α7 Neuronal nicotinic acetylcholine receptors are one of two major classes of receptors responsible for cholinergic neurotransmission in the central nervous system. The existence of α7 neuronal nAChRs in different regions of the nervous system suggests their involvement in certain essential physiological functions as well as in disorders such as Alzheimer’s disease (AD), drug dependence, and depression. This project was aimed toward the discovery and development of small–molecule arylguanidines that modulate α7 nAChR function with improved subtype-selectivity through an allosteric approach. Identifying the required structural features of these small molecules allowed optimization of their negative allosteric modulator (NAM) actions at α7 neuronal nAChRs. MD-354 (3-chlorophenylguanidine) was the first small–molecule NAM at α7 nAChRs; however, it also binds at 5-HT3 receptors. The N-methyl analog of MD-354 appeared to be more selective toward α7 nAChRs than 5-HT3 receptors. Comparative studies using two series of novel compounds based on MD-354 and its N-methyl analog explored the aryl 3-position and investigated whether or not the MD-354 series and the N-methyl series bind in the same manner. Biological potencies of the MD-354 series and the N-methyl series of compounds, obtained from electrophysiological assays with Xenopus laevis oocytes expressing human α7 nAChRs in two-electrode voltage-clamp assays, showed that N-(3-iodophenyl)-N- methylguanidine (28) is the most potent analog at α7 nAChRs. Our comparative study and Hansch analyses indicated different binding modes of the two series. In addition, we investigated: i) the length/size of the aliphatic side chain at the anilinic nitrogen, ii) the effect of alkylating the guanidine nitrogen atoms, and iii) the necessity of the presence of these nitrogen atoms for the inhibitory effects of arylguanidines at α7 nAChRs. In efforts to explain the varied functional activity of these arylguanidines, homology models of the extracellular domain and the transmembrane domain of human α7 nAChRs were developed, allosteric sites identified, and docking studies and hydropathic analysis conducted. The 3D quantitative structure-activity relationships for our compounds were also analyzed using CoMFA. A pharmacophore for arylguanidines as α7 nAChR NAMs was identified. Together, these data should be useful for the subsequent design of novel arylguanidine analogs for their potential treatment of neurological disorders.

Au cours de ce travail, nous nous sommes intéressés à développer des voies de synthèse convergentes et diastéréosélectives en vue de préparer des analogues pyrrolidiniques des alcaloïdes de Lobelia comme nouveaux ligands des récepteurs nicotiniques centraux à l’acétylcholine. Ainsi, nous avons mis au point une méthode « bidirectionnelle » basée sur des réactions de double aza-Michael et donnant accès à des pyrrolidines 2,5-disubstituées. Une étude de réactivité a également été mené afin d’améliorer la chimiosélectivité des différents processus réactionnels impliquant des réactions d’aza-Michael dans des séquences de cyclisation tandem. Dans un second temps, nous avons décrit une voie dite « d’élongation monodirectionnelle » permettant d’accéder à des 2,5-trans-pyrrolidines énantiopures. Enfin, la dernière partie de ce manuscrit aborde une étude prospective de réductions désymétrisantes pour la synthèse d’homologues pyrrolidiniques de la lobéline. / The present work has been dedicated to the development of convergent and diastereoselective routes for the preparation of pyrrolidine Lobelia alkaloid analogues as novel neuronal nicotinic receptors. We have settled a selective bidirectional strategy based on chain homologation by double olefination followed by aza-Michael reactions as a straightforward access to 2,5-cis-disubstituted pyrrolidines that was extended to the synthesis of 2,6-cis-piperidines. Additional studies have been carried out in order to drive chemoselectivities in the course of competitive tandem aza-Michael-induced ring closure reactions. In the same time, we also described a monodirectional route to access 2,5-trans-disubstituted pyrrolidines. The last part of this manuscript has been finally dedicated to a prospective reductive desymmetrization study for the rapid and enantioselective synthesis of pyrrolobeline homologues.

