués dans sa réponse calcique dans les kératinocytes différenciés suggèrent des mécanismes d’action de PAR-2 différents pour ceux-ci. Dans le but d’étudier le rôle pro-inflammatoire de PAR-2 au cours des dermatoses prurigineuses, nous avons analysé l’effet de son activation sur des monocultures de neurones sensoriels issus de ganglions rachidiens dorsaux (GRD) de rat et de kératinocytes humains différenciés (DhPK), en criblant l’expression de médiateurs de l’inflammation. Pour approfondir, les voies calciques de PAR-2 sous-jacente à la modulation d’expression dans les kératinocytes différenciés, des expériences d’imagerie calcique ont été réalisées et différents antagonistes ont été utilisés pour analyser les acteurs impliqués.Dans le cadre d’un partenariat avec les laboratoires dermatologiques d’Uriage, nous avons testé les effets de l’eau thermale d’Uriage sur la modulation de gènes induite par PAR-2 dans les DhPK. Nous avons également utilisé une lignée de PC12 différenciables en neurones par le NGF afin de les utiliser comme alternatives des neurones sensoriels issus des GRD de rat pour l’étude de l’INC.L’ensemble des résultats obtenus au cours du criblage des gènes modules par PAR-2 confirme le rôle pro-inflammatoire de PAR-2 dans les neurones sensoriels de rat et dans les DhPK. La découverte d’une nouvelle voie calcique de PAR-2 dans les DhPK offre de nouvelles pistes thérapeutiques pour les dermatoses prurigineuses telles que le psoriasis, la DA et le NS. Les résultats obtenus avec l’eau thermale d’Uriage peuvent présenter une perspective thérapeutique pour les patients souffrants de dermatoses prurigineuses réfractaires aux traitements conventionnels. L’utilisation d’une lignée neuronale comme lesPC12 pour l’étude de l’INC serait une alternative utile dans le développement des tests cosmétiques avec les industriels pour notre laboratoire. / Cutaneous neurogenic inflammation (CNI) is an inflammation of the skin induced by the activation of intraepidermal nerve fibers that release neuropeptides such as substance P (SP). CNI is involved in pruritic inflammatory skin disorders such as psoriasis, atopic dermatitis (AD) and Netherton syndrome (NS). A new concept is growing, suggesting that keratinocytes could also trigger INC. The proteases activated receptor 2 (PAR-2) is strongly incriminated in CNI associated with these dermatoses, which allow to understand the histamine-independent itching pathways. The therapeutic stakes are high since there is currently no effective treatment allowing the specific management of histamine-independent pruritus during skin disorders associated with CNI.Although the role of PAR-2 in the secretion of neuropeptides from sensory neurons is clearly established, its involvement in the modulation of genes involved in the maintenance or amplification of CNI remains unknown. The inflammatory role of PAR-2 on keratinocytes has also been demonstrated through the production of cytokines in a Ca2+-dependent mechanisms. The overexpression of PAR-2 and the loss of ORAI1 expression, a calcium channel following keratinocytes differentiation suggest different signaling pathways downstream to PAR-2 activation between undifferentiated and differentiated keratinocytes.In order to study the pro-inflammatory role of PAR-2 during pruritic dermatoses, we analyzed the effect of its activation on rat primary sensory neurons from dorsal spinal ganglia (DRG) and on differentiated human primary keratinocytes (DhPK) by screening the expression of inflammatory mediators. To deepen the Ca2+ pathways underlying PAR-2-mediated inflammatory mediator modulation in DhPK, we performed Ca2+ imaging experiments and different antagonists were used to analyze the involvement of intracellular actors. In a partnership with the dermatological laboratories of Uriage, we tested the effects of Uriage thermal water on PAR-2-induced gene modulation in DhPK. We also used a PC12 cell line differentiable in neurons by the NGF in order to use them as alternatives of rat primary sensory neurons from DRG for the study of INC. We also used a PC12 cell line differentiable in neurons by the NGF use them as alternatives of rat primary sensory neurons from DRG for the study of INC.The results obtained during the screening of the PAR-2-modulated genes confirmed the proinflammatory role of PAR-2 in rat primary sensory neurons and in DhPK. The discovery of a new PAR-2-mediated Ca2+ pathway in DhPK offers new therapeutic pathways for pruritic dermatoses such as psoriasis, AD and NS. The results obtained with the thermal water of Uriage can present a therapeutic perspective for patients suffering from pruritic dermatoses refractory to conventional treatments. The use of a neuronal cell line as the PC12 for the study of INC would be an useful alternative in the development of cosmetic tests.