La protéine kinase Ca²⁺/calmoduline-dépendante II (CaMKII) est une enzyme clé dans le processus de la mémoire. Elle est impliquée dans les mécanismes qui régissent la plasticité synaptique et le recrutement de protéines à la synapse. Certaines hypothèses suggèrent que pour y parvenir, la CaMKII se fixe à la densité post-synaptique de manière persistante. Cette localisation pourrait faciliter son rôle de kinase vis-à-vis de ses partenaires et faciliter la transmission synaptique, en permettant l'exocytose des récepteurs AMPA par exemple. L'enzyme peut également se lier aux microtubules de manière plus brève lors des entrées calciques. Cette liaison pourrait à son tour phosphoryler certaines protéines associées aux microtubules et permettre la libération de cargos aux endroits spécifiques. Plusieurs questions sont présentement posées par la communauté scientifique au sujet des rôles de l'enzyme face à la plasticité synaptique. De quelles manières est-elle retenue à la synapse, quelles sont ses mécanismes et ses modes de diffusion, est-elle impliquée dans certains processus d'étiquetage synaptique ? Ce sont là quelques questions auxquelles j'ai tenté de répondre au cours de mes travaux de maîtrise. J'ai donc étudié les dynamiques spatiales et temporelles de la CaMKII de façon à mieux comprendre comment elle intervient au niveau synaptique. J'ai utilisé des cultures d'hippocampe de rats, dans lesquelles j'ai surexprimé la GFP-aCaMKII. J'ai fait du FRAP (fluorescence recovery after photobleaching) et du FLAPA (Fluorescence loss after Photo Activation) ce qui m'a permis d'obtenir de l'information sur la mobilité et la rétention de la CaMKII. J'ai également adapté une méthode d'analyse de régression sur le profil d'intensité en utilisant la méthode des maxima d'entropie. Mes résultats démontrent que suite à une activation synaptique, une plus grande fraction de CaMKII est retenue à la synapse, pour une durée de temps prolongée. Cette rétention, observée sur une longue période, pourrait avoir un rôle important dans les changements plastiques à long terme. J'ai aussi démontré que l'enzyme doit d'être activée via le complexe Ca²⁺/CaM. D'autres résultats m'ont permis de constater que lors des entrées calciques, la CaMKII se fixe aux microtubules. Mes travaux proposent donc que plusieurs dynamiques spatiales et temporelles de la CaMKII sont mises à contribution pour le bon fonctionnement du système de transmission synaptique.

Regulator of K+ conductance (RCK) domains control the activity of a variety of K+ channels and transporters, including the prokaryotic TrkA/H K+ transport complex and the eukaryotic BK channel, through binding of cytoplasmic ligands such as ATP, H+, and Ca2+. Thus RCK domains transduce ligand binding to gate transmembrane K+ flux in response to signaling events and cellular metabolism, in organisms ranging from bacteria to humans. In this work, I utilize the prokaryotic RCK domain containing K+ channel, MthK as a model system to provide insight toward the structural basis of ion channel gating by RCK domains. In MthK, binding of Ca2+ to an octameric ring of RCK domains (the gating ring) which is tethered to the pore of the channel, leads to a series of conformational changes that facilitates channel opening and K+ conduction. Using electrophysiology and X-ray crystallography, I identify the presence of additional Ca2+ binding sites in the MthK RCK domain, showing that each RCK domain contributes to three different regulatory Ca2+ binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca2+ binding sites, resulting in a stoichiometry of 24 Ca2+ ions per channel, is consistent with the steep relation between [Ca2+] and MthK channel activity. Comparison of Ca2+ bound and unliganded RCK domains suggests a physical mechanism for Ca2+-dependent conformational changes that underlie gating in this class of channels. To gain insight toward mechanisms of RCK domain activation, I crystallized and solved the structure of the RCK domain of MthK bound with Ba2+. The Ba2+-bound RCK domain was assembled as an octomeric gating ring, as observed in structures of the full-length MthK channel, and shows Ba2+ bound at several