Os receptores nicotínicos de acetilcolina (colinérgicos) (nAChRs) neuronais são proteínas integrais de membrana e pertencem à família de canais iônicos controlados por ligante, compostos por subunidades α e β. Esses receptores desempenham um papel-chave na transmissão de sinal entre os neurônios nos sistemas nervoso central e periférico. O subtipo α3β4, por exemplo, é o nAChR neuronal mais expresso no sistema nervoso autônomo; nAChRs contendo a subunidade α3 estão presentes em alta densidade no gânglio cervical superior, glândulas pineal e adrenais. Também estão presentes na substancia nigra, striatum, hipocampo, locus ceruleus, tracto habênulo-interpeduncular e cerebelo. Os nAChRs são inibidos por uma variedade de substâncias químicas, incluindo toxinas naturais, anestésicos locais, drogas de abuso (por, exemplo, cocaína) e compostos clinicamente importantes (tranqüilizantes, por exemplo). O mecanismo de inibição desses receptores tem sido investigado intensivamente. Neste estudo, nós investigamos o mecanismo pelo qual a tacrina (9-1,2,3,4-tetraidroaminoacridina), um agente usado clinicamente no tratamento da doença de Alzheimer, inibe o nAChR α3β4 de rato recombinante expresso nas células KXα3β4R2, utilizando uma técnica de cinética química rápida. A constante de dissociação da nicotina do sítio que controla a ativação do receptor, Kd, é 23 µM e a constante de equilíbrio de abertura do canal, Φ-1, é 4. A tacrina inibe o receptor competitivamente, com um KI de 0,77 µM. / Neuronal nicotinic acetylcholine (cholinergic) receptors (nAChRs) are integral membrane proteins and belong to the family of ligand-gated cation channels composed by α and β subunits. These receptors play a key role in the signal transmission between neurons in the central and peripheral nervous system. The α3β4 subtype, for example, is the most expressed neuronal nAChR in autonomic ganglia; α3-containing nAChRs are present at particularly high density in the superior cervical ganglia, pineal, and adrenal glands. They are also present in the substancia nigra, striatum, hippocampus, locus ceruleus, habenulo-interpeduncular tract and cerebellum. The nAChRs are inhibited by a variety of chemical substances, including natural toxins, local anesthetics, abused drugs (e.g., cocaine) and clinically important compounds (e.g., tranquilizers). The mechanism of inhibition of these receptors has been intensively investigated. In this study, we investigated the mechanism by which tacrine (9-1,2,3,4-tetahydroaminoacridine), an agent used clinically to treat Alzheimers disease, inhibits the recombinant rat α3β4 nAChR expressed in KXα3β4R2 cells, using a rapid chemical kinetic technique. The nicotine dissociation constant for the site controlling receptor activation, Kd, is 23 µM and the channel-opening equilibrium constant, Φ-1, is 4. Tacrine inhibits the receptor competitively, with a KI of 0.77 µM.

Introdução: Colite Ulcerativa (UC) é uma Doença Inflamatória Intestinal (DII) caracterizada por uma resposta imune exacerbada, com sintomas como diarreia, perda de peso e sangue nas fezes. Apesar dos medicamentos disponíveis, a remissão da doença nem sempre consegue ser alcançada e há a necessidade de terapias alternativas. A colite induzida por DSS (Dextran Sulfate Sodium) é um modelo animal utilizado na investigação de novas terapias por sua semelhança à UC humana. DSS provoca dano à barreira epitelial do cólon, induzindo uma resposta imune exacerbada; entretanto, o exato mecanismo não está totalmente esclarecido. O Ultrassom Terapêutico (TUS) foi utilizado para tratamento de injúria renal em modelo experimental, sua ação se dá através da estimulação do nervo vago (VN) e consequente ativação da via antiinflamatória colinérgica (CAIP). Uma vez que pacientes com DII podem exibir atividade disfuncional do VN, TUS pode ser investigado como terapia alternativa. Objetivos: Investigar temporalmente o perfil clínico, proteômico, histológico e imunológico da colite aguda induzida por DSS; e determinar os efeitos de TUS na colite induzida por DSS. Métodos: No primeiro estudo, a severidade da colite foi avaliada pela administração de DSS 1-3%, observando a resposta clínica e histológica. A análise temporal de DSS 3% incluiu uma avaliação proteômica e histológica do cólon, e a resposta imune celular no baço, linfonodo mesentérico (MLN) e cólon. No segundo estudo, utilizando o modelo de DSS 2%, TUS foi aplicado no abdômen dos animais e foram observados os sintomas clínicos, dano histológico, proteômica do cólon e respostas imunes celulares no baço, MLN e cólon. Animais esplenectomizados ou knockout para a7nAChR (marcador clássico para ativação de CAIP) foram utilizados. Resultados: No primeiro estudo, observou-se que a