L'objectif de mon travail de thèse était d'une part, de mieux comprendre le rôle des différentes familles de phosphodiestérases (PDEs) dans la régulation de la signalisation dépendante de l'AMPc (PDE-AMPc) dans les cellules musculaires lisses vasculaires (CMLVs), et d'autre part, d'évaluer leur implication fonctionnelle dans la réactivité vasculaire et leur altération potentielle dans un modèle physiopathologique, l'insuffisance cardiaque (IC). Mon travail s'est articulé autour de deux modèles de muscle lisse vasculaire : (1) des CMLVs isolées en culture ayant acquis un phénotype synthétique sur lesquelles une approche d'imagerie en temps réel (FRET : Transfert d'Energie de Fluorescence par Résonance) a été appliquée afin de visualiser in situ la dynamique spatiotemporelle des signaux dépendants de l'AMPc. Nos résultats indiquent que, dans ces cellules, l'augmentation des niveaux d'AMPc provoquée par la stimulation β-adrénergique (β-AR) implique différents récepteurs suivant le compartiment intracellulaire considéré (β1- et β2-ARs dans le cytosol et seulement β2-ARs dans le compartiment sous-membranaire). Nous avons par ailleurs observé que l'expression des ARNm des différentes isoformes de PDE-AMPc et la contribution fonctionnelle de ces enzymes dans la régulation des signaux AMPc intracellulaires étaient dépendantes de la densité des CMLVs en culture.(2) des anneaux d'artères intactes issues de deux lits vasculaires différents (aorte et artère mésentérique) isolées à partir de rats sains et IC, permettant d'étudier leur fonction contractile et donc la régulation de celle-ci par la voie de l'AMPc. Nous avons montré que les familles de PDE-AMPc contribuent de façon différente au contrôle du tonus vasculaire dans l'aorte thoracique (PDE3 = PDE4 sans participation de la PDE2) et dans l'artère mésentérique (PDE4 > PDE2 sans participation de la PDE3), l'endothélium exerçant un rôle essentiel dans la régulation de l'activité de ces PDEs musculaires lisses, notamment par le biais de la production de NO. Nous avons également mis en évidence des altérations de la réactivité vasculaire, et notamment de son contrôle par la voie de l'AMPc/PDE, dans notre modèle de rat IC. Dans l'aorte, la dysfonction endothéliale liée à l'altération de la voie du NO est à l'origine d'une augmentation de l'activité PDE3 masquant l'activité PDE4 et la relaxation β-adrénergique. Dans l'artère mésentérique des rats IC, dont la fonction endothéliale apparaît préservée, les PDE2, 3 et 4 restent fonctionnelles.L'ensemble de nos travaux souligne le rôle essentiel des PDEs dans la régulation de la signalisation AMPc vasculaire, et montre que l'activité et la fonction des différentes familles de PDE-AMPc sont finement modulées par de nombreux paramètres (phénotype et densité cellulaire des CMLVs) ou situations physio-pathologiques (nature du lit vasculaire, présence de l'endothélium, situation d'IC).

O óxido nítrico (NO) é um mensageiro químico gerado pela atividade da enzima óxido nítrico sintase (NOS) a qual foi detectada em vários órgãos incluídos os do sistema reprodutor e parece estar envolvido na maturação oocitária. No entanto, há poucos estudos sobre o papel desse sistema em oócitos da espécie bovina. Sabe-se que o NO atua pela via da guanilato ciclase (GC) estimulando a produção do nucleotídeo GMPc, que por sua vez é capaz de influenciar nos níveis de outro nucleotídeo (AMPc) via fosfodiesterases (PDE). O AMPc é um importante elemento da via de sinalização do FSH nos complexos cumulus-oócitos e no controle da maturação oocitária. O objetivo do presente projeto foi investigar a influência do FSH na via do NOS/NO e seus componentes em oócitos bovinos maturados in vitro e o envolvimento das células do cumulus (CC) na via de sinalização. Para tanto, complexos cumulus-oócito (CCO) e oócitos desnudos (OD - maturados sem células do cumulus) foram maturados in vitro por 24h na presença ou ausência de FSH. As amostras foram avaliadas quanto a: 1) taxa da maturação nuclear; 2) níveis de produção de NO; 3) níveis de AMPc e GMPc; 4) abundância relativa de RNAm de NOS2, PDE5A, PDE6C, PKG1, PKG2, ADCY6, ADCY9, PDE3A e PKA1. O FSH, na concentração de 0,05UI/mL, estimulou positivamente a maturação nuclear em CCO e OD, com 80,6 e 89% de oócitos maturados, respectivamente. Quando comparados diretamente os grupos CCO e OD, o FSH não influenciou as taxas