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The transient receptor potential vanilloid 1 (TRPV1) is a calcium permeable channel responsible for the transduction and modulation of acute and chronic pain signaling, being a potential target for treatment of different pain disorders. In spite of that, AMG517, a TRPV1 antagonist, presents several clinical limitations, such as the development of severe hypertermia. The aim of this study was to investigate the possible interaction of the flavonoid eriodictyol with the TRPV1 receptor and its putative antinociceptive and hyperthermic effect. Eriodictyol was able to displace the [3H]-resiniferatoxin binding (IC50 = 47 (21 - 119) nM) and to inhibit the calcium influx mediated by capsaicin (IC50 = 44 (16 125) nM), suggesting that eriodictyol acts as a TRPV1 antagonist. Moreover, eriodictyol induces antinociception in the intraplantar capsaicin test, with maximal effect of 49±10 and 64±4% of inhibition for oral (ED50 = 2 (1-5) mg/kg) and intrathecal (ED50 = 2 (1-3) nmol/site) routes, respectively. Concomitantly, eriodictyol did not induce any alteration on body temperature or locomotor activity. Orally administered eriodictyol (4.5 mg/kg) prevented the nociception induced by intrathecal injection of capsaicin (72±6% of inhibition), the non-protein thiol loss and the 3-nitrotyronise (3-NT) formation induced by capsaicin in spinal cord. Eriodictyol (4.5 mg/kg, p.o.) also reduced the thermal hyperalgesia (100% of inhibition) and mechanical allodynia (62±9% of inhibition) elicited by complete Freund s adjuvant (CFA) paw injection. In conclusion, Eriodictyol acts as an antagonist of TRPV1 receptor and an antioxidant, inducing antinociception without some side effects and limitations expected for TRPV1 antagonists, as hyperthermia. / O receptor de potencial transiente vanilóide 1 (TRPV1) é um canal iônico permeável a cátions ativado por uma série de estímulos nocivos, como calor, acidificação e agentes irritantes como a capsaicina. Este receptor é responsável pela detecção e transmissão da dor aguda e crônica. Devido a isso, substâncias que modulem a atividade deste receptor apresentam um potencial clínico para o tratamento da dor. Assim, este trabalho objetiva a possível interação do flavonóide eriodictiol com o receptor TRPV1. Inicialmente, observamos que o eriodictiol foi capaz de deslocar o radioligante [3H]-resiniferatoxina, em ensaio de união específica, do receptor TRPV1 com uma concentração inibitória 50% (IC50) de 46.9 (20.70 - 118.9) nM. Ao mesmo tempo, o eriodictiol também inibiu o influxo de cálcio estimulado por capsaicina com IC50 de 44,4 (15,6 125,1) nM, sugerindo que este aja como um antagonista do receptor. Além disso, também observamos que o eriodictiol induz antinocicepção no teste da capsaicina intraplantar com efeito máximo de 49,0±10.5 e 63,9±4.0 % de inibição máxima para o tratamento oral e intratecal, respectivamente, e com uma dose efetiva 50% (DE50) de 2,4 (1,0 5,5) mg/kg 2,2 (1,6 2,9) nmol/site, respectivamente. Além disso, não observamos alterações na atividade locomotora ou temperatura corporal dos animais. A administração oral de eriodictiol também foi capaz de prevenir a nocicepção induzida por capsaicina intratecal (71,7±5,7 % de inibição). Ao mesmo tempo, o eriodictiol também aboliu a hiperalgesia térmica e reduziu a alodínia mecânica (62,4±9,2 %) induzidas por adjuvante completo de Freund. Da mesma forma, o eriodictiol também preveniu totalmente a diminuição de tiois não protéicos e formação de 3-nitrotirosina (3-NT) espinhais induzidas por capsaicina, ao passo que apresentou atividade antioxidante direta no texto de neutralização do radical ABTS. Em conclusão, nossos resultados mostram que o eriodictiol age como um antagonista do receptor TRPV1, com atividade antioxidante, induzindo antinocicepção sem os efeitos colaterais e limitações esperados para antagonistas do receptor TRPV1.