severidade da doença foi aumentada seguindo concentrações de 1-3% DSS. A análise temporal de DSS 3% demonstrou que os macrófagos (F4/80+) se apresentam como a primeira resposta celular, seguidos por células T CD25+, CD4+ e CD8+. A piora clínica da doença correspondeu ao aumento progressivo de fatores pró-inflamatórios e dano tecidual no cólon, exceto no dia 8. Foram observados menores níveis dos marcadores de células T CD25+, CD4+ e CD8+ no MLN e/ou baço, sugerindo a ocorrência de tropismo destas células para o intestino. No segundo estudo, a aplicação de TUS diminuiu a severidade da doença através da melhora de sintomas clínicos, danos teciduais e encurtamento do cólon. A proteômica do cólon demonstrou uma resposta anti-inflamatória durante a fase de injúria (D0-7), induzindo uma resolução acelerada da doença na fase de recuperação (D8-14). TUS diminuiu os níveis de células T CD8+ e normalizou os níveis de células T CD25+ no cólon. Animais esplenectomizados não demonstraram melhora clínica ou histológica, enquanto animais a7nAChR KO apresentaram piora da colite experimental. Além disso, TUS aumentou os níveis de células F4/80+a7nAChR+ no intestino de animais WT DSS 2%. Conclusão: Nossos resultados demonstram que a severidade da doença depende da concentração de DSS, relacionada com as respostas clínica, proteômica e imune no modelo animal de DSS 3%; e TUS diminuiu a severidade da colite induzida por DSS presumidamente pela da estimulação do VN e consequente ativação de CAIP através do baço. / Introduction: Ulcerative Colitis (UC) is an Inflammatory Bowel Disease (IBD) characterized by uncontrolled immune response, presenting with symptoms of diarrhea, weight loss and bloody stools. Despite available treatments, UC sustained remission is not achievable and there is still the need for alternative therapies. Dextran Sulfate Sodium (DSS)-induced colitis is a mouse model used to investigate novel therapies, since it closely mimics human UC. DSS damages the colonic epithelial barrier, leading to an exacerbated immune response. However, the exact mechanism is not totally understood. Previous studies showed the use of Therapeutic Ultrasound (TUS) to prevent kidney injury in mice through stimulation of the vagus nerve (VN) and activation of the cholinergic anti-inflammatory pathway (CAIP). Since IBD patients can present with dysfunctional VN activity, TUS could be studied as an alternative therapy. Objectives: To investigate the temporal clinical, proteomic, histological and cellular immune profiles of DSS-induced acute colitis; and to determine the effects of TUS directed toward the VN and spleen in the course of DSS-induced colitis. Methods: First, we analyzed DSS-induced colitis severity by administration of 1-3% DSS, observing the clinical course and histological damage. A time course analysis was performed at 3% DSS, including colon proteomics, colon histology and immune cell responses in the spleen, MLN (mesenteric lymph node) and colon. Next, utilizing 2% DSS in drinking water, we applied TUS over the mice abdomen and analyzed clinical symptoms, histological damage, colon proteomics and immune cell responses in the spleen, MLN and colon. Splenectomized and a7nAChR (key indicator of CAIP activation) KO animals were also used. Results: In the first study, we observed worsening of the disease when increasing DSS concentrations from 1 to 3%. Time course analysis of 3% DSS revealed macrophages to be the first responders, followed by CD25+, CD4+ and CD8+ T cells. Worsening of the disease corresponded to a progressive increase in pro-inflammatory colonic factors and histological damage, except at day 8. Lower levels of CD25+, CD4+ and CD8+ T cells in MLN and/or spleen suggest an immune cell tropism to the gut. In the second study, TUS attenuated DSS induced colitis through amelioration of clinical symptoms, histological damage and colon shortening. Proteomic colon analysis demonstrated an antiinflammatory profile during the injury phase (D0-7), whilst inducing an early resolution of the disease during the recovery phase (D8-14). TUS decreased CD8+ and normalized CD25+ T cell levels in the gut. Splenectomized animals demonstrated no improved clinical and pathological outcomes, and a7nAChR KO mice presented with worsening of the disease. Furthermore, there were increased levels of F4/80+a7nAChR+ cells in the colon of 2% DSS WT mice under TUS treatment. Conclusion: Our results demonstrate that the severity of colitis is dependent on DSS concentration, correlated with clinical, proteomic and cellular immune responses on 3% DSS; and TUS significantly improved DSS-induced acute colitis presumably through stimulation of the VN and consequent activation of CAIP through the spleen.

