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Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Swelling-activated Cl- current (ICl,swell) is an outwardly rectifying Cl- current that influences cardiac electric activities and acts as a potential effector of mechanoelectrical feedback that antagonizes the effects of stretch-activated cation channels. Persistent activation of ICl,swell has been observed in multiple models of cardiovascular diseases. Previously we showed that angiotensin II (AngII) signaling and reactive oxygen species (ROS) produced by NADPH oxidase (NOX) are involved in the activation of ICl, swell by both beta1-integrin stretch and osmotic swelling. Because endothelin-1 (ET-1) is a potential downstream mediator of AngII and ETA receptor blockade abrogates AngII-induced ROS generation, we studied how ET-1 signaling regulates ICl,swell and the relationship between AngII and ET-1 signaling. Under isosmotic conditions, ET-1 elicited an outwardly rectifying Cl- current that was fully blocked by the highly selective ICl,swell inhibitor DCPIB and by osmotic shrinkage. Selective ETA blockade (BQ123), but not ETB blockade (BQ788), fully suppressed the ET-1-induced current. ET-1-induced ICl,swell was abolished by blockade of EGFR kinase (AG1478) and PI-3K inhibitors (LY294002 and wortmannin), which also suppress beta1-integrin stretch- and swelling-induced ICl,swell. ET-1-induced ICl,swell was abrogated by ebselen, a membrane-permeant glutathione peroxidase mimetic that dismutates H2O2 to H2O, suggesting that ROS were required intermediates in ET-1-induced activation of ICl,swell. Both NOX and mitochondria are important sources of ROS in cardiomyocytes. Blocking NOX with apocynin or mitochondrial complex I with rotenone both completely suppressed ET-1-induced ROS generation and activation of ICl,swell, indicating that ROS from both NOX and mitochondria were required to activate ICl,swell, and complete block by inhibitors of either ROS source suggests mitochondrial and NOX must act in series rather than in parallel. ICl,swell elicited by antimycin A, which stimulates superoxide production by mitochondrial complex III, was insensitive to NOX inhibitor apocynin and the NOX fusion peptide inhibitor gp91ds-tat. Activation of ICl,swell induced by diazoxide, which stimulates mitochondrial ROS production by opening mitochondrial KATP channels, was not affected by gp91ds-tat. These data suggests that mitochondrial ROS is downstream from NOX in the regulation of ICl,swell. Mitochondrial ROS production that is enhanced by NOX ROS is likely to be responsible for the activation of ICl,swell by ET-1. In order to determine the role of ERK in the proposed signaling pathway that regulates ICl,swell, we examined the effect of ERK inhibitors (PD 98059 and U0216) on the activation of ICl,swell elicited by ET-1, EGF, and H2O2. ERK inhibitors partially blocked ET-1-induced ICl,swell but fully inhibited activation of ICl,swell in response to EGF. However, ERK inhibitors did not affect ICl,swell elicited by exogenous H2O2. We also established the the relationship of ET-1 to AngII and osmotic swelling in the regulation of ET-1 ICl,swell. ETA blockade abolished ICl,swell elicited by both AngII and osmotic swelling, whereas AT1 blockade did not effect ET-1-induced ICl,swell, suggesting that ET-1 signaling is downstream from AngII and osmotic swelling. HL-1 cell is a murine atrial cell line that retain phenotypic characteristics of adult cardiomyocytes. We showed that osmotic swelling and ET-1 turned on DCPIB-sensitive outwardly rectifying Cl- current in HL-1 cells with both physiological and symmetrical Cl- gradients. The swelling-induced current was suppressed by gp91ds-tat and rotenone but insensitive to apocynin. Blockade of ETA receptor (BQ123) and NOX (gp91ds-tat) completely inhibited ET-1-induced ICl,swell in HL-1 cells. These data indicate that ICl,swell is present in HL-1 cell and regulated by similar mechanisms as in native cells. Finally, we confirmed the production of ROS by ET-1 signaling by flow cytometry of HL-1 cells using the nominally H2O2-selective fluorescent probe C H2DCFDA-AM. Exposure to ET-1 increased ROS production, as did H2O2, a positive control. ET-1-induced ROS production was fully suppressed by both gp91ds-tat and rotenone. HL-1 cell ROS production also was stimulated by the mitochondrial complex III inhibitor antimycin A, and antimycin A-induced ROS production was blocked by rotenone but not by gp91ds-tat. These data suggest that ET-1 ETA receptor signaling elicits ICl,swell by sequentially stimulating ROS production by NOX and mitochondria. ETA receptor signaling is down stream from AngII in the osmotic swelling-induced activation of ICl,swell and is upstream from EGFR kinase and PI-3K. Endothelin signaling is likely to be an important means of activating ROS production and ICl,swell in a variety of cardiovascular diseases.