positions, one of which overlaps with a known Ca2+ binding site. Functionally, I determined that Ba2+ could activate reconstituted MthK channels as observed in electrophysiological recordings. These results suggest a working hypothesis for a sequence of ligand-dependent conformational changes that may underlie RCK domain activation and channel gating. In an effort to more accurately describe the Ca2+-dependent gating process in MthK, I crystallized and solved structures of mutant and wild-type RCK domains, and found that distinct Ca2+ activation sites near the N- and C-termini of the RCK domain (termed C1 and C3, respectively) are allosterically coupled to one another, to affect tuning of Ca2+ affinity and Ca2+-dependent channel activation. These results define a structural mechanism of allosteric modulation in a ligand-gated K+ channel, and provide a framework for understanding similar mechanisms in related RCK-containing channels and transporters. / Biochemistry

Este estudo descreve um novo método para a produção da Ca2+-ATPase do retículo sarcoplasmático de coelho em levedura utilizando um vetor de expressão regulado por choque térmico. Após solubilização das membranas de levedura com lisofosfatidilcolina, a introdução de um \"tag\" de 6 histidinas na extremidade amino-terminal da Ca2+-ATPase permitiu a sua purificação por cromatografia de afinidade utilizando uma resina carregada com níquel. Utilizando essa estratégia, foi possível obter frações enriquecidas em até 75% de Ca2+-ATPase recombinante, algo não descrito ainda na literatura. A 6xHis Ca2+-ATPase solubilizada em LPC e purificada em coluna de níquel se mantém estável desde que seja introduzido DOPC juntamente com o detergente nas etapas de lavagem e eluição. Nessas condições, a enzima purificada possui elevada atividade ATPásica cálcio-dependente (1.5-2.0 µmol/mg proteína.min) durante vários minutos de reação. A titulação da atividade ATPásica em função do cálcio livre demonstrou que a 6xHis Ca2+-ATPase purificada possui alta afinidade para o íon (K0.5= 0.15 µlM) e manteve uma forte cooperatividade na ativação por cálcio (nH = 2.07). A quantidade e o grau de pureza obtidos são suficientes para permitir a caracterização bioquímica e espectroscópica de mutantes pontuais da Ca2+-ATPase já construídos e expressos em levedura. A conversão da energia presente em ligações químicas em gradiente eletroquímico é um tema central da bioenergética. Espera-se que o estudo dos mutantes pontuais de triptofano da Ca2+-ATPase gerados nesse trabalho contribua para uma melhor compreensão do mecanismo de acoplamento entre a hidrólise de ATP e o transporte vetorial de íons nesse modêlo de estudo de proteínas de transporte. / We describe in this work a new method for the production of SERCA-l Ca2+-ATPase in yeast using a heat-shock regulated expression vector. Following solubilization of yeast membranes with lysophospholipids, the presence of an hexahistidine tag introduced at the Nterminal end of the Ca2+-ATPase allowed its purification by metal chelating affinity chromatography using a nickel-NTA resin. Using this procedure highly enriched ftactions (75% oftotal protein in the ftaction) of yeast-expressed rabbit Ca2+-ATPase were obtained. Detergent-solubilized 6xHis-Ca2+-ATPase retained highly active (1.5 - 2 µmol/mg protein .min) calcium-dependent, vanadate inhibitable ATPase activity as determined by 32P-γ-ATP hydrolysis. Titration of ATPase activity as a function of ftee calcium revealed high Ca2+ affinity (K0.5 =~ 0.15 µM) and the persistence of a strong cooperative pattem of calcium activation (Hill number of 2.07). The yield and purity of 6xHis Ca2+-ATPase fractions produced with this method allows the biochemical and spectroscopic characterization of Ca2+-ATPase mutants produced in the course of this work. Conversion of the energy present in chemical bonds to electrochemical gradient is a central theme of bioenergetics. It is hoped that the study of the Ca2+-ATPase tryptophan mutants generated in this work will contribute to a better understanding of the coupling mechanism between ATP hydrolysis and the vectorial transport of ions across membranes that occur in this model system.