Introdução: A inflamação constitui um dos fatores mais importantes da fisiopatologia da asma brônquica, caracterizada por uma resposta eosinofílica com produção de citocinas de perfil Th2. A persistência desta inflamação induz no pulmão um processo de reparo associado à redução progressiva da função pulmonar, que nem sempre é revertida pelos tratamentos disponíveis. O sistema colinérgico anti-inflamatório é descrito como um mecanismo neural que suprime a resposta imune e controla a inflamação principalmente pelo efeito da acetilcolina em receptores nicotínicos do tipo alfa7 (alfa7nAChR) encontrados em células do sistema imune. A acetilcolina (ACh) exerce um importante efeito na asma e recentemente demonstramos que a redução parcial da liberação da acetilcolina induz per se a inflamação pulmonar. Embora se saiba que os receptores muscarínicos (mAChRs) exercem um efeito pró-inflamatório e broncoconstritor na asma, a ativação de receptores nicotínicos (nAChRs) poderia ter um efeito benéfico reduzindo a inflamação pulmonar, fato demonstrado em modelos de inflamação sistêmica e aguda. O efeito da ativação do alfa7nAChR na fisiopatologia da asma ainda não está claramente elucidado. Objetivo: Investigar o efeito do tratamento com PNU-282987 (PNU), um agonista específico do alfa7nAChR, em um modelo murino de inflamação alérgica crônica das vias aéreas. Metodologia: Camundongos BALB/c foram submetidos ao protocolo de indução alérgica crônica das vias aéreas com ovoalbumina (OVA) ou salina intraperitoneal (i.p.) e posterior desafios inalatórios. A partir do 22° dia, os animais receberam diariamente tratamento com PNU ou veículo (Ve) até o 28° dia. Foram testadas três doses de PNU (0,5, 1,0 e 2,0 mg/Kg). A fim de evidenciar se o efeito obtido no tratamento com PNU era dependente do receptor alfa7nAChR, um grupo de animais foi tratado com MLA (antagonista do alfa7nAChR), previamente ao tratamento com PNU. No 29° dia do protocolo, os animais foram eutanasiados e foram avaliados o número de células inflamatórias no lavado broncoalveolar (LBA) e no sangue, os níveis de citocinas no LBA, a expressão do alfa7nAChR e mAchRs do tipo 3 (M3) e a ativação do fator de transcrição nuclear kB (NF-kB) no pulmão. O remodelamento brônquico foi avaliado por morfometria. As análises estatísticas foram realizadas por meio do programa SigmaStat (Jandel Scientific, San Rafael, CA), onde um P < 0,05 foi considerado estatisticamente significativo. Resultados: Houve expressão do alfa7nAChR e M3 no homogenato de pulmão de animais controle e sensibilizados. Determinamos por meio da redução de eosinófilos que a dose de 0,5 mg/Kg do tratamento com PNU foi a mais efetiva. Assim, observamos que o tratamento com PNU0,5 nos animais sensibilizados reduziu o número de células totais, eosinófilos, neutrófilos, macrófagos e linfócitos no LBA, assim como número de eosinófilos no sangue periférico e ao redor das vias aéreas. O tratamento com PNU reduziu os níveis de IgE no sangue e as citocinas IL-4, IL-13 e IL-17 no LBA. Todos estes efeitos foram revertido com o pré-tratamento com MLA, exceto para a citocina IL-17. Alem disso, o tratamento com PNU reduziu o remodelamento brônquico (área de edema, de epitélio e de músculo liso e deposição de fibras colágenas) assim como o número de células positivas para MMP-9 e TIMP-1 ao redor das vias aéreas. No pulmão a expressão do p-65-NF-kB, STAT3 fosforilado e o SOCS3 foram inibidas pelo PNU. Conclusão: Estes dados claramente demonstram que o alfa7nAChR está envolvido no controle da resposta inflamatória pulmonar alérgica e de remodelamento brônquico em modelo experimental de asma alérgica e portanto é um novo alvo com potencial terapêutico a ser explorado na fisiopatologia da asma brônquica / Background: Inflammation is one of the most important features in asthma pathophysiology, characterized by eosinophilic response with production of Th2 cytokine profile. The persistence of this inflammation can induce a lung repair process associated with a progressive reduction in lung function, which is not always reversed by available treatments. The anti-inflammatory cholinergic system was described as a neural mechanism that suppresses the immune response and controls inflammation mainly by the activaction of acetylcholine alfa7 nicotinic receptors (alfa7nAChR) found on immune cells. Acetylcholine (ACh) is an important mediator in asthma and we recently demonstrated that