L'insuffisance cardiaque (IC) est l'une des principales causes de décès dans le monde. Les maladies cardiovasculaires sont devenues une préoccupation majeure de santé publique et le resteront probablement à l'avenir avec le vieillissement de la population et l'augmentation du taux de survie des patients atteints de maladies cardiovasculaires. L'infarctus du myocarde (IM) est le principal facteur de risque favorisant le développement de l’IC. L'une des principales caractéristiques de l'IC est une dérégulation du fonctionnement du système nerveux autonome (SNA), en particulier une hyperactivité du système nerveux sympathique (SNS) qui contribue largement à la progression de la maladie et à l'augmentation de la morbidité. Le mécanisme de l'hyperactivité du SNS n'est que partiellement connu. En ce qui concerne la progression de l'IM à l'IC, des études suggèrent un engagement concerté du cerveau (médulla), du nerf vague et des nerfs sympathiques, en plus du tissu cardiaque qui serait à l'origine de la maladie systémique. En plus des altérations du SNA, des exemples de comorbidités de l'IC comprennent des troubles cognitifs tel que l’anxiété et la dépression ainsi que des modifications atrophiques des régions cérébrales chez les patients atteints d'IC. L’ensemble, de ces données montrent l'importance du système nerveux central et périphérique dans l'IC. En plus du système nerveux cardiaque intrinsèque, qui comprend un réseau de ganglions intracardiaques et de neurones interconnectés, le coeur, en particulier l'épicarde, possède des milliers de neurones intégrés, dont beaucoup expriment le récepteur vanilloïde 1 (TRPV1). Au cours des dernières années, des études scientifiques ont montré que l'application épicardique de résinifératoxine (RTX), un agoniste spécifique de TRPV1, au moment de l'IM induit, conduit à une réduction de la fibrose cardiaque, prévient l'hyperactivation du SNS et améliore la fonction cardiaque dans plusieurs modèles. La thèse visait à mieux caractériser la fonction de ces fibres exprimant TRPV1 dans l'IM et l'IC qui en découle. Les principaux objectifs de présente étude sont les suivants : 1) Identifier si les fibres épicardiques exprimant TRPV1 entraînent des modifications des fonctions cérébrales. 2) Élucider les mécanismes moléculaires sous-jacents dans les tissus du système nerveux en aval des traitements IM et RTX en utilisant la protéomique ; et 3) Déterminer si les stimuli nociceptifs dans un modèle alternatif, C. elegans, via la modulation des récepteurs vanilloïdes orthologues par RTX, peuvent entraîner une modification du comportement et des mécanismes moléculaires associés aux effets induits par l'exposition à RTX. Pour répondre à ces 4 objectifs, nous avons combiné la dénervation des afférences sympathiques cardiaques, via l'application épicardique de RTX, avec un modèle IM validé. Des études comportementales ont été menées pour évaluer la dépression et l'anxiété des animaux après le début de l’IC. L'analyse protéomique a été réalisée sur plusieurs tissus dont le cortex frontal, le ventricule gauche, le bulbe rachidien (médulla), la moelle épinière et le nerf vague. Les principaux résultats de cette thèse ont montré que la dénervation afférente cardiaque sympathique par RTX atténue le remodelage cardiaque et restaure la fonction cardiaque lors d’un IM dans un modèle murin. L'analyse comportementale a démontré que les souris IM sont déprimées et anxieuses et que le traitement RTX réduit significativement l'expression du phénotype anxieux. La protéomique réalisée sur des cortex frontaux isolés a identifié des signatures protéiques uniques pour chacun des groupes (IM, RTX et IM/RTX), indiquant des voies partagées et uniques attribuées par IM et RTX. Les analyses bio-informatiques ont montré un enrichissement significatif des voies métaboliques dans tous les tissus et traitements, et à tout moment, suggérant un rôle central de la fonction mitochondriale après les traitements IM et RTX. Des voies fonctionnelles enrichies dans ces tissus, y compris le cytosquelette, les vésicules et la transduction du signal, peuvent être en aval des réponses initiées par les mitochondries en raison de modifications du taux d'impulsion neuronale après un IM ou d'une altération de la communication coeur-cerveau après l'application de RTX. Certaines voies et molécules communes ont aussi été observées chez C. elegans, comme la voie de signalisation de Wnt, ce qui suggère des effets semblable de RTX. La thèse contribue à une meilleure compréhension des mécanismes physiologiques des nerfs exprimant TRPV1 et offre des informations