A atividade nervosa simpática está aumentada na insuficiência cardíaca (IC) e relacionada com a gravidade e o prognóstico da doença. Camundongos com deleção dos receptores adrenérgicos α2A e α2C (α2A/α2CARKO) desenvolvem IC induzida pela hiperestimulação simpática e apresentam 50% de mortalidade aos sete meses de idade. A diminuição na contratilidade cardíaca, a perda de cardiomiócitos e a intolerância à realização de esforço físico sugerem esse modelo genético para o estudo de possíveis terapias farmacológicas e não-farmacológicas para o tratamento da IC. O presente estudo avalia o possível efeito terapêutico do treinamento físico (TF) na cardiomiopatia induzida pela hiperatividade simpática. Os benefícios sobre a tolerância ao esforço e a função sistólica observados nos camundongos α2A/α2CARKO com o TF foram acompanhados pelo aumento do pico do transiente de Ca2+ intracelular e da expressão de proteínas cardíacas que regulam o transiente de Ca2+ SERCA2 (20 %), fosfo-PLB-Ser16 (92 %), fosfo-PLB-Tre17 (285 %), bem como pela redução na expressão do NCX e da PP1. Portanto, esse estudo fornece evidências de alterações na sinalização intracelular de Ca2+ nos camundongos α2A/α2CARKO que contribuem para o agravamento da IC nesse modelo, e que o TF melhora a função ventricular associada ao aumento no transiente de Ca2+ intracelular no cardiomiócito. / The sympathetic nervous activity is increased in heart failure (HF) and is associated with the severity and prognosis of disease. Mice lacking both α2A and α2C adrenergic receptors (α2A/α2CARKO) develop sympathetic hyperactivity- induced HF and present 50% mortality rate by seven mo of age. The decreased cardiac contractility, cardiomyocytes degradation and exercise intolerance suggest that these mice are a good genetic model to unravel molecular mechanisms involved in the improvements of ventricular function by different pharmacological and non-pharmacological therapies for HF. The present study was underlined to test the possible therapeutic effect of exercise training in sympathetic hyperactivity- induced HF. The improved exercise tolerance and systolic function after exercise training in α2A/α2CARKO was accompanied by increased intracellular Ca2+ transient and the expression of cardiac proteins which regulate Ca2+ transients, such as expression SERCA2 (20%), phospho-PLB-Ser16 (92%), phospho-PLB-Tre17 (285%), paralleled by reduction in NCX and PP1 expression. Therefore, this study provide direct evidence for the altered intracellular Ca2+ signaling in α2A/α2CARKO mice and that exercise training improves the ventricular function associated with an increase in intracellular Ca2+ transient in cardiomyocyte.

L'hippocampe est une région du cerveau importante pour la formation de mémoire. Des études récentes ont montré que la zone CA2 de l'hippocampe, longtemps ignorée, joue un rôle clef dans certaines formes de mémoire et notamment dans la mémoire sociale. De plus, des études post-mortem ont révélé des altérations spécifiques à la région CA2 chez les patients schizophrènes. Cependant, l’implication des neurones de CA2 dans les circuits de l'hippocampe reste peu connu, tant dans des conditions physiologiques que pathologiques. En combinant pharmacologie, génétique et électrophysiologie sur tranches d’hippocampe de souris, nous avons étudié comment les neurones pyramidaux (NP) CA2 sont recrutés dans les circuits hippocampiques après des changements d’inhibition et comment le recrutement des NP CA2 pourrait moduler l’information sortant de l'hippocampe. D’autre part, nous avons examiné les altérations fonctionnelles de la zone CA2 dans le modèle murin Df(16)A+/- de la microdélétion 22q11.2, le facteur génétique de risque de schizophrénie le plus élevé. Dans la région CA2 de l’hippocampe, les synapses inhibitrices contrôle les afférences des collatérales de Schaeffer (CS) et expriment une dépression à long-terme (DLTi) unique qui dépendant des récepteurs delta-opioïdes (RDO). Contrairement aux synapses CS-CA1, les synapses excitatrices CS-CA2 n’expriment pas de potentialisation à long-terme après application des protocoles d'induction. Cependant, nous avons constaté que différents types d'activités induisent une augmentation durable de l’amplitude des potentiels post-synaptiques (PPS) évoqués aussi bien par une stimulation des CS que des afférences distales des NP CA2, et ceci via une modulation de la balance excitation/inhibition. Nous avons démontré que ces augmentations du rapport excitation/inhibition sont les conséquences directes