de maturação (71 e 71,3%, p>0,05), nem os níveis de produção de NO (12,8 e 7,4 µM/mL, p>0,05). Os níveis de GMPc em CCO aumentaram após 1 e 3 h de MIV na presença de FSH (266,3 e 187,2 pmol/pool com FSH e 240,5 e 168,5 pmol/pool sem FSH, respectivamente, p<0,05). Após 6 h os níveis de GMPc declinaram de forma mais acentuada no grupo sem FSH (46,3 e 106,9 pmol/pool, com e sem FSH, respectivamente, p<0,05). Os níveis de AMPc em CCO também foram mais elevados na presença de FSH à 1 e 3 h de MIV (7,60 e 7,81 pmol/pool, respectivamente) em comparação com CCO maturados sem FSH (0.30 e 0,76 pmol/pool, respectivamente, p<0,05). Após 6h, os níveis declinaram e foram similares para ambos os grupos (0,43 pmol/pool, p>0,05). Em relação à expressão dos genes selecionados, todos foram detectados nos oócitos (CCO e OD), porém, em células do cumulus, foram detectados apenas PDE5A, ADCY6, ADCY9 e PKA1. Quando observados os resultados do grupo CCO, apenas os genes PKG1, ADCY6 e PDE3A sofreram influência do FSH (p<0,05), apresentando um aumento destes transcritos. No grupo OD, apenas o gene PKG1 sofreu influência do FSH, também apresentando um aumento destes transcritos (p<0,05). Em células do cumulus, os genes ADCY6 e ADCY9 sofreram influência do FSH, sendo que para a ADCY6 provocou um aumentos destes transcritos, e para a ADCY9 provocou uma queda dos mesmos (p<0,05). Em conclusão, o FSH pode exercer influência positiva na maturação nuclear de oócitos bovinos, agindo sobre os níveis de GMPc e AMPc, mas não sobre o NO. O FSH pode influenciar a expressão gênica em oócitos e em células do cumulus de bovinos. / Nitric oxide (NO) is a chemical messenger generated by the nitric oxide synthase (NOS) enzyme, which was detected in several organs including the reproductive system and appears too involved in oocyte maturation. However, there are few studies on the role of this system in bovine oocytes. NO is known to act via guanylate cyclase (GC) stimulating the production of the nucleotide cGMP, which in turn is capable of influencing the levels of another nucleotide, cAMP via phosphodiesterases (PDE). cAMP is an important factor in FSH signaling in cumulus-oocyte complexes (COC) for the control of maturation. The aim of the present work was to investigate the influence of FSH on the NOS/NO pathway and its components in bovine oocytes matured in vitro and the involvement of cumulus cells (CC) in the signaling pathway. COC and denuded oocytes (DO - matured without cumulus cells) were matured in vitro for 24 h with or without FSH. Samples were assessed for: 1) maturation rate; 2) levels of NO production; 3) levels of cGMP and cAMP; 4) relative abundance for mRNA of NOS2, PDE5A, PDE6C, PKG1, PKG2, ADCY6, ADCY9, PDE3A and PKA1. FSH positively stimulated oocyte maturation at 0.05UI/mL concentration for both COC and OD (80.6 and 89% maturation rates, respectively). When COC and OD were compared directly, FSH did not affect maturation rates (71 and 71.3%, p>0.05) nor NO production levels (12,8 and 7,4 µM/mL), p>0.05). cGMP levels increased after 1 and 3 h in vitro maturation (IVM) with FSH (266.3 and 187.2 pmol/pool with FSH and 240.5 and 168.5 pmol/pool without FSH, respectively, p<0.05). After 6 h IVM, cGMP levels in COC declined more in the group cultured with FSH (46.3 and 106.9 pmol/pool, with and without FSH, respectively, p<0.05). cAMP levels in COC were also increased in the presence of FSH at 1 and 3 h IVM (7.60 and 7.81 pmol/pool, respectively) in comparison to COC cultured without the hormone (0.30 and 0.76 pmol/pool, respectively, p<0.05). After 6 h, the levels declined and were similar for both groups (0.43 and 0.02 pmol/pool, p>0.05). Regarding mRNA expression for the selected genes, all of them were detected in oocytes, but only four of them were detected in cumulus cells: PDE5A, ADCY6, ADCY9 and PKA1. For COC only PKG1, ADCY6 and PDE3A were influenced by FSH (p<0.05), with an increase in transcript relative abundance, For DO, only PKG1 was influenced by FSH and also showed an increase in these transcripts (p<0.05). In cumulus cells, ADCY6 and ADCY9 were affected by FSH, with an increase for ADCY6 and a decrease in ADCY9 transcripts (p<0.05). In conclusion, FSH may positively influence nuclear maturation, acting on cGMP and cAMP levels, but not on NO. FSH may also influence gene expression in bovine oocytes and cumulus cells.