partial reduction on ACh release induced lung inflammation per se. Although it is known that muscarinic receptors (mAChRs) has a pro-inflammatory action and causes bronchoconstriction in asthma, the activation of nicotinic receptors (nAChRs) could have a beneficial effect reducing pulmonary inflammation as demonstrated in models of acute and systemic inflammation. The effects of alfa7nAChR activation in the pathophysiology of asthma have not been clearly elucidated. Aim: To investigate the effects of PNU- 282987 (PNU) treatment, a specific alfa7nAChR agonist, in a murine model of chronic allergic airway inflammation. Methods: BALB/c mice were subjected to a protocol of chronic allergic inflammation induced by intraperitoneal ovalbumin (OVA) or saline and subsequent challenges with inhalation. From the 22th day, the animals daily received PNU or vehicle (Ve) until the 28th day. PNU were tested in three differents doses (0.5, 1.0 and 2.0 mg/kg). In order to demonstrate that the effects obtained by PNU treatment was dependent on alfa7nAChR, a group of animals was treated with MLA (antagonist of alfa7nAChR) prior to the PNU treatment. On the 29th day of the protocol, the animals were euthanised and the number of inflammatory cells in the bronchoalveolar lavage fluid(BALF) fluid and blood, cytokine levels in BALF, the expression of alfa7nAChR and mAChRs type 3 (M3), and activation of nuclear transcription factor kB (NF-kB) in the lung were evaluated. Bronchial remodeling was assessed by morfometric methods. Statistical analyses were performed using the SigmaStat (Jandel Scientific, San Rafael, CA) and P < 0.05 is considered statistically significant. Results: ?7nAChR and M3 expression was detected in control and sensitized lung homogenate. The most effective dose of PNU treatment was 0.5 mg/kg evaluated by the effects on eosinophil reduction. Thus, we observed that treatment with PNU0,5 reduced the number of total cells, eosinophils, neutrophils, macrophages and lymphocytes in BALF, as well as number of eosinophils in peripheral blood and around the airways of sensitized animals. The treatment with PNU also reduced IgE levels in the blood, and cytokines IL-4, IL-13 and IL-17 in BALF. All these effects were reversed by pretreatment with MLA, except for IL-17 cytokine. Furthermore, treatment with PNU reduced bronchial remodeling (edema, epithelium and smooth muscle area and airway collagen deposition) as well as the number of positive cells for MMP-9 and TIMP-1 around the airways. The lung p-65-NF-kB, phosphorylated STAT3 and the SOCS3 expression were inhibited by PNU-282987. Conclusion: These data clearly demonstrate that the alfa7nAChR is involved in the control of allergic pulmonary inflammatory response and in bronchial remodeling in an experimental model of allergic asthma and it can be a new target with therapeutic potential to be explored in the pathophysiology of asthma

Introdução: A inflamação constitui um dos fatores mais importantes da fisiopatologia da asma brônquica, caracterizada por uma resposta eosinofílica com produção de citocinas de perfil Th2. A persistência desta inflamação induz no pulmão um processo de reparo associado à redução progressiva da função pulmonar, que nem sempre é revertida pelos tratamentos disponíveis. O sistema colinérgico anti-inflamatório é descrito como um mecanismo neural que suprime a resposta imune e controla a inflamação principalmente pelo efeito da acetilcolina em receptores nicotínicos do tipo alfa7 (alfa7nAChR) encontrados em células do sistema imune. A acetilcolina (ACh) exerce um importante efeito na asma e recentemente demonstramos que a redução parcial da liberação da acetilcolina induz per se a inflamação pulmonar. Embora se saiba que os receptores muscarínicos (mAChRs) exercem um efeito pró-inflamatório e broncoconstritor na asma, a ativação de receptores nicotínicos (nAChRs) poderia ter um efeito benéfico reduzindo a inflamação pulmonar, fato demonstrado em modelos de inflamação sistêmica e aguda. O efeito da ativação do alfa7nAChR na fisiopatologia da asma ainda não está claramente elucidado. Objetivo: Investigar o efeito do tratamento com PNU-282987 (PNU), um agonista específico do alfa7nAChR, em um modelo murino de inflamação alérgica crônica das vias aéreas. Metodologia: Camundongos BALB/c foram submetidos ao protocolo de indução alérgica crônica das vias aéreas com ovoalbumina (OVA) ou salina