Nous avons étudié l'influence de la voie du cGMP sur le comportement d'auto-administration de cocaïne chez le rat. Les injections, dans le cortex préfrontal médian, de trois activateurs différents de cette voie diminuent le nombre d'injections que les rats déclenchent, indiquant une réduction de l'effet renforçant de la cocaïne et de leur motivation pour la drogue. Des études immunohistochimiques nous ont permis de mettre en évidence que cette effet comportemental s'accompagnait d'une diminution de l'expression de marqueurs épigénétiques (MeCP2, HDAC2) et d'une augmentation des niveaux d'acétylation des histones. Des résultats complémentaires confirment que la voie du cGMP est bien en mesure de réguler des protéines impliquées dans les mécanismes épigénétiques. La découverte d'une action via ces régulations nous permet de suggérer des pistes originales quant aux phénomènes mis en jeu dans la diminution observée des propriétés renforçantes de la cocaïne. / We studied the influence of the cGMP pathway on cocaine self-administration by rats. When injected in the medial prefrontal cortex, three distinct activators of this pathway reduced the number of self-injections triggered by rats, suggesting a reduction of the reinforcing properties of cocaine and a lesser motivation of the animals for the drug. Immunohistochemical studies revealed that this behavioural effect was accompanied by a reduced expression of epigenetic markers (MeCP2, HDAC2), as well as inceased levels of histone acetylation. Complementary results indicate that the cGMP pathway is indeed able to regulate proteins implied in epigenetic mechanisms. The uncovering of an implication of these types of regulations leads us to suggest original hypotheses about the processes underlying the reduction of the reinforcing properties of cocaine.

Made available in DSpace on 2014-07-29T16:11:50Z (GMT). No. of bitstreams: 1 Dissertacao DAniella Ramos Martins.pdf: 914272 bytes, checksum: 038f97a7fcd95721aab346f4a3bd0587 (MD5) Previous issue date: 2012-02-28 / The inhibition of phosphodiesterases (PDEs) increases intracellular levels of cyclic nucleotides 3 ': 5'-cyclic adenosine monophosphate (cAMP) and 3 ': 5'-cyclic guanosine monophosphate (cGMP), which has many physiological and biochemical effects, especially in cardiovascular system. The objective of this study was to analyze the pharmacological effects of a new compound derived from pyrazole, LQFM 021, which was indicated by molecular modeling studies as a possible inhibitor of PDE-3. For this purpose, aortas were isolated of rats and mounted in organ baths for isometric tension recording of the relaxing effect of LQFM 021, in preparations pre-contracted with phenylephrine. We analyzed the involvement of the vascular endothelium, soluble guanylate cyclase (sGC) and adenylate cyclase (AC), the role of K+ channels and Ca2+, besides the contribution of Ca2+ uptake by the sarcoplasmic reticulum. As a result, was demonstrated that the LQFM 021 induces vascular relaxation (Emax: 54.9 ± 6.0%), being this relaxation potentiated by endothelium (Emax: 88.1 ± 2.1%). The inhibition of AC with MDL-12.330A (10 μM) or of the sGC with ODQ (1 μM) reduced the relaxation of 88.1 ± 2.1%, to 48.35 ± 3.01% and 19.95 ± 2.32%, respectively. The pre-contraction with KCl 45 mM or treatment of preparations with TEA (5 mM), reduced almost completely the relaxing effect of the compound. Inhibition of Ca2+ / ATPase reticular with CPA (10 mM) reduced the relaxation stimulated by 021 LQFM approximately 66.5%. Concentration-response curve contractile induced by phenylephrine (0.1 nM to 1 μM) or by CaCl2 (0-3 mM, zero-calcium + phenylephrine) were reduced by pretreatment of preparations with LQFM 021 (EC50). In conclusion, this study showed that the new synthetic derivative of pyrazole LQFM 021 is a potential