de la DLTi RDO-dépendante. De plus, la DLTi permet le recrutement des NP CA2 par les NP CA3 alors qu’une inhibition intacte empêche complètement leur activation en réponse aux stimulations des CS. Par ailleurs, le recrutement des pyramides de CA2 par les CS après disinhibition activité-dépendante ajoute une composante polysynaptique (SC-CA2-CA1) au PPS monosynaptique (SC-CA1) dans les NP CA1 et augmente leur activité. De plus, l’inactivation des interneurones exprimant la parvalbumine à l’aide d’outils pharmacogénétiques, a montré que ces cellules inhibitrices contrôlent fortement l'amplitude du PPS et l’activité des NP CA2 en réponse à la stimulation des CS et qu’elles sont nécessaires à l'augmentation RDO-dépendante du rapport excitation/inhibition entre CA3 et CA2. Enfin, l'étude de la zone CA2 chez les souris Df(16)A+/- a révélé plusieurs modifications dépendantes de l'âge dont une réduction de l'inhibition, une altération de la plasticité du rapport excitation/inhibition entre CA3 et CA2 et une hyperpolarisation NP CA2. Ces modifications cellulaires peuvent expliquer les déficiences de mémoire sociale que nous observons chez les souris Df(16)A+/- adultes. L’ensemble de nos études a permis de mettre en évidence le rôle des neurones CA2 dans les circuits de l'hippocampe. Enfin pour conclure, nous postulons que le recrutement des neurones CA2 dans les réseaux neuronaux sous-tend des aspects particuliers de la fonction de l'hippocampe. / The hippocampus is a region of critical importance for memory formation. Recent studies have shown that the long-overlooked hippocampal region CA2 plays a role in certain forms of memory, including social recognition. Furthermore, post-mortem studies of schizophrenic patients have revealed specific changes in area CA2. As yet, the role of CA2 neurons in the hippocampal circuitry remains poorly understood under both normal physiological and pathological conditions. By combining pharmacology, mouse genetics and electrophysiology, we investigated how CA2 pyramidal neurons (PNs) could be recruited in hippocampal circuits in mice hippocampal slices following an activity-dependent change in the strength of their inhibitory inputs. We further investigated how subsequent recruitment of CA2 PNs could modulate hippocampal output. Moreover, we examined the functional alterations of area CA2 in the Df(16)A+/- mouse model of the 22q11.2 microdeletion, a spontaneous chromosomal deletion that is the highest known genetic risk factor for developing schizophrenia. In area CA2, inhibitory synapses exert a powerful control of Schaffer collateral (SC) inputs and undergo a unique long-term depression (iLTD) mediated by delta-opioid receptor (DOR) activation. Unlike SC-CA1 synapses, SC-CA2 excitatory synapses fail to express long-term potentiation after classical induction protocols. However, we found that different patterns of activity persistently increase both the SC and the distal input net excitatory drive onto CA2 PNs via a modulation of the balance between excitation and inhibition. We demonstrated that increases in the excitatory/inhibitory ratio are direct consequences of the DOR-mediated iLTD. Interestingly, we found that the inhibition in area CA2 completely preventing CA3 PNs to activate CA2 PNs, and following iLTD, SC stimulation allows CA2 PNs to fire action potentials. Moreover, the recruitment of CA2 PNs by SC intra-hippocampal inputs after their activity-dependent disinhibition adds a delayed SC-CA2-CA1 response to the SC-CA1 monosynaptic post-synaptic potential (PSP) in CA1 and increases CA1 PN activity. Furthermore, pharmaco-genetic silencing of parvalbumin-expressing interneurons revealed that these inhibitory cells control the PSP amplitude and the firing of CA2 PNs in response to SC stimulation and are necessary for the DOR-mediated increase in excitatory/inhibitory balance between CA3 and CA2. Finally, we found several age-dependent alterations in area CA2 in Df(16)A+/- mouse model of the 22q11.2 microdeletion. These included a reduction in inhibition, an impaired activity-dependent modulation of the excitatory drive between CA3 and CA2 and a more hyperpolarized CA2 PN resting potential. These cellular disruptions may provide a potential mechanism for the social memory impairment that we observe in Df(16)A+/- adult mice. Altogether, our studies highlight the role of CA2 neurons in hippocampal circuitry. To conclude, we postulate that the recruitment of CA2 neurons in neuronal networks underlies key aspects of hippocampal function.