Estudos anteriores de nosso laboratório demonstraram que a estimulação aguda dos receptores 2-adrenérgicos (2-AR) atenua a perda de massa muscular induzida pela desnervação motora (DEN) por meio de uma via dependente de AMPc/PKA. No entanto os mecanismos moleculares envolvidos na ativação crônica destes receptores ainda são pouco conhecidos. Por outro lado, a ativação desta via de sinalização também está envolvida no controle da estabilidade dos receptores nicotínicos (AChR) na junção neuromuscular (JNM), sugerindo que a densidade dos AChR possa estar sob controle neuro-humoral. Desta forma, aventou-se a possibilidade de que além dos efeitos protetores na massa muscular, a ativação dos receptores 2-AR pudesse mediar a estabilização dos AChR na placa motora. Para testar essa hipótese, camundongos foram submetidos à DEN através da secção do nervo ciático, um protocolo clássico de indução de atrofia muscular e desestabilização dos AChR, e tratados com salina ou clembuterol (CB), um 2-agonista seletivo, por até 14 dias. Após 3 dias de DEN, observou-se redução da massa muscular e aumento do conteúdo proteico e expressão do RNAm de genes relacionados à ativação do sistema Ubiquitina-Proteassoma (atrogina-1 e MuRF1) e do sistema autofágico/lisossomal (catepsina L e LC3). A DEN também promoveu aumento no turnover dos AChR, no número de vesículas endocíticas e na expressão do RNAm para a subunidade 1 dos AChR. Após 7 dias, a DEN reduziu a expressão dos genes relacionados à atrofia e aumentou a atividade da via do AMPc/PKA independentemente do tratamento com CB. Na tentativa de elucidar os sinais extracelulares que produziam esta resposta adaptativa, foi demonstrado que neurônios catecolaminérgicos trafegam ao longo do nervo ciático e sua ablação pela DEN reduziu o conteúdo de noradrenalina muscular. Baseados nestes resultados, foi postulado a existência de uma hipersenbilidade às catecolaminas em músculos desnervados cronicamente. O tratamento com CB por 3 dias aboliu o aumento da expressão dos atrogenes induzido pela DEN e este efeito foi associado ao maior conteúdo de AMPc e de substratos fosforilados pela PKA. Além disso, o CB diminuiu a hiperexpressão do RNAm para catepsina L e LC3 induzida pela DEN de 7 dias. Embora o CB não tenha alterado a meia-vida dos AChR em músculos inervados e desnervados, houve um total bloqueio do aumento do número de vesículas endocíticas contendo o AChR em músculos desnervados e tratados com CB. Corroborando estes dados, o CB aumentou a incorporação de AChR novos nas JNM e este efeito foi também associado à maior expressão do RNAm para a subunidade 1-AChR em músculos desnervados. Esta ação do CB no turnover dos AChR parece ser direta uma vez que neuroniôs catecolaminérgicos presentes no nervo ciático ativam receptores 2-ARe a produção de AMPc especificamente na JNM. Em estudos in vitro, foi demonstrado que a estimulação colinérgica produzida pelo carbacol (10-4M) diminuiu a velocidade de síntese de proteínas, aumentou a proteólise total e a atividade do sistema proteolítico Ca2+-dependente em músculos soleus de ratos por meio da ativação dos receptores nicotínicos. Este efeito catabólico do carbacol foi completamente bloqueado pela adição de CB (10-4M) ao meio de incubação. Os dados obtidos no presente estudo permitem sugerir que a estimulação crônica dos 2-AR no músculo esquelético induz um efeito anti-catabólico pela supressão dos sistemas proteolíticos proteassomal e lisossomal, provavelmente através da via de sinalização do AMPc/PKA. A inibição destes sistemas pode estar relacionada ao aumento do turnover dos AChR, uma vez que a velocidade de incorporação destes receptores na JNM foi aumentada pelo CB. Além disso, os achados que mostram a associação entre neurônios noradrenérgicos e colinérgicos no nervo ciático, que conjuntamente inervam as JNM, e a co-localização de receptores 2-AR e AChR na sinapse permitem sugerir a existência de uma interação funcional entre o sistema colinérgico e adrenérgico na manutenção da massa muscular e da placa motora. / Previous studies from our laboratory have shown that the acute stimulation of 2-adrenergic receptor (2-AR) attenuates the muscle loss induced by motor denervation (DEN) through a cAMP/PKA dependent pathway. However, the molecular mechanisms involved in the chronic activation of these receptors are poorly understood. Furthermore, the activation of this signaling pathway is also involved in controlling the stability of nicotinic receptors (AChR) at the neuromuscular junction (NMJ), suggesting that the density of AChR may be under neurohumoral control. Thus, we postulated that besides the protective effects on muscle mass the activation of 2-AR receptors could mediate the stabilization of AChR in the motor plate. To test this hypothesis, mice were submitted to DEN through of the sciatic nerve section, a classical protocol of induction muscle atrophy and destabilization of AChR, and were treated with saline or clenbuterol (CB), a selective 