inhibitor of PDE-3 and has vasorelaxant activity. The endothelium potentiates the relaxation stimulated by the compound. The route of sGC and AC are involved in the mechanism of action of LQFM 021. Was also evidenced by participation from sarcoplasmatic reticulum, well as the flow of K+ and Ca2+ through the cell membrane. / A inibição das fosfodiesterases (PDEs) aumenta os níveis intracelulares de nucleotídeos cíclicos 3' : 5'-monofosfato cíclico de adenosina (AMPc) e 3' : 5'-monofosfato cíclico de guanosina (GMPc), os quais tem muitos efeitos fisiológicos e bioquímicos, sobretudo no sistema cardiovascular. O objetivo deste estudo foi analisar os efeitos farmacológicos de um novo composto derivado do pirazol, LQFM 021, o qual foi apontado por estudos de modelagem molecular como possível inibidor de PDE-3. Para tanto, artérias aortas de ratos foram isoladas e montadas em banhos de órgão para registro da tensão isométrica do efeito relaxante do LQFM 021, em preparações pré-contraídas com fenilefrina. Foi analisada a participação do endotélio vascular, da guanilato ciclase solúvel (GCs) e da adenilato ciclase (AC), o papel dos canais de K+ e de Ca2+, além da contribuição da captação de Ca2+ pelo retículo sarcoplasmático. Como resultado, foi demonstrado que o LQFM 021 induz relaxamento vascular (Emax: 54.9 ± 6.0%), sendo este relaxamento potencializado pelo endotélio (Emax:88.1 ± 2.1%). A inibição da AC com MDL-12.330A (10 μM) ou da GCs com ODQ (1 μM), reduziram o relaxamento de 88,1 ± 2,1%, para 48,35 ± 3,01% e 19,95 ± 2,32%, respectivamente. A pré-contração com KCl 45 mM ou o tratamento das preparações com TEA (5 mM), reduziram quase que por completo o efeito relaxante do composto. A inibição da Ca2+/ATPase reticular com CPA (10 μM), reduziu o relaxamento estimulado pelo LQFM 021 em aproximadamente 66,5%. Curva concentração-resposta contrátil induzida pela fenilefrina (0,1 nM a 1 μM) ou pelo CaCl2 (0 a 3 mM, em meio zero-cálcio + fenilefrina) foram reduzidas pelo pré-tratamento das preparações com LQFM 021 (EC50). Em conclusão, este estudo mostrou que o novo derivado sintético de pirazol LQFM 021 é um possível inibidor de PDE-3 e possui atividade vasorelaxante. O endotélio participa e potencializa o relaxamento estimulado pelo composto. A via da GCs e AC estão envolvidas no mecanismo de ação do LQFM 021. Também foi evidenciada a participação do retículo sarcoplasmático, bem como o fluxo de K+ e de Ca2+ através da membrana celular.

A maturação in vitro do oócito é um dos fatores limitantes na produção in vitro de embriões. In vivo, esta maturação é um processo altamente orquestrado no qual a meiose é retomada pela onda de gonadotrofina que antecede a ovulação e que induz à queda dos níveis de AMPc no oócito. No entanto, os oócitos aspirados ao serem retirados dos folículos ovarianos retomam espontaneamente a maturação comprometendo a competência de seu desenvolvimento. O AMPc é sintetizado pela adenilato ciclase (AC) e degradado pelas fosfodiesterases (PDE), existindo algumas relacionadas à degradação do AMPc e outras do GMPc. Sendo assim, a proposição deste trabalho foi averiguar a contribuição de diferentes isoformas de fosfodiesterases na retomada da meiose e nos níveis de GMPc, AMPc e ainda, determinar quando há manutenção de AMPc em níveis elevados observando sua influência na competência oocitária e ativação da MAPK. Para isso, os complexos cumulus-oócito (CCOs) foram maturados in vitro na ausência, presença ou associação de inibidores de PDEs-AMPc e GMPc específicas e FSHr. As amostras foram avaliadas em relação a: 1) taxa de maturação; 2) níveis intracelulares de AMPc e GMPc nos CCOs; 3) taxa de desenvolvimento de blastocistos ; 4) ativação da MAPK em oócitos e células do cumulus. Os resultados obtidos no primeiro experimento indiaram que o inibidor da PDE3 foi o mais eficaz (p<0,05) em