Made available in DSpace on 2015-04-11T13:38:25Z (GMT). No. of bitstreams: 1 francianny.pdf: 1582573 bytes, checksum: 107d67128bf43813bf5591f4c2d0e26f (MD5) Previous issue date: 2012-06-25 / Quassia amara L. (Simaroubaceae) is a pan american plant known as pau amargo and quassia. It is used in the folk medicine to treat gastrointestinal disorders, hypertension, malaria, and as insecticide and vermifuge. Several compounds were isolated from the plant leaves and barks as β-carbonile alkaloids, 6-cantin, steroids and quassinoids (neoquassin and quassin). In this work, the effects of the standardized extracts from Q. amara collected in Manicoré/AM were studied on the cardiovascular system of rodents. The aqueous extract (AE) was prepared from the dried leaves infusion and the butanolic fraction (BuF) was obtained from the AE partition in butanol. The BuF standardization and purification in HPLC isolated 6 fractions (F1 to F6). The AE oral administration (1.0 g/kg) did not produce evident signals of pharmacological activity in rats; BuF caused sedation, anesthesia and cyanosis after 1 h. Death was not observed after 24 h of the treatment. However, BuF (1.0 g/kg) intraperitoneal administration produced intense sedation, anesthesia and cyanosis after 1 h of the treatment; all animals died after 2 h. In anesthetized rats, BuF produced fast and transient hypotension, without altering the hypertensive effect of noradrenalin, or the hypotension induced by acetylcholine. The hypotension caused by BuF (10.0 mg/kg, e.v.) was unchanged by previous treatment with atropine or prazosin. The fractions responsible for the hypotensive effect were: F1, F3 and F6. All fractions were tested on intact endothelium- and denuded- aorta rings contracted by noradrenalin. F1, F3, F4, F5 and F6 fractions relaxed the endothelium intact aorta tonus, indicating a mechanism dependent on NO cascade. The chronotropic and inotropic effects of AE and BuF were also evaluated in isolated right and left rat atrium. Neither AE (30 to 300 μg/mL) nor BuF (10 to 100 μg/mL) alter the chronotropism, however, the force of contraction increased in a concentration dependent manner. BuF potentiated the diaphragm muscle contraction induced by direct and indirect electrical stimulation. BuF (1 to 300 μg/mL) inhibited the activity of the Ca2+-ATPase isolated from rabbit skeletal muscle; this enzyme inhibition may be related to the potentiation of the diaphragm contraction. In rat vas deferens preparations, BuF inhibited the maximal contraction induced by noradrenalin without affecting the drug affinity to the α1-adrenergic receptors. The purified fractions responsible for this effect were F1, F3, F4 e F5. BuF did not inhibit the calcium influx in cardiomyocytes and cultured rat uterus cells. / A Quassia amara L. (Simaroubaceae) é planta pan-americana conhecida como pau-amargo e quassia. É utilizada na medicina popular para o tratamento de distúrbios gastrintestinais, hipertensão, malária, e também como inseticida e vermífuga. Das folhas e cascas foram isolados vários compostos químicos como alcaloides β-carbonila, 6-cantin, esteroides e quassinoides (neoquassina e quassina). Neste trabalho, foram estudados a farmacologia geral e os efeitos no sistema cardiovascular de roedores de extratos padronizados de Quassia amara coletada em Manicoré, AM. O extrato aquoso (EA) foi obtido por infusão de folhas secas, e a fração butanólica, por partição do EA em butanol. A padronização e purificação da FBut