2-agonist for 14 days. DEN decreased the muscle mass and increased the protein content and mRNA expression of genes related to the activation of the ubiquitin-proteasome system (atrogin-1 and MuRF1) and autophagic/lysosomal system (cathepsin L and LC3). DEN also promoted an increase in the turnover of AChR, number of endocytic vesicles and the expression of mRNA for the 1 subunit of AChR. Interestingly, chronic DEN induced down-regulation of atrophy related-genes, and increased the activity of cAMP/PKA pathway independently of CB treatment. In an attempt to elucidate the extracellular signals that produced this adaptive response, it was demonstrated that catecholaminergic neurons travels along the sciatic nerve and its ablation by DEN reduces muscle norepinephrine content. Based on these results, it was postulated the existence of a muscle adrenergic hypersensitivity to circulating catecholamines induced by chronic DEN. CB treatment for 3 days completely abolished the higher expression of atrogenes and this effect was associated with increased Camp content and PKA phosphorylated substrates. Furthermore, CB decreased the DEN-induced hyperexpression of cathepsin L and LC3 mRNA at 7 days. Although CB has not altered the half-life of AChR in innervated and denervated muscles, it produced a total blockage of the increased number of endocytic vesicles containing the AChR in denervated muscles. Consistently, CB increased the incorporation of new AChR and this effect was associated with an increased expression of the 1-subunit AChR mRNA in denervated muscles. This action of CB on AChR turnover appears to be direct, since catecholaminergic neurons are present in the sciatic nerve stimulating 2-AR and cAMP production specifically in the NMJ. Furthermore, in vitro studies demonstrated that cholinergic stimulation produced by carbachol (10-4M) decreased the rate of protein synthesis and increased the proteolytic activity of Ca2+-dependent system in rat soleus muscle through activation of nicotinic receptors. This catabolic effect of carbachol was completely blocked by the addition of CB (10-4M) to the incubation medium. These data suggest that chronic stimulation of 2-AR in skeletal muscle induces an anti-catabolic effect by suppressing proteasomal and lysosomal proteolytic systems, probably through the cAMP/PKA signaling. The inhibition of these systems seems to be related to the increased AChR incorporation into NMJ induced by CB treatment. Moreover, the association between noradrenergic and cholinergic neurons in the sciatic nerve, both of which innervate the motor endplates, and the co-localization of AChR and 2-ARat the synapse suggest the existence of a functional interaction between cholinergic and adrenergic systems in the maintenance of muscle mass and motor endplate.

A transmissão sináptica é essencial para o funcionamento do sistema nervoso. A neuromodulação permite regular esse processo de forma precisa. Um desses mecanismos modulatórios é a regulação da liberação de neurotransmissores. A adenosina é um importante modulador da transmissão sináptica. Além disso, a ativação do subtipo A2a dos receptores para adenosina está envolvida com a facilitação da liberação de neurotransmissores no sistema nervoso central. O presente trabalho teve como objetivo avaliar os efeitos modulatórios da ativação do receptor A2a de adenosina sobre a liberação de neurotransmissores e sua via de sinalização intracelular em modelo in vitro. Além disso, a tese contempla a construção histórica dos conceitos abordados no trabalho permitindo uma visão clara de sua evolução. Esse projeto foi o pioneiro no Brasil a utilizar o sensor biossintético fluorescente de liberação de vesículas sinápticas (supereclipse sinapto-pHluorina), o qual foi gentilmente cedido pelo professor Gero Miensenboeck do Sloan-Kettering Institute for Cancer Research. Nossos resultados demonstraram que o tratamento com o agonista do receptor A A2a de adenosina aumentou a fluorescência do supereclipse sinapto-pHluorina, assim como os níveis de glutamato e noradrenalina. Além disso, foi demonstrado que o inibidor da proteína cinase dependente de AMPc aboliu o aumento nos níveis do glutamato e noradrenalina, tal como a fosforilação da proteína sináptica sinapsina I evocado pelo agonista do receptor A2a de adenosina. Desta forma, nossos dados sugerem que a ativação do receptor A2a de adenosina em cultura de células do bulbo de ratos Wistar modula a liberação de neurotransmissores e a fosforilação da sinapsina I, assim como a proteína cinase dependente do AMPc pode ser o modus operandi desse fenômeno modulatório / Synaptic transmission is a sine qua non process for nervous system physiology. Such precise process is accomplished in part due to modulation of neurotransmitter release. Adenosine is a putative synaptic transmission modulator. Moreover, adenosine A2a receptor facilitates neurotransmitter release in the Central Nervous System. The present study focuses on the