atrasar a retomada da meiose, às nove horas de maturação, porém, isolado ou em associação com o inibidor da PDE8, não foi capaz de alterar (p>0,05) os níveis de AMPc. No experimento dois, o inibidor da PDE5 isolado não influenciou a retomada da meiose (p>0,05), porém, quando associado aos inibidores da PDE3 e 8 houve atraso na retomada (p<0,05) e ainda alteraram os níveis de GMPc e AMPc (p<0,05) nas primeiras horas de maturação. O experimento três mostrou a influencia do FSHr durante a MIV, o qual estimulou a retomada da meiose, mas em associação com inibidores da PDE5 e 8 atrasa a retomada (p<0,05). Além disso, o FSHr provoca aumento do nível de AMPc e sua associação com inibidores de PDE5 e PDE8 ocasionou elevação adicional (p<0,05). As condições de cultivo estudadas no experimento quatro mostraram que a maturação induzida (pré-MIV de duas horas com agentes para elevar AMPc seguindo de 22 horas de MIV com FSH associado a inibidores de PDEs) atrasaram a retomada da meiose às nove horas de maturação, mas não afetaram progressão da meiose às 24, 28 e 30 horas. Os tratamentos, porém, não melhoraram a competência oocitária após a fertilização in vitro e ocasionaram pequenas variações na ativação da MAPK em oócitos e células do cumulus. / In vitro maturation of oocytes is a limiting factor in the in vitro production of bovine embryos. In vivo, this maturation is a highly orchestrated process in which meiosis resumption by the gonadotropin surge that precedes ovulation induces the decrease in cAMP levels in the oocyte. However, when oocytes are removed from follicles, they spontaneously resume maturation compromising the competence for its development. cAMP is synthesized by adenylyl cyclase (AC) and degraded by phosphodiesterases (PDE), and there are some PDEs related to degradation of cAMP and/or cGMP. Thus, the purpose of this work was to investigate the contribution of different isoforms of phosphodiesterases in the resumption of meiosis and levels of cAMP and, also, to determine differences in signaling pathways when maintaining high levels of cAMP and its influence on oocyte competence. For this purpose, cumulus-oocyte complexes (COC) were matured in vitro in the presence, absence or combination of inhibitors of cAMP- and cGMP-specific PDEs and FSH. Samples be were evaluated in relation to: 1) maturation rate, 2) intracellular levels of cAMP and cGMP in COCs, 3) rate of blastocyst development and 4) activation of MAPK in oocytes and cumulus cells. The results of the first experiment showed that the PDE3 inhibitor is more effective (p <0.05) in delaying meiosis resumption, at nine hours of maturation, but was not capable of altering cAMP levels (p> 0.05) either alone or in combination with the PDE8 inhibitor. In experiment two, the PDE5 inhibitor alone did not affect the meiosis resumption (p> 0.05), however, when associated with PDE3 and PDE8 inhibitors it delayed their resumption (p <0.05) and also altered cGMP and cAMP levels of (p <0.05) in the early hours of maturation. The third experiments showed the influence of FSHr during IVM, which stimulated the resumption of meiosis, but in combination with PDE5 and PDE8 inhibitors meiosis was delayed (p <0.05). Furthermore, FSHr causes increased levels of cAMP and its association with PDE5 and PDE8 inhibitors caused an additional increase (p <0.05). Culture conditions studied in experiment four showed that induced maturation (pre-IVM for two hours with agents to elevate cAMP followed by 22 hours IVM with FSH associated with PDE inhibitors) delayed the resumption of meiotic maturation at nine hours, but has no effect on meiosis progression at 24, 28 and 30 hours. The treatments, however, did not improve oocyte competence after in vitro fertilization and caused minor variations in the activation of MAPK in oocytes and cumulus cells.