intraperitoneal (i.p.) e posterior desafios inalatórios. A partir do 22° dia, os animais receberam diariamente tratamento com PNU ou veículo (Ve) até o 28° dia. Foram testadas três doses de PNU (0,5, 1,0 e 2,0 mg/Kg). A fim de evidenciar se o efeito obtido no tratamento com PNU era dependente do receptor alfa7nAChR, um grupo de animais foi tratado com MLA (antagonista do alfa7nAChR), previamente ao tratamento com PNU. No 29° dia do protocolo, os animais foram eutanasiados e foram avaliados o número de células inflamatórias no lavado broncoalveolar (LBA) e no sangue, os níveis de citocinas no LBA, a expressão do alfa7nAChR e mAchRs do tipo 3 (M3) e a ativação do fator de transcrição nuclear kB (NF-kB) no pulmão. O remodelamento brônquico foi avaliado por morfometria. As análises estatísticas foram realizadas por meio do programa SigmaStat (Jandel Scientific, San Rafael, CA), onde um P < 0,05 foi considerado estatisticamente significativo. Resultados: Houve expressão do alfa7nAChR e M3 no homogenato de pulmão de animais controle e sensibilizados. Determinamos por meio da redução de eosinófilos que a dose de 0,5 mg/Kg do tratamento com PNU foi a mais efetiva. Assim, observamos que o tratamento com PNU0,5 nos animais sensibilizados reduziu o número de células totais, eosinófilos, neutrófilos, macrófagos e linfócitos no LBA, assim como número de eosinófilos no sangue periférico e ao redor das vias aéreas. O tratamento com PNU reduziu os níveis de IgE no sangue e as citocinas IL-4, IL-13 e IL-17 no LBA. Todos estes efeitos foram revertido com o pré-tratamento com MLA, exceto para a citocina IL-17. Alem disso, o tratamento com PNU reduziu o remodelamento brônquico (área de edema, de epitélio e de músculo liso e deposição de fibras colágenas) assim como o número de células positivas para MMP-9 e TIMP-1 ao redor das vias aéreas. No pulmão a expressão do p-65-NF-kB, STAT3 fosforilado e o SOCS3 foram inibidas pelo PNU. Conclusão: Estes dados claramente demonstram que o alfa7nAChR está envolvido no controle da resposta inflamatória pulmonar alérgica e de remodelamento brônquico em modelo experimental de asma alérgica e portanto é um novo alvo com potencial terapêutico a ser explorado na fisiopatologia da asma brônquica / Background: Inflammation is one of the most important features in asthma pathophysiology, characterized by eosinophilic response with production of Th2 cytokine profile. The persistence of this inflammation can induce a lung repair process associated with a progressive reduction in lung function, which is not always reversed by available treatments. The anti-inflammatory cholinergic system was described as a neural mechanism that suppresses the immune response and controls inflammation mainly by the activaction of acetylcholine alfa7 nicotinic receptors (alfa7nAChR) found on immune cells. Acetylcholine (ACh) is an important mediator in asthma and we recently demonstrated that partial reduction on ACh release induced lung inflammation per se. Although it is known that muscarinic receptors (mAChRs) has a pro-inflammatory action and causes bronchoconstriction in asthma, the activation of nicotinic receptors (nAChRs) could have a beneficial effect reducing pulmonary inflammation as demonstrated in models of acute and systemic inflammation. The effects of alfa7nAChR activation in the pathophysiology of asthma have not been clearly elucidated. Aim: To investigate the effects of PNU- 282987 (PNU) treatment, a specific alfa7nAChR agonist, in a murine model of chronic allergic airway inflammation. Methods: BALB/c mice were subjected to a protocol of chronic allergic inflammation induced by intraperitoneal ovalbumin (OVA) or saline and subsequent challenges with inhalation. From the 22th day, the animals daily received PNU or vehicle (Ve) until the 28th day. PNU were tested in three differents doses (0.5, 1.0 and 2.0 mg/kg). In order to demonstrate that the effects obtained by PNU treatment was dependent on alfa7nAChR, a group of animals was treated with MLA (antagonist of alfa7nAChR) prior to the PNU treatment. On the 29th day of the protocol, the animals were euthanised and the number of inflammatory cells in the bronchoalveolar lavage fluid(BALF) fluid and blood, cytokine levels in BALF, the expression of alfa7nAChR and mAChRs type 3 (M3), and activation of nuclear transcription factor kB (NF-kB) in the lung were evaluated. Bronchial remodeling was assessed by