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2013-05-20T22:24:50Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DistribuicaoEnzimasNADPH.pdf: 3742931 bytes, checksum: aff3cba8ac3c63bc8f6a64965ce54c8b (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2013-05-27T13:21:18Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DistribuicaoEnzimasNADPH.pdf: 3742931 bytes, checksum: aff3cba8ac3c63bc8f6a64965ce54c8b (MD5) / Made available in DSpace on 2013-05-27T13:21:18Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DistribuicaoEnzimasNADPH.pdf: 3742931 bytes, checksum: aff3cba8ac3c63bc8f6a64965ce54c8b (MD5) Previous issue date: 2010 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / O Toxoplasma gondii é um protozoário parasita intracelular obrigatório de prevalência mundial e importante causador de doenças em humanos e animais domésticos. No Brasil, até 80% da população pode estar infectada, dependendo da região. Os pacientes infectados agudamente geralmente apresentam infecção assintomática com posterior desenvolvimento de cistos teciduais, que são mais comumente encontrados nos músculos estriados, retina e cérebro. A infecção latente pode alterar o comportamento dos animais hospedeiros e provocar sintomas psicóticos em humanos. Estudos sugerem que esta infecção pode contribuir para a ocorrência de desordens neurológicas e psiquiátricas, como por exemplo, a doença de Parkinson e esquizofrenia, que são associadas com anormalidades do sistema dopaminérgico. Neste estudo, avaliou-se a imunoreatividade contra a enzima tirosina hidroxilase (TH) e a atividade da NADPH-diaforase na substância negra do cérebro de camundongos infectados. Camundongos machos da linhagem Swiss Webster (Mus musculus) receberam por gavagem 10 cistos contendo bradizoítos da cepa Me-49 do Toxoplasma gondii. Os cérebros destes camundongos foram removidos após eutanásia por decapitação após os períodos de 30 e 60 dias de inoculação. A análise das secções mostrou uma reduzida marcação histoquímica para NADPHdiaforase na substância negra dos animais infectados quando comparados com os animais controle. Esta redução foi observada também na imunoreatividade contra a enzima TH na substância negra. Estes resultados indicam que a presença do T. gondii modifica o metabolismo na região da substância negra, modulando os níveis de óxido nítrico (NO) e dopamina, o que pode ser responsável pelas alterações comportamentais presentes nos hospedeiros intermediários infectados. / Toxoplasma gondii is a worldwide intracellular protozoan parasite and an important cause of disease in both humans and domestic animals. In Brazil, up to 80% of the population may be infected, depending on the region. Acute infection is generally asymptomatic, with posterior development of tissues cysts located mostly in muscles, retina and the central nervous system. The latent infection can alter behavior in animals and provoke psychotic symptoms in humans. Studies suggest that this infection may contribute to the occurrence of both schizophrenia and Parkinson’s disease, which are associated with abnormalities of the dopaminergic system. In this study we evaluated immunoreactivity for tyrosine hydroxylase (TH) and NADPH-diaphorase activities in the substantia nigra of infected male swiss mice. The animals were inoculated with bradyzoites (10 cysts) from a Toxoplasma gondii (Me-49 strain). The brains were removed after 30 and 60 days, sectioned and submitted to protocols to reveal NADPH-diaforase activity and immunohistochemistry anti-TH. The densitometric analysis showed a lower reactivity to NADPH-diaforase in the substantia nigra of infected animals when comparated with non-infected controls. A similar reduction was also observed in immunoreactivity against TH in the substantia nigra. These results indicate that T. gondii may change the dopaminergic system in infected brains.

Le complexe de la NADPH oydase des neutrophiles catalyse la formation des ions<br />superoxydes nécessaires à la destruction des pathogènes. Il est constitué de deux protéines<br />membranaires (gp91phox, p22phox), de trois facteurs cytosoliques (p47phox, p67phox, p40phox) et<br />d'une petite protéine G, Rac. Afin de comprendre les interactions et les réarrangements<br />structuraux mis en jeu lors de l'activation du complexe, des études structurales des facteurs<br />cytosoliques entiers seuls et en complexe ont été menées. Deux approches ont été choisies : la<br />microscopie électronique et la diffusion des rayons X aux petits angles. Cette dernière<br />approche a permis de calculer une enveloppe pour p47phox et p67phox.

Lors de la phagocytose d'un micro-organisme, le complexe de la NADPH Oxydase des neutrophiles est activé. Il permet alors la production d'espèces réactives de l'oxygène qui contribuent à la destruction du pathogène. Ce complexe est constitué de facteurs cytosoliques (p67phox, p40phox, p47phox), d'une petite protéine G Rac et du flavocytochrome b558 membranaire, lui-même composé de p22phox et gp91phox. Des mutations dans les gènes codant pour certaines de ces protéines conduisent à une maladie génétique rare mais grave!: la granulomatose septique chronique (CGD). Au sein du complexe ternaire formé par les facteurs cytosoliques, il existe des interactions de type domaine SH3/motif polyproline et une interaction entre domaines PB1. Par Résonance Magnétique Nucléaire, nous avons caractérisé d'un point de vue structural l'interaction entre les domaines PB1 de p67phox et p40phox. Nous avons également étudié les conséquences de l'activation sur les interactions entre le motif polyproline C-terminal de p47phox et ses domaines SH3 partenaires. Ainsi, nous avons combiné l'analyse des structures des domaines SH3 de p40phox et SH3 C-terminal de p67phox, en complexe avec le polyproline C-terminal de p47phox, avec nos mesures d'affinité entre ces partenaires à différents stades de l'activation. Ces données ont été obtenues par fluorescence intrinsèque du tryptophane présent au sein des domaines SH3.