L'ANP est une hormone cardiaque libérée lors de l'insuffisance cardiaque. Les fibroblastes cardiaques, responsables de la synthèse des composants de la matrice extracellulaire (MEC), acquièrent dans les conditions pathologiques la capacité de se différencier en myofibroblastes, conduisant ainsi à une fibrose cardiaque. Les mécanismes de régulation impliquant l'ANP et ses récepteurs (NPR) restent peu connus et font l'objet de ce travail. Les fibroblastes ventriculaires ont été isolés à partir de coeurs de rats Wistar et mis en culture afin d'induire leur différenciation. Les cultures ont ensuite été soumises à différents traitements impliqués dans la voie ANP/NPR. L'ANP diminue le taux de prolifération, la migration cellulaire, et la sécrétion de collagène des myofibroblastes. Cet effet est mimé par le 8-Br-GMPc. L'analyse protéomique et génomique a permis de confirmer la présence des récepteurs natriurétiques A et B dans nos cellules. Par ailleurs, l'expression de dix isoformes de phosphodiestérases dans les myofibroblastes a été révélée par un criblage génomique. L'inhibition non sélective de ces phosphodiestérases provoque une diminution de la prolifération et de la sécrétion de collagène. Enfin, les concentrations intracellulaires de GMPc et d'AMPc ont été trouvées augmentées en présence de l'ANP. En parallèle, la caractérisation des courants ioniques présents sur les myofibroblastes a montré une absence des courants sodique rapide et potassique ATP-dépendant. Cette étude montre le rôle de la voie ANP/NPR/GMPc dans la modulation des propriétés fibroblastiques et illustre la complexité des processus de différenciation cellulaire au cours de la fibrogenèse cardiaque. / ANP is a cardiac hormone released during heart failure and acts as a regulator of the extracellular matrix (ECM). Cardiac fibroblasts are responsible for the synthesis of ECM components and acquire under pathological conditions the capacity to differentiate into myofibroblasts, leading to cardiac fibrosis. Regulatory mechanisms involving ANP and its receptors (NPR) are poorly known and make the subject of our work. Ventricular fibroblasts were isolated from Wistar rat hearts and cultured to induce differentiation. The cultures were then subjected to various treatments involved in the ANP/NPR pathway. ANP decreases the proliferation rate, cell migration and collagen secretion. This effect was mimicked by 8-Br-cGMP. In addition, genomic and proteomic analysis confirmed the presence of the natriuretic receptor A and B in our cells. Furthermore, the expression of ten phosphodiesterases isoforms in the myofibroblasts was revealed by genomic screening. The non-selective inhibition of these phosphodiesterases causes a decrease in the proliferation and secretion of collagen. Finally, the intracellular concentrations of cAMP and cGMP were increased in the presence of ANP. In parallel, the characterization of ionic currents present in myofibroblasts revealed the absence of rapid sodium and potassium ATP-dependent currents. This study shows the role of the ANP/NPR/cGMP pathway in modulating fibroblast properties and exposes the complexity of the cell differentiation process during cardiac fibrogenesis.

No description available.

O segundo mensageiro cGMP está envolvido em diversas funções celulares incluindo a visão em mamíferos. Embora trabalhos anteriores mostrassem variações nos níveis de cGMP durante o ciclo de vida de Blastocladiela emersonii e evidências da existência de enzimas específicas envolvidas na sua síntese (guanilato ciclase) e degradação (cGMP fosfodiesterase), nenhum genoma de fungo publicado até o momento mostrou a existência de genes codificando estas enzimas. Este fato é atribuído por evolucionistas à completa perda de motilidade dos fungos em geral, já que cGMP está primordialmente associado a células com cílios. Blastocladiomicetos, como Blastocladiella, apresentam células móveis em pelo menos um estágio do seu ciclo de vida, o que poderia explicar a existência dessa via nesses fungos. Uma investigação no banco de ESTs de B. emersonii revelou a existência de cDNAs codificando parte de prováveis guanilato ciclases (BeGC1, BeGC2 e BeGC3) e uma possível cGMP fosfodiesterase (BePDE). Assim, este trabalho buscou confirmar a existência destas enzimas e caracterizar a sinalização por cGMP em B. emersonii. A proteína recombinante selvagem correspondente ao domínio catalítico de BePDE mostrou atividade de degradação sobre cGMP e a mutação E389A foi capaz de alterar a especificidade por cGMP. Com o sequênciamento do genoma de B. emersonii obteve-se as sequências completas das guanilato ciclases. Em BeGC2 não foi possível identificar o ligante responsável por sua ativação. Em BeGC3, a presença de um domínio Heme-Pas sugeriu sua ativação por óxido nítrico. A presença de um domínio rodopsina em BeGC1 