modulation of neurotransmission by adenosine A2a receptor and its intracellular signaling pathway in in vitro model. Here, we provided evidence that adenosine A2a receptor agonist increases an optical biosynthetic sensor of synaptic vesicle release (supereclipct synapto-pHluorin), as well as glutamate and noradrenaline. Furthermore, it was demonstrated that cAMP-dependent protein kinase inhibitor abolished glutamate and norepinephrin increase, as well as synapsin I phosphorylation evoked by adenosine A2a receptor agonist. Therefore, our data suggest that adenosine A2a receptor activation modulates neurotransmitter release and synapsin I phosphorylation in cultured cells from medulla oblongata of Wistar rats, as well as cAMP-dependent protein kinase might be the modus operandi of this modulatory phenomenon

Les cellules ganglionnaires de la rétine (CGR) projettent dans le corps genouillé latéral dorsal (CGLd) et le colliculus supérieur (CS). Initialement, les projections établissent des connexions imprécises avec les cibles puis elles se raffinent grâce à des mécanismes activité-dépendant. D’abord, j’ai cherché à déterminer la contribution de la libération synaptique dans le raffinement des cartes visuelles en utilisant un modèle où Rim 1 et 2, protéines essentielles à la libération synaptique calcium-dépendante, sont délétées dans les CGRs. J’ai montré que la libération synaptique était nécessaire à la ségrégation œil spécifique dans le CGLd mais pas dans le CS, qu’elle était impliquée dans le raffinement de la topographie de la projection ipsilatérale mais qu’elle n’était pas impliquée dans la rétinotopie des projections controlatérales dans le CS. Ensuite, j’ai cherché à identifier un microdomaine de signalisation AMPc au niveau des cônes de croissance impliqué dans le raffinement des projections rétiniennes. La compartimentation spatiale des signaux AMPc pourrait expliquer qu’ils soient capables de réguler des voies de signalisation distinctes de façon spécifique. J’ai montré que la perturbation des signaux AMPc dans les radeaux lipidiques entraine des défauts de raffinement des projections dans le CS tandis que leur perturbation à la membrane mais en dehors des radeaux lipidiques n’entraine pas de défauts. J’ai aussi montré que la perturbation de la signalisation AMPc dans une petite fraction de CGRs d’un œil suffit à perturber largement la carte œil spécifique, suggérant une coopération entre les projections nécessaire à leur raffinement. / Retinal ganglion cells (RGCs) project to the dorsal lateral geniculate nucleus (dLGN) and to the superior colliculus (SC). Initially, retinal projections establish imprecise connections with the target cells then refine through activity-dependent mechanisms.First, I studied the role of synaptic release in the visual maps refinement, using a mouse model where Rim 1 and 2, which are proteins important for calcium-dependent synaptic release, are deleted in the RGCs. I found that synaptic release is important for eye specific segregation in the dLGN but not in the SC, that it is involved in the refinement of the ipsilateral projection topography but that it is not involved in the contralateral projection retinotopy in the SC.Second, I identified a microdomain of cAMP signaling in growth cones involved in the refinement of retinal projections. The spatial compartmentation of cAMP signals could explain the fact that cAMP signals are able to regulate specifically different signaling pathways. I found that cAMP signals perturbation within the lipid rafts of the RGCs induces defects in the refinement of retinal projections in the SC while their perturbation at the membrane level but outside the lipid rafts does not induce defects.I also showed that cAMP signaling perturbation only in a fraction of RGCs in one eye is sufficient to extensively disturb the eye specific map, suggesting that a cooperation between the projections is necessary for their refinement.

Les deux nucléotides cycliques, AMPc et GMPc, sont des seconds messagers importants qui régulent une grande variété de fonctions cellulaires, en particulier la fonction contractile cardiovasculaire, la croissance des cardiomyocytaires et la prolifération des cellules musculaires lisses vasculaires. Les phosphodiestérases (PDE) dégradent les nucléotides cycliques et exercent un contrôle local de leur concentration intracellulaire. Une altération de la voie de signalisation des nucléotides cycliques est impliquée dans plusieurs situations pathologiques telles que l’hypertension artérielle systémique ou pulmonaire, l’athérosclérose et l'hypertrophie cardiaque. Ainsi, les PDE constituent de puissantes cibles thérapeutiques pour restaurer un contrôle correct des nucléotides cycliques. Onze familles de PDEs sont actuellement décrites, les PDE1-6 étant les plus étudiées et les PDE 7-11 représentant de nouvelles familles.L'objectif de cette thèse était d'étudier le rôle respectif de 4 familles de PDEs, la PDE1, famille stimulée par