morfometric methods. Statistical analyses were performed using the SigmaStat (Jandel Scientific, San Rafael, CA) and P < 0.05 is considered statistically significant. Results: ?7nAChR and M3 expression was detected in control and sensitized lung homogenate. The most effective dose of PNU treatment was 0.5 mg/kg evaluated by the effects on eosinophil reduction. Thus, we observed that treatment with PNU0,5 reduced the number of total cells, eosinophils, neutrophils, macrophages and lymphocytes in BALF, as well as number of eosinophils in peripheral blood and around the airways of sensitized animals. The treatment with PNU also reduced IgE levels in the blood, and cytokines IL-4, IL-13 and IL-17 in BALF. All these effects were reversed by pretreatment with MLA, except for IL-17 cytokine. Furthermore, treatment with PNU reduced bronchial remodeling (edema, epithelium and smooth muscle area and airway collagen deposition) as well as the number of positive cells for MMP-9 and TIMP-1 around the airways. The lung p-65-NF-kB, phosphorylated STAT3 and the SOCS3 expression were inhibited by PNU-282987. Conclusion: These data clearly demonstrate that the alfa7nAChR is involved in the control of allergic pulmonary inflammatory response and in bronchial remodeling in an experimental model of allergic asthma and it can be a new target with therapeutic potential to be explored in the pathophysiology of asthma

Os receptores nicotínicos de acetilcolina (colinérgicos) (nAChRs) neuronais são proteínas integrais de membrana e pertencem à família de canais iônicos controlados por ligante, compostos por subunidades &#945; e &#946;. Esses receptores desempenham um papel-chave na transmissão de sinal entre os neurônios nos sistemas nervoso central e periférico. O subtipo &#945;3&#946;4, por exemplo, é o nAChR neuronal mais expresso no sistema nervoso autônomo; nAChRs contendo a subunidade &#945;3 estão presentes em alta densidade no gânglio cervical superior, glândulas pineal e adrenais. Também estão presentes na substancia nigra, striatum, hipocampo, locus ceruleus, tracto habênulo-interpeduncular e cerebelo. Os nAChRs são inibidos por uma variedade de substâncias químicas, incluindo toxinas naturais, anestésicos locais, drogas de abuso (por, exemplo, cocaína) e compostos clinicamente importantes (tranqüilizantes, por exemplo). O mecanismo de inibição desses receptores tem sido investigado intensivamente. Neste estudo, nós investigamos o mecanismo pelo qual a tacrina (9-1,2,3,4-tetraidroaminoacridina), um agente usado clinicamente no tratamento da doença de Alzheimer, inibe o nAChR &#945;3&#946;4 de rato recombinante expresso nas células KX&#945;3&#946;4R2, utilizando uma técnica de cinética química rápida. A constante de dissociação da nicotina do sítio que controla a ativação do receptor, Kd, é 23 &#181;M e a constante de equilíbrio de abertura do canal, &#934;-1, é 4. A tacrina inibe o receptor competitivamente, com um KI de 0,77 &#181;M. / Neuronal nicotinic acetylcholine (cholinergic) receptors (nAChRs) are integral membrane proteins and belong to the family of ligand-gated cation channels composed by &#945; and &#946; subunits. These receptors play a key role in the signal transmission between neurons in the central and peripheral nervous system. The &#945;3&#946;4 subtype, for example, is the most expressed neuronal nAChR in autonomic ganglia; &#945;3-containing nAChRs are present at particularly high density in the superior cervical ganglia, pineal, and adrenal glands. They are also present in the substancia nigra, striatum, hippocampus, locus ceruleus, habenulo-interpeduncular tract and cerebellum. The nAChRs are inhibited by a variety of chemical substances, including natural toxins, local anesthetics, abused drugs (e.g., cocaine) and clinically important compounds (e.g., tranquilizers). The mechanism of inhibition of these receptors has been intensively investigated. In this study, we investigated the mechanism by which tacrine (9-1,2,3,4-tetahydroaminoacridine), an agent used clinically to treat Alzheimers disease, inhibits the recombinant rat &#945;3&#946;4 nAChR expressed in KX&#945;3&#946;4R2 cells, using a rapid chemical kinetic technique. The nicotine dissociation constant for the site controlling receptor activation, Kd, is 23 &#181;M and the channel-opening equilibrium constant, &#934;-1, is 4. Tacrine inhibits the receptor competitively, with a KI of 0.77 &#181;M.