sugeriu sua ativação por luz. Experimentos de microscopia por imunofluorescência localizaram BeGC1 no \"eyespot\", BeGC2 no capacete nuclear e BeGC3 no citoplasma de zoósporos de B. emersonii. Verificamos também que zoósporos realizam fototaxia em direção à luz verde e que a adição de hidroxilamina, inibidor de rodopsina, ou do inibidor de guanilato ciclase LY83583 tem efeito negativo na fototaxia, bem como impede o aumento dos níveis de cGMP observado em zoósporos expostos à luz verde. O bloqueio da síntese de retinal por Norflurazon também inibiu a fototaxia sendo esta restaurada quando adicionamos retinalA1. Estes dados, juntamente com o fato de o domínio rodopsina de BeGC1 ser a única rodopsina presente no genoma, indicam que BeGC1 é responsável pela fototaxia nos zoósporos de B. emersonii. O genoma do fungo apresenta ainda um possível canal de potássio ativado por cGMP (BeCNG1) localizado na membrana plasmática de zoósporos, similar ao canal regulado por cGMP envolvido na visão em humanos. Ensaios de microfluorimetria também evidenciaram a presença de um canal ativado por cGMP relacionado com o influxo de potássio e a motilidade dos zoósporos. Um modelo para a via de sinalização da fototaxia em B.emersonii foi proposto e comparado com a sinalização presente na visão de mamíferos, destacando a existência de cGMP e rodopsina em ambos os processos e sugerindo uma possível origem comum. Portanto, os resultados obtidos suportam a existência da sinalização por cGMP em B. emersonii, além de indicar o papel dessa sinalização na fototaxia dos zoósporos, sendo esta a primeira via de sinalização por cGMP caracterizada em fungos. / The second messenger cyclic GMP is involved in a wide array of cellular processes including vision in mammals. Although previous studies demonstrated changes in cGMP levels during the life cycle of Blastocladiela emersonii and evidences of specific enzymes involved in its synthesis (guanylyl cyclase) and hydrolysis (cGMP-phosphodiesterase), no fungal genome published so far shows the presence of genes encoding these enzymes. Evolutionists attribute the absence of cGMP signaling pathways in higher fungi to the sedentary life style of these organisms, since cGMP is primarily associated with ciliated cells. However, blastocladiomycetes like Blastocladiella, have motile cells in at least one stage of their life cycle, which could explain the existence of this pathway in these primitive fungi. Inspection of B. emersonii EST data bank, revealed cDNAs encoding part of three putative guanylyl cyclases (BeGC1, BeGC2 e BeGC3) and one possible cGMP phosphodiesterase (BePDE). Thus, the purpose of this study was to confirm the existence of these enzymes and characterize the cGMP signaling pathway in this model. The recombinant protein containing the wild type catalytic domain of BePDE presented activity towards hydrolysis of cGMP and the E389A mutation of this domain changed the cGMP specificity of this enzyme. The complete nucleotide sequence of the guanylyl cyclases were obtained by sequencing of B. emersonii genome. In BeGC2 we were unable identify the ligand responsible for its activation, but in BeGC3, the presence of a Heme-Pas domain suggested its activation by nitric oxide. The presence of a rhodopsin domain in BeGC1 suggested its activation by light. Immunofluorescence microscopy localized BeGC1 in the \"eyespot\" structure, BeGC2 in the nuclear cap and BeGC3 in the cytoplasm of zoospores of B. emersonii. We found that Blastocladiella zoospores performed phototaxis toward green light and photobleaching of rhodopsin function using hydroxylamine prevented both phototaxis and the increased cGMP levels observed when zoospores were exposed to green light. The same effect was observed using the guanylyl cyclase inhibitor LY83583. Inhibition of retinal synthesis using Norflurazon prevented the phototaxis response, which could be restored by zoospore complementation with retinalA1. The BeGC1 gene is the only rhodopsin found in the draft assembly of B. emersonii genome, which indicates that BeGC1 is responsible for phototaxis observed in zoospores. We also found in the genome a possible cGMP-activated potassium channel (BeCNG1), localized in the plasma membrane of the zoospores, which is similar to the cGMP-activated channel involved in human vision. In addition, microfluorimetry assays revealed the presence of a cGMP-activated potassium channel involved in potassium influx and zoospore motility. The signaling model of B. emersonii phototaxis was proposed and compared with the mammalian vision system, with cGMP and rhodopsin acting in both signaling pathways, suggesting a common origin. Altogether our data indicate that Blastocladiella emersonii has a cGMP signaling system involved in phototaxis, being the first cGMP signaling pathway characterized in fungi.