le complexe Ca2+/calmoduline, les PDE5 et PDE9 spécifiques du GMPc, et la PDE8 spécifique de l'AMPc, dans le contrôle des concentrations intracellulaires d'AMPc ([AMPc]i) et de GMPc ([GMPc]i) dans les cellules musculaires lisses aortiques de rat (CMLARs) et les myocytes cardiaques de rat en utilisant une approche pharmacologique facilitée par le développement de nouveaux inhibiteurs sélectifs de PDEs. Les activités d'hydrolyse d’AMPc et de GMPc ont été mesurées par dosage enzymatique, tandis que les [AMPc]i et [GMPc]i ont été suivies sur cellules isolées, in situ, en temps réel, grâce à l'utilisation de l'imagerie FRET (Fluorescence Resonance Energy Transfer). Dans les CMLARs en culture, une activité d'hydrolyse des nucléotides cycliques via les PDE1, PDE5 et PDE9 a été observée. Nous avons montré un rôle fonctionnel de la PDE1 non stimulée dans le contrôle de l’augmentation de la [GMPc]i induite par le peptide natriurétique de type C (CNP). Il est intéressant de noter que, lors de l’élévation de la concentration intracellulaire en Ca2+, la PDE1 exerce également un contrôle de la réponse GMPci induite par le monoxyde d’azote (NO) et de la réponse AMPc médiée par la stimulation des récepteurs β-adrénergiques (β-AR). La PDE5 exerce un rôle majeur dans la réponse GMPc provoquée par l'activation de la guanylyl cyclase (GC) soluble par le NO ou des GC membranaires par les peptides natriurétiques, CNP et ANP. En revanche, la PDE9 ne régule que la réponse GMPc induite par le NO dans les RASMC cultivées. Aucune activité ou fonction hydrolytique de l'AMPc n'a été révélée avec l'inhibiteur de la PDE8 dans les CMLARs ou les cardiomyocytes de rat. Dans ces cellules cardiaques, l'activité d'hydrolyse médiée par la PDE1 n'a été détectée que sur la réponse GMPc et uniquement en présence de Ca2 +/Calmoduline. L'inhibiteur de la PDE1 n'a que légèrement affecté la réponse AMPc médiée par les récepteurs β-AR, par augmentation du pic du signal FRET.En conclusion, notre travail démontre que dans les cellules musculaires lisses vasculaires, les PDE1, PDE5 et PDE9 exercent une régulation spécifique et locale des [AMPc]i et [GMPc]i, renforçant le rôle clé des PDEs dans la compartimentation subcellulaire de la signalisation des nucléotides cycliques. / The two cyclic nucleotides cAMP and cGMP are important second messengers that regulate a large variety of cellular functions, in particular cardiovascular contractile function, cardiomyocyte cell growth and vascular smooth muscle cell proliferation. Phosphodiesterases (PDEs) degrade cyclic nucleotides, and exert a fine local control of their intracellular concentration. Alteration of cyclic nucleotides signaling pathway is involved in several pathological situations such as systemic and pulmonary arterial hypertensions, atherosclerotic lesions and cardiac hypertrophy. Thus, PDEs constitute potent therapeutic targets to restore a right cyclic nucleotide function. Eleven families of PDEs are now described, PDE1-6 being the most studied and PDE 7-11 representing the new families.The aim of the present thesis was to investigate the respective role of 4 PDE families, the Ca2+/calmodulin-stimulated PDE1, the cGMP-specific PDE5 and PDE9, and the cAMP-specific PDE8, in controlling intracellular cAMP ([cAMP]i) and intracellular cGMP ([cGMP]i) concentrations in both rat aortic smooth muscle cells (RASMCs) and cardiac myocytes by using a pharmacological approach taken advantage of the development of new selective PDE inhibitors. Cyclic AMP- and cGMP-hydrolyzing activities were measured by enzymatic assay on cell lysate, whereas real-time [cAMP]i and [cGMP]i were followed in situ in isolated cells using Fluorescence Resonance Energy Transfer (FRET) imaging. In cultured RASMCs, PDE1, PDE5 and PDE9 hydrolyzing activities were observed. We showed a functional role of basal PDE1 in controlling [cGMP]i increased by the C-type Natriuretic Peptide (CNP). Interestingly, upon high intracellular Ca2+ concentration, PDE1 also regulated the Nitric Oxide (NO)-mediated [cGMP]i response and the β-adrenoceptor (β-AR)-mediated [cAMP]i response. PDE5 exerted a major role in degrading [cGMP]i produced by the activation of either the soluble guanylyl cyclase (GC) elicited by NO or the particulate GCs by the natriuretic peptides, CNP and ANP. By contrast, PDE9 only regulated NO-induced [cGMP]i increase in cultured RASMCs. No cAMP-hydrolyzing activity or function was revealed with the PDE8 inhibitor in RASMCs or cardiac myocytes. In rat cardiomyocytes, PDE1-mediated hydrolyzing activity was only detected on cGMP in the presence of Ca2+/calmodulin. Unexpectedly, PDE1 inhibition slightly affected the β-AR-mediated [cAMP]i response by increasing the peak of FRET signal.In conclusion, our work underscores the distinct role of PDE1, PDE5, and PDE9 in locally regulating the [cAMP]i and [cGMP]i, in vascular smooth muscle cells, strengthening the concept of PDEs as key actors of cyclic nucleotide subcellular compartmentation.