por cromatografia líquida de alta eficiência (CLAE) levou ao isolamento de 6 frações (F1 a F6). A administração oral do EA (1,0 g/kg) não produziu sinais evidentes de atividade farmacológica em ratos; a FBut produziu sinais de sedação, anestesia e cianose após 1 h. Não houve morte dos animais após 24 h do tratamento. No entanto, a administração da FBut (1,0 g/kg) por via intraperitoneal produziu sinais de sedação, anestesia e cianose intensos após 1 h de tratamento; todos os animais morreram após 2 h. Na pressão arterial em ratos anestesiados, a FBut produziu hipotensão rápida e passageira, sem alterar o efeito hipertensor da noradrenalina, ou a hipotensão produzida pela acetilcolina. A hipotensão provocada pela FBut (10,0 mg/kg, e.v.) também não foi alterada pelo tratamento prévio do animal com atropina ou prazosin. As frações responsáveis pela ação hipotensora são a F1, F3 e F6. As frações foram testadas em preparações de aorta de rato com e sem endotélio. As frações F1, F3, F4, F5 e F6 relaxaram o tônus muscular da aorta dependente de endotélio, indicando um provável mecanismo via cascata do NO. As atividades cronotrópica e inotrópica do EA e da FBut foram avaliadas em preparações de átrios direito e esquerdo isolados de rato. O EA (30 a 300 μg/mL) e a FBut (10 a 100 μg/mL) não alteraram o cronotropismo, no entanto, aumentaram o inotropismo de maneira concentração-dependente. A FBut potenciou a contração do músculo diafragma de rato sob estímulo direto e indireto. A FBut (1 a 300 μg/mL) inibiu a atividade da Ca2+-ATPase de músculo esquelético de coelho; essa inibição pode estar relacionada à potenciação da contração do músculo diafragma. Em ducto deferente de rato, a FBut diminuiu a contração máxima produzida pela noradrenalina sem alterar a afinidade da droga pelos receptores α1-adrenérgicos. As frações purificadas responsáveis por este efeito foram as F1, F3, F4 e F5. A FBut não inibiu o influxo de cálcio

Ankstesni tyrimai, susiję su laikinėmis sinapsinio efektyvumo pasikeitimo taisyklėmis per nuo veikimo potencialų poravimo priklausantį plastiškumą, praktiškai nekreipė jokio dėmesio į neuronų erdvines savybes. Mes nagrinėjame nuo kalcio/kalmodulino priklausančią kinazę II (CaMKII) ir kalcineuriną (CaN) artimuosiuose ir tolimuosiuose spygliuose bei jų įtaką ilgalaikei potenciacijai ir ilgalaikei depresijai. Tyrimų metu buvo atskleista, kad esant neigiamam veikimo potencialų laiko poravimui tolimoje sinapsėje gali vykti ilgalaikė potenciacija, tuo tarpu artimajame spyglyje visi procesai vyksta remiantis klasikinėmis sinapsinio plastiškumo taisyklėmis. Gauti rezultatai teigia, kad sinapsės vieta dendritiniame medyje yra lemiamas veiksnys nuo veikimo potencialų laikų poravimo priklausančiame sinapsiniame plastiškume. / Previous studies focusing on the temporal rules governing changes in synaptic efficacy during spike timing-dependent plasticity (STDP) have paid little attention to spatial characteristics of neurons. We analyze the activity of calcium-calmodulin dependent protein kinase II (CaMKII) and phosphatase calcineurin (CaN) in proximal and distal spines and their impact on long-term potentiation (LTP) and long-term depression (LTD). During tests we found that at negative timing of action potentials in distal spine synapse can undergo LTP while in proximal spine the processes follow the classic STDP rules. Our results suggest that synapse location within the dendritic tree is a crucial determinant of STDP.

No description available.