L’AMP cyclique (AMPc) et le GMP cyclique (GMPc) sont des seconds messagers essentiels pour la régulation de la fonction cardiaque. La concentration de l’AMPc intracellulaire est régulée par les activités d'au moins deux familles d'enzymes: les cyclases et guanylyl cyclases, responsable de la synthèse de l'AMPc et du GMPc, et les phosphodiestérases (PDE) qui interviennent dans l’hydrolyse de l'AMPc et du GMPc.Parmi la superfamille des PDEs, la PDE2 est une enzyme à double substrat qui hydrolyse à la fois l'AMPc et le GMPc et a la propriété unique d'être stimulée par le GMPc. Il a été récemment montré que la PDE2 du myocarde est augmentée dans l'insuffisance cardiaque humaine et expérimentale (IC), tandis que d'autres (par exemple PDE3 et PDE4) sont réduites. Cependant, les conséquences physiopathologiques de l'activité PDE2 renforcée dans le cœur sont inconnues.Dans ce contexte, nous avons généré des souris transgénique (TG) avec une surexpression spécifique cardiaque de l’isoforme PDE2A3 (souris PDE2 TG).Grace à l’utilisation de Western blot et de dosage radioenzymatique nous avons montré que l'AMPc cardiaque et l'activité PDE cGMP et l'activité spécifique de PDE2 sont fortement augmentées dans les PDE2 TG par rapport à des souris de type sauvage (WT).Le raccourcissement cellulaire, les transitoires calciques et le courant calcique de type L (ICa, L) ont été enregistrés dans les myocytes ventriculaires adultes de souris WT et PDE2 TG et l'isoprénaline (ISO) a été utilisée pour examiner et comparer la réponse β-adrénergique (β-AR) de ces paramètres. Nous avons montré que lors de la stimulation β-AR, la contractilité cellulaire, la transitoire Ca2+ et l’amplitude du courant ICa,L sont fortement diminués. En conséquence, la surexpression de la PDE2 dans les cardiomyocytes a réduit les taux d'AMPc et abolit l'effet inotrope après une stimulation β-AR aiguë. L'ECG mesuré par télémétrie chez la souris PDE2 TG a montré une réduction marquée de la fréquence cardiaque au repos ainsi que de la fréquence cardiaque maximale, tandis que le débit cardiaque a est entièrement préservé en raison d'une contractilité plus forte. Fait important, les souris TG PDE2 sont résistantes à des arythmies ventriculaires déclenchées et à des arythmies induites par isoprénaline.En conclusion, ce travail montre que PDE2 joue un rôle essentiel dans la régulation du couplage excitation-contraction cardiaque. La surexpression de PDE2 semble protéger les cardiomyocytes contre une stimulation excessive β-AR et réduit le risque d'arythmie lors de l'activation sympathique.L’activation de la PDE2 peut donc représenter une nouvelle stratégie thérapeutique anti-adrénergique et anti-arythmique subcellulaire dans l’insuffisance cardiaque. / Cyclic AMP (cAMP) and cyclic GMP (cGMP) are critical second messengers for the regulation of cardiac function. Intracellular cAMP concentration is regulated by the activities of at least two families of enzymes: adenylyl and guanylyl cyclases, responsible for cAMP and cGMP synthesis and cyclic nucleotide phosphodiesterases (PDEs) that mediate cAMP and cGMP hydrolysis.Among the PDE superfamily, PDE2 is a dual substrate enzyme that hydrolyzes both cAMP and cGMP and has the unique property to be stimulated by cGMP. It was recently showed that myocardial PDE2 is increased in human and experimental heart