L’AMPc est un messager important de la régulation neurohormonale du cœur. En activant ses effecteurs, l’AMPc régule de nombreuses fonctions cellulaires telles que l'expression de gènes, le couplage excitation-contraction et le métabolisme cellulaire. Chez les mammifères, l'AMPc est produit par une famille d’adénylate cyclases au sein de plusieurs compartiments subcellulaires solubles ou membranaires. L'existence et le rôle de la signalisation des nucléotides cycliques dans les mitochondries ont été postulés, mais n'ont pas encore été démontrés. De plus, son implication dans la régulation de la mort cellulaire est encore inconnue. Dans cette thèse, nous avons démontré l'expression locale de plusieurs acteurs de la signalisation de l'AMPc dans les mitochondries cardiaques, à savoir une forme tronquée soluble AC (sACt) et la protéine d'échange directement activées par AMPc 1 (Epac1). Nous avons montré un rôle protecteur pour sACt contre la mort cellulaire, l'apoptose, ainsi que la nécrose de cardiomyocytes primaires. Lors de la stimulation par du bicarbonate (HCO3-) et du Ca2+, la sACt produit de l’AMPc, qui à son tour stimule la consommation d'oxygène, une augmentation du potentiel mitochondrial de membrane (ΔΨm) et la production d'ATP. L’AMPc est limitant pour l’entrée matricielle de Ca2+ via l’uniport calcique mitochondrial (MCU) et, en conséquence, prévient la transition de perméabilité mitochondriale (MPT). En outre, dans les mitochondries isolées de cœurs de rats défaillants, la stimulation de la voie de l'AMPc par le HCO3- prévient la sensibilisation des mitochondries au Ca2+. Nous avons également constaté que les familles de phosphodiestérases (PDE), PDE2, 3 et 4, sont exprimées dans les mitochondries cardiaques régulant le taux d’AMPc. Ainsi, ces protéines forment une voie de signalisation locale dans la matrice régulant la fonction mitochondriale cardiaque. Finalement, notre étude a permis d’identifier un lien entre l'AMPc mitochondrial, le métabolisme, certaines PDEs et la mort cellulaire dans le cœur, qui est indépendant de la signalisation AMPc cytosolique. Ceci pourrait constituer un nouveau mécanisme cardioprotecteur via la préservation de la fonction mitochondriale dans un contexte physiopathologique. / CAMP is an important messenger in neurohormonal regulation of the heart. By activating its effectors, cAMP regulates many cellular functions such as gene expression, excitation-contraction coupling and cellular metabolism. In mammals, cAMP is produced by a family of adenylyl cyclase with various subcellular locations and membrane anchorage. The existence and role of cyclic nucleotide signaling in mitochondria has been postulated, but has not yet been demonstrated. Moreover, its implication in the regulation of cell death is still unknown. In this thesis, we demonstrated the local expression of several actors of cAMP signaling within cardiac mitochondria, namely a truncated form of soluble AC (sACt) and the exchange protein directly activated by cAMP 1 (Epac1) and showed a protective role for sACt against cell death, apoptosis as well as necrosis, in primary cardiomyocytes. Upon stimulation with bicarbonate (HCO3-) and Ca2+, sACt produces cAMP, which in turn stimulates oxygen consumption, increased the mitochondrial membrane potential (∆Ψm) and ATP production. cAMP is rate-limiting for matrix Ca2+ entry via the mitochondrial calcium uniporter (MCU) and, as a consequence, prevented mitochondrial permeability transition (MPT). In addition, in mitochondria isolated from failing rat hearts, stimulation of the mitochondrial cAMP pathway by HCO3- rescued the sensitization of mitochondria to Ca2+-induced MPT. We also found that PDE2, 3 and 4 families are located in cardiac mitochondria. They form a local signaling pathway with soluble AC in the matrix, which regulates cardiac mitochondrial functions. Thus, our study identifies a link between mitochondrial cAMP, mitochondrial metabolism, some PDEs and cell death in the heart, which is independent of cytosolic cAMP signaling. This might constitute a novel cardioprotective mechanism through mitochondrial function preservation in pathophysiological conditions.