Pentameric ligand-gated ion channels (pLGICs) are membrane-embedded receptors found extensively in pre- and post-synaptic membranes throughout the nervous system where they play an important role in neurotransmission. The function of the prototypic pLGIC, the nicotinic acetylcholine receptor (nAChR) is highly sensitive to changes in its lipid environment, while other pLGICs display varying lipid sensitivities. This thesis presents a multidisciplinary investigation into the features of the transmembrane domain (TMD) that determine the unique functional and physical traits of different pLGICs. Using two prokaryotic homologues of the nAChR, ELIC and GLIC, as models, I focus on the outermost, lipid-exposed α-helix, M4, which, despite being distant from the primary allosteric pathway coupling agonist binding to channel gating, exercises significant control over channel function. Here, I present evidence that M4 acts as a lipid sensor, detecting changes in the surrounding lipids and transmitting these changes to the channel pore via contacts with the adjacent TMD α-helices, M1 and M3, and/or with structures in the extracellular domain. Using ELIC and GLIC chimeras, I first show that the TMD is the main driver of pLGIC thermal stability. I then demonstrate that the M4 α-helices in each channel play different roles in channel maturation and function, which suggests a divergent evolutionary path. Following this, I show that the M4 C-terminus is essential to both maturation and function in GLIC, while in ELIC its role is less defined, again showcasing possible evolutionary differences. Building on these findings, I examined the role of aromatic residues at the M4 – M1/M3 interface, and found that they predictably determine the interactions between M4 and M1/M3. Notably, the addition of aromatic residues to enhance M4-M1/M3 interactions in ELIC promotes channel function, while the elimination of aromatic residues at the M4-M1/M3 interface in GLIC is detrimental to channel function. Furthermore, I show that these same aromatics alter the strength of pLGIC lipid sensing and the sensitivity to certain disease-causing mutations, both indicating that aromatic residues are key players in channel function, stability and modulation. Finally, I and my collaborators identified and characterized a novel desensitization-linked lipid binding site in ELIC. Extensive mutagenesis studies coupled with biophysical measurements allowed us to develop a model describing how lipid binding influences the rates of ELIC desensitization to shape the agonist-induced response.

Alcohol, recently named the most dangerous drug in the world, contributes to nearly 40% of violent crimes and fatal traffic accidents, increases risk of roughly 60 different diseases and injuries, and is responsible for 2.5 million deaths each year worldwide. Despite these staggering figures, treatments remain ineffective and riddled with adverse side effects, making successful use of even the most effective treatments unlikely. Moreover, many of the treatments, and the supporting research, have focused only on male subjects, despite sex differences in various alcohol-related behaviors. Human alcohol use is frequently accompanied by nicotine use, and vice versa, suggesting a common mechanism of the two drugs. In fact, alcohol may act through the same family of receptors as nicotine, the nicotinic acetylcholine receptors (nAChRs), eliciting similar activation of the reward pathway as nicotine and other drugs of abuse. Studies have shown that nAChRs containing the α4 and/or α6 subunits are involved in nicotine-induced activation of the reward pathway, leading to the hypothesis that these same receptor subtypes may be important for alcohol effects in the brain as well. Using male and female genetic mouse models and various behavioral assays, we have shown not only that these α4 and/or α6-containing nAChRs are involved in alcohol- related behaviors and activation of the reward pathway, but also show sex differences in this involvement. Uncovering the mechanism of alcohol in the brain, in males as well as in females, is an important step in developing targeted treatments for alcohol abuse.

Indiana University-Purdue University Indianapolis (IUPUI) / Nicotine use among individuals with schizophrenia occurs at extremely high rates. The prevailing theory is that individuals with schizophrenia smoke as a form of self-medication to ameliorate sensory and cognitive deficits. However, these individuals also have enhanced rates of addiction to several drugs of abuse and may therefore smoke as a result of enhanced addiction liability. The experiments described herein explored these two hypotheses by assessing the effect that nicotine has on working memory, addiction vulnerability (locomotor sensitization and self-administration), and nicotinic acetylcholine receptor (nAChR) expression as well as the developmental expression of these characteristics in the neonatal ventral hippocampal (NVHL) neurodevelopmental animal model of schizophrenia. The results from these studies indicate that NVHLs had working memory impairments in both adolescence and adulthood, with nicotine having a negligible effect. Additionally, NVHLs displayed enhanced locomotor sensitization to nicotine which emerged in adulthood as well as an enhanced acquisition of nicotine self-administration, administering more nicotine overall. These behavioral differences cannot be attributed to nAChR expression as nicotine upregulated nAChR to a similar extent between NVHL and SHAM control animals. These data indicate that the enhanced rates of nicotine use among individuals with schizophrenia may occur as a result of an enhanced vulnerability to nicotine addiction.