failure (HF), while other PDEs (e.g. PDE3 and PDE4) are reduced. However, the pathophysiological consequences of enhanced PDE2 activity in the heart are unknown.In this context, we generated a transgenic (TG) mouse with a heart specific overexpression of the PDE2A3 isoform (PDE2 TG mouse). Using immunoblotting and radioenzymatic assay we showed that total cardiac cAMP and cGMP PDE activity and specific PDE2 activity was strongly increased in PDE2 TG compared to wild type (WT) mice. Sarcomere shortening, Ca2+ transients and the whole L-type Ca2+ current (ICa,L) were recorded in adult ventricular myocytes from WT and PDE2 TG mice and isoprenaline (ISO) was used to examine and compare the β-adrenergic (β-AR) response of these parameters. We showed that upon β-AR stimulation, cell contractility, Ca2+ transient and ICa,L were severely blunted. Accordingly, PDE2 overexpression in cardiomyocytes reduced the cAMP levels and abolished the inotropic effect following acute β-AR stimulation. ECG telemetry in PDE2 TG mice showed a marked reduction in resting as well as in maximal heart rate, while cardiac output was completely preserved due to greater contractility. Importantly, PDE2 TG mice were resistant to triggered ventricular arrhythmias and to isoprenaline-induced arrhythmias.In conclusion, this work demonstrates that PDE2 plays a critical role in the regulation of cardiac excitation-contraction coupling. PDE2 overexpression appears to protect the cardiomyocytes against excessive β-AR drive and reduces the risk of arrhythmias during sympathetic activation. PDE2 activation may thus represent a new subcellular anti-adrenergic and anti-arrhythmic therapeutic strategy in HF.

This thesis focuses on the optimazation of the electrochemical method, which characterizes products of untemplated nonenzymatic polymerization of 3',5' -cyclic guanosine monophosphate (cGMP) under conditions modeling prebiotic environment. An adsorptive transfer stripping techniques on carbon electrode and gel electrophoresis were used. The method was optimized on the model system of oligonucleotides located in solution of cGMP on carbon electrode, where DNA and RNA adsorb. This technique allows simple removing of interfering substances such as cGMP, which are not present in the original sample, but they do not adsorb on the surface of electrode or they adsorb weaker than oligonucleotides or polynucleotides. Analyses are based on the selective desorption of cGMP from the surface of the carbon electrode by the chemical and physical methods before the measurement of linear voltammetry itself. Detergents, such as SDS, Tween 20 and Triton x-100 with different concentrations and electrostatic repulsions of cGMP with different negative potentials on the carbon electrode were used for the selective desorption of cGMP. The selective desorption of cGMP was observed for all detergents and inserted negative potentials. Used methods were compared and the most effective detergent for selective desorption of cGMP was SDS. Desorption of oligonucleotides was minimalized by inserted positive potential on washed carbon electrode in 0,01% SDS in basic medium. This optimized method was used on electrochemical analysis of preliminary samples of untemplated nonenzymatic polymerization of 3',5' -cGMP and compared to the analysis of gel electrophoresis.