clés pour comprendre les mécanismes sous-jacents aux troubles neurologiques d'origine cardiaque. Le modèle de C. elegans peut servir de futur modèle pour tester des molécules pharmacologiquement actives pour de futures thérapeutiques. / Heart failure (HF) is one of the leading causes of death worldwide. Cardiovascular diseases are therefore becoming a major health problem and will probably continue to be so in the future with the aging of the population and the increase in the survival rate of patients with cardiovascular disease. Myocardial infarction (MI) is the main risk factor for developing HF. One of the prominent features of HF is a dysregulation in the functioning of the autonomic nervous system (ANS), in particular a sympathetic nervous system (SNS) hyperactivity that largely contributes to disease progression and increased morbidity. The mechanism for the SNS hyperactivity is only partially known. Regarding the progression from MI to HF, studies suggest a concerted engagement of the brain (medulla oblongata), the vagus nerve and the sympathetic nerves, in addition to cardiac tissue that are thought to instigate systemic disease. In addition to the alterations in the ANS, examples of HF comorbidities include cognitive impairment and atrophic changes in brain regions in HF patients. Together these data show the importance of the central and peripheral nervous system in HF. In addition to the intrinsic cardiac nervous system, which includes a network of intracardiac ganglia and interconnecting neurons, the heart, especially the epicardium, has thousands of embedded neurons, many of which express the transient receptor potential cation channel subfamily V member 1 (TRPV1). Over recent years studies have shown that the epicardial application of resiniferatoxin (RTX), a specific agonist of TRPV1, at the time of induced MI, leads to a reduction of cardiac fibrosis, prevents hyperactivation of the SNS and improves the heart function in several model systems. The thesis was aimed to better characterize the function of these TRPV1-positive fibers in MI and resulting HF. The main objectives of the current study were : 1) To identify whether the TRPV1 expressing epicardial fibers lead to changes in brain activity and function. 2) To elucidate the underlying molecular mechanisms in nervous system tissue downstream from MI and RTX treatments using proteomics; and 3) To determine if nociceptive stimuli in an alternate model, C. elegans, via the modulation of orthologous vanilloid receptors by RTX, can lead to altered behavior and molecular mechanisms associated with RTX exposureinduced effects. To meet these objectives, we combined denervation of cardiac sympathetic afferents, via epicardial application of RTX, with a validated MI model. Behavioral studies were carried out to evaluate the depression and anxiety of the animals after the onset of HFt. Proteomic 6 analysis was carried out on several tissues including the frontal cortex, left ventricle, medulla oblangata, spinal cord, and vagus nerve. The major findings of this thesis are that sympathetic cardiac afferent denervation by RTX attenuates cardiac remodeling and restores cardiac function during MI in a mouse model. Behavioral analysis demonstrated that MI mice are depressed and anxious and that RTX treatment significantly reduced the expression of the anxious phenotype. Proteomics performed on isolated frontal cortices identified unique protein signatures for each of the groups (MI, RTX and MI/RTX), indicating shared and unique pathways attributed by MI and RTX. Bioinformatic analyses showed a significant enrichment for metabolic pathways in all tissues and treatments, and at all time points, suggesting a central role of mitochondria function following MI and RTX treatments. Enriched functional pathways in these tissues, including cytoskeleton, vesicles, and signal transduction, may be downstream of mitochondria-initiated responses due to changes in neural impulse rate after MI or altered heart-brain communication following RTX application. Some common pathways and molecules were observed in C. elegans, such as the Wnt signaling pathway, suggesting similar effects of RTX. The current thesis contributes to a better understanding of the physiological mechanisms of the TRPV1 expressing nerves and offers key information to understand the mechanisms underlying neurological disorders of cardiac origin. The C. elegans model may serve as a future model for testing pharmacologically active molecules for future therapeutics.

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