Au cours du développement, les axones des cellules ganglionnaires de la rétine (CGRs) voyagent sur de longues distances pour établir des connexions avec leurs cellules cibles. La navigation des cônes de croissance est guidée par différentes molécules chimiotropiques présentes dans leur environnement. Les endocannabinoïdes (eCB) sont d’importants neuromodulateurs qui régulent de manière rétrograde la fonction de nombreuses synapses du cerveau. Ils agissent principalement par le biais de leurs récepteurs liés à une protéine Gi/o CB1 (CB1R) et CB2 (CB2R). La présence des eCBs durant le stade fœtal et la période postnatale suggère leur implication dans des événements régulant le développement du système nerveux. Cette thèse confirme l’expression des récepteurs aux cannabinoïdes CB1 et CB2 ainsi que l’enzyme dégradant les eCBs lors du développement embryonnaire et perinatal des CGRs et de la voie rétinothalamique in vivo. La manipulation pharmacologique de l’activité de CB1R et CB2R réorganise la morphologie du cône de croissance des CGRs et des neurones corticaux in vitro. De plus, la stimulation locale avec un agoniste de CB1R ou de CB2R modifie le comportement du cône de croissance entraînant sa répulsion. CB1R et CB2R modulent par le biais de la voie de signalisation AMPc/PKA, la mobilisation de DCC à la membrane plasmique. Par ailleurs, les résultats de cette recherche démontrent également l’implication de CB1R et CB2R dans la ségrégation des projections ipsi- et controlatérales et le développement de la voie rétinothalamique. / Following differentiation, retinal ganglion cell (RGC) axons, tipped at their distal end by the growth cone (GC), navigate through relatively long distances in a highly directed manner in order to establish functional synapses with thalamic and superior colliculus (SC) neurons. This is achieved with the help of extracellular guidance molecules which steer RGC axon growth by regulating GC morphology by means of attractive and/or repulsive mechanisms. In the adult brain, endocannabinoids (eCBs) exert an important neuromodulatory function by acting as retrograde messengers to regulate the function of many synapses. Endocannabinoids act mainly via their Gi/o protein coupled receptors CB1 (CB1R) and CB2 (CB2R). Due to their presence at the fetal and early postnatal periods, it has been proposed that eCBs and their receptors might be involved in several developmental events, such as cell proliferation and migration, axon guidance and synaptogenesis. We observed that during early postnatal development, components of the eCB system are expressed along the visual pathway (the optic chiasm, the lateral geniculate nucleus and the SC). To assess the implication of the eCB system, in vitro, embryonic retinal explant and primary neuron cultures were treated with pharmacological agonists and inverse agonists of CB1R and CB2R. These experiments demonstrated that these cannabinoid receptors modify the GC’s morphology. Most importantly, CB1R and CB2R act through the cAMP/PKA pathway to modulate the presence of DCC at the plasma membrane. In vivo, CB1R and CB2R play a major role and the absence of either one of them induces a decrease in eye-specific segregation of retinal projections. These results show an implication of CB1R and CB2R during RGC growth and retinothalamic development.

À partir des ovocytes de la palourde Spisula solidissima, un ADNc codant un récepteur nommé Spi-OAR a été cloné et séquencé. Une analyse de la séquence en acides aminés a indiqué que ce nouveau récepteur possède une forte similarité avec les récepteurs β-adrénergiques et les récepteurs à octopamine. En effet, il est étroitement lié à la classe des récepteurs à octopamine « β-adrénergique-like » couplés à une protéine Gs. L’ADNc de Spi-OAR a été introduit dans un vecteur d'expression (pCEP4) et un épitope reconnaissable par un anticorps commercial a été ajouté au segment N-terminal. Cette construction a été transfectée dans des cellules hôtes (HEK 293) et des études d’immunofluorescence ont montré une expression efficace du récepteur au niveau membranaire. Également, des mesures d'AMPc pour les cellules exprimant Spi-OAR ont révélé une augmentation de ce messager secondaire lors de l'ajout de l'octopamine, et dans une moindre mesure, la tyramine, tandis que la dopamine, la sérotonine et l'histamine n’ont engendré aucun effet. Une légère activité constitutive de ce récepteur dans les cellules hôtes a été observée. De plus, une analyse RT-PCR avec des oligonucléotides spécifiques a révélé l'ARNm de Spi-OAR non seulement dans les ovocytes, mais aussi dans les gonades, le cœur, les muscles adducteurs, les branchies et les ganglions suggérant que ce récepteur soit exprimé de façon ubiquitaire dans divers tissus et dans différents stades embryonnaires chez la palourde. En outre, des études avec des ovocytes isolés n'ont montré aucun effet de l’octopamine sur la réactivation méiotique. Des études éventuelles pourront finalement confirmer le rôle fonctionnel de Spi-OAR. / A cDNA encoding for an octopamine receptor named Spi-OAR was cloned and sequenced from the surf clam Spisula solidissima oocytes. An analysis of its predicted amino acid sequence showed a high degree of similarity with β-adrenergic and octopamine receptors. This receptor qualifies as a novel receptor closely related to the proposed class of insect octopamine « β-adrenergic–like » receptors coupled to Gs protein. This cDNA was introduced into an expression vector (pCEP4), with an added N-terminal FLAG tag sequence, and transfected in host cells (HEK 293). Immunofluorescence studies showed expression of the receptor with a proper localization to the plasma membrane. Measurements of cAMP in transfected cells revealed that addition of octopamine, and to a lower extent, tyramine induced a rise in cAMP while dopamine, serotonine and histamine had no effect. Overexpression of Spi-OAR in mammalian cells induced slight constitutive increase of cAMP. An RT-PCR analysis with specific oligonucleotides revealed the presence of the receptor mRNA not only in oocytes but also in whole gonads, heart, adductor muscle, gills and ganglia suggesting that this receptor is likely ubiquitously expressed. Expression of Spi-OAR was also detected at different embryonic stages. Despite the demonstrated expression of Spi-OAR in oocytes, octopamine had no effect on meiotic reinitiation. Further studies will examine the function of Spi-OAR.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.