Nitric oxide (NO) is an important signaling molecule in plants. In the pollen of Arabidopsis thaliana, NO causes re-orientation of the growing tube and this response is mediated by 3′,5′-cyclic guanosine monophosphate (cGMP). However, in plants, NO-sensors have remained somewhat elusive. Here, the findings of an NO-binding candidate, Arabidopsis thaliana DIACYLGLYCEROL KINASE 4 (ATDGK4; AT5G57690) is presented. In addition to the annotated diacylglycerol kinase domain, this molecule also harbors a predicted heme-NO/oxygen (H-NOX) binding site and a guanylyl cyclase (GC) catalytic domain which have been identified based on the alignment of functionally conserved amino acid residues across species. A 3D model of the molecule was constructed, and from which the locations of the kinase catalytic center, the ATP-binding site, the GC and H-NOX domains were estimated. Docking of ATP to the kinase catalytic center was also modeled. The recombinant ATDGK4 demonstrated kinase activity in vitro, catalyzing the ATP-dependent conversion of sn-1,2-diacylglycerol (DAG) to phosphatidic acid (PA). This activity was inhibited by the mammalian DAG kinase inhibitor R59949 and importantly also by the NO donors diethylamine NONOate (DEA NONOate) and sodium nitroprusside (SNP). Recombinant ATDGK4 also has GC activity in vitro, catalyzing the conversion of guanosine-5'-triphosphate (GTP) to cGMP. The catalytic domains of ATDGK4 kinase and GC may be independently regulated since the kinase but not the GC, was inhibited by NO while Ca2+ only stimulates the GC. It is likely that the DAG kinase product, PA, causes the release of Ca2+ from the intracellular stores and Ca2+ in turn activates the GC domain of ATDGK4 through a feedback mechanism. Analysis of publicly available microarray data has revealed that ATDGK4 is highly expressed in the pollen. Here, the pollen tubes of mis-expressing atdgk4 recorded slower growth rates than the wild-type (Col-0) and importantly, they showed altered NO responses. Specifically, the mis-expressing atdgk4 pollen tubes have growth rates that were less affected by NO and showed reduced bending angles when challenged by an NO source. Further works on atdgk4 knockout/knockdown mutants will reveal the biological functions of ATDGK4 in NO and/or cGMP signaling in the pollen, and in the broader fertilization process.

Cyclic nucleotides such as cAMP and cGMP serve as intracellular second messengers in diverse signaling pathways that control a wide range of cellular functions. Such pathways are regulated by key cyclic nucleotide receptor proteins including protein kinase A (PKA), the exchange protein directly activated by cAMP (EPAC), the hyperpolarization-activated cyclic-nucleotide-modulated (HCN) ion channels, and protein kinase G (PKG), and malfunction of these proteins has been linked to a number of pathologies. While it is known that cyclic nucleotide binding to these proteins leads to structural perturbations that promote their activation, the role played by dynamics in auto-inhibition and cyclic-nucleotide-dependent activation is not fully understood. Therefore, in this thesis we examined dynamics within the cyclic-nucleotide receptor proteins EPAC, HCN and PKG, and found that dynamics are critical for allosteric control of activation and/or autoinhibition of all three proteins. In particular, our findings for EPAC and HCN have highlighted dynamics as a key modulator of the entropic and enthalpic components, respectively, of the free-energy landscape for cAMP-dependent allostery, while our findings for PKG have highlighted dynamics as a key determinant of the cGMP-vs.-cAMP selectivity necessary to minimize cross-talk between signaling pathways. Ultimately, we envision that the methods outlined in this thesis will reveal key differences in the regulatory mechanisms of human cyclic nucleotide receptors that can eventually be exploited in the development of novel therapeutics to selectively target a single receptor, and thus treat physiological conditions/diseases linked to malfunction of the target receptor. / Thesis / Doctor of Philosophy (PhD) / In this thesis, we examined cyclic-nucleotide-responsive proteins that regulate key physiological processes, and whose malfunction has been linked to cardiovascular and neurological disorders. In particular, in three such proteins we examined dynamics, whose role in cyclic-nucleotide-responsive function is not fully understood. We found that cyclic-nucleotide-dependent variations in dynamics play a critical role in the function of these proteins, with the results for each protein highlighting a different role played by dynamics. Ultimately, we envision that the methods outlined in this thesis will reveal key functional differences among human cyclic-nucleotide-responsive proteins that can eventually lead to the development of novel therapeutics to treat certain diseases such as arrhythmias or epilepsy by selectively targeting a single cyclic-nucleotide-responsive protein.