Made available in DSpace on 2015-05-14T12:59:34Z (GMT). No. of bitstreams: 1 arquivototal.PDF: 7069577 bytes, checksum: 994737d813443da6d7f127b17873faf9 (MD5) Previous issue date: 2012-03-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The flavonoid galetin 3,6 dimethyl ether (FGAL) was isolated from the aerial parts of Piptadenia stipulacea (Benth.) Ducke and was investigated its possible hemolytic activity in rat erythrocytes and spasmolytic activity in several isolated smooth muscles as rat uterus, guinea pig ileum and trachea, and rat aorta. FGAL showed no hemolytic effect on rat erythrocytes, which is suggestive of a low toxicity. In preliminary pharmacological screening performed in smooth muscle models, FGAL showed non-selective spasmolytic effect in the four organs tested, with a higher potency to relax the guinea pig trachea pre-contracted with carbachol and rat aorta pre-contracted with phenylephrine (FEN) in a functional epithelium and endothelium independent manner, respectively. The aim of this study was to investigate the action mechanism of FGAL relaxing effect in guinea pig trachea and rat aorta. The observation that FGAL was more potent in relaxing both pre-contracted organs with moderate increases in extracellular concentration of KCl than when contracted with larger increases of KCl is suggestive that FGAL modulates the channels positivety. This hypothesis was confirmed by decreasing of relaxant potency of FGAL in both organs in the presence of tetraethylammonium (TEA+) 10 mM, non-selective blocker of K+ channels. To determine the subtypes of K+ channel involved, were used selectives blockers: in trachea the effect of FGAL was not altered in the presence of TEA+ 1 mM, blocker of large conductance calcium-activated K+ channels (BKCa); glibenclamide, blocker of sensitive-ATP K+ channels (KATP); BaCl2, blocker of inward rectifier K+ channels (Kir) or 4-AP, blocker of voltage activated K+ channels (KV), but was reduced in the presence of apamin, blocker of small conductance calcium-activated K+ channels (SKCa). In aorta, the relaxant effect of FGAL was not altered in the presence of TEA+ 1 mM, but was reduced in the presence of apamin, glibenclamide, BaCl2 and 4-AP, suggesting the involvement of SKCa, KATP, Kir and KV in vasorelaxant action of flavonoid. The fact of FGAL rightward shifted, with Emax reduced the CaCl2-induced contractions in depolarizing medium, and CaCl2 in the presence of verapamil, a voltage activated calcium channel (CaV) blocker, and FEN, suggests the involvement of CaV and ROCs (receptor-operated calcium channel), respectively. Also in the aorta, FGAL inhibited FEN induced contractions in Ca2+-free medium, suggesting inhibition of Ca2+ release from the sarcoplasmic reticulum SR. We also evaluated the participation of the cyclic nucleotides pathway, and observed that the trachea and aorta relaxation induced by aminophylline, non selective inhibitor of phosphodiesterases (PDEs), was more potent in the presence of FGAL, suggesting the involvement of cAMP and/or cGMP. On the aorta was assessed FGAL effect on relaxation induced by selective inhibitors of PDE-3 (milrinone, cAMP selective) and PDE-5 (sildenafil, cGMP selective), and FGAL only potentiated the relaxation induced by sildenafil, suggesting the participation of cGMP. Since K+ channels are modulated negatively by PKC, we investigated a possible inhibition of PKC by FGAL and the flavonoid relaxed the aorta pre-contracted with a PKC activator (PMA), suggesting inhibition of this enzyme. In conclusion, the spasmolytic mechanism of FGAL in trachea involves positive modulation of SKCa and cyclic nucleotides, and in the aorta involves the positive modulation of KATP, SKCa, Kir, Kv and inhibition of CaV, ROCs, Ca2+ release of SR, PDE-5 and PKC. / O flavonoide 3,6-dimetil éter galetina (FGAL) foi isolado das partes aéreas de Piptadenia stipulacea (Benth.) Ducke e foi investigada sua possível atividade hemolítica em eritrócitos de rato e espasmolítica em vários músculos lisos isolados como útero de rata, íleo e traqueia de cobaia, e aorta de rato. FGAL não causou efeito hemolítico em eritrócitos de ratos, o que é sugestivo de baixa toxicidade. Na triagem farmacológica preliminar realizada em músculos lisos, FGAL apresentou efeito espasmolítico não seletivo nos 4 órgãos testados, apresentando uma maior potência em relaxar a traqueia de cobaia pré-contraída com carbacol e a aorta de rato pré-contraída com fenilefrina (FEN), de maneira independente de epitélio e endotélio funcional, respectivamente. Assim, o objetivo deste trabalho foi investigar o mecanismo de ação relaxante de FGAL em traqueia de cobaia e aorta de rato. A observação de que FGAL foi mais potente em relaxar ambos os órgãos pré-contraídos com aumentos moderados na concentração extracelular de KCl do que quando contraídos com aumentos maiores de KCl é sugestivo de que FGAL está agindo por modular positivamente os canais de K+, hipótese esta confirmada pela diminuição da potência relaxante de FGAL em ambos os órgãos na presença de tetraetilamônio (TEA+) 10 mM, bloqueador não seletivo dos canais de K+. Para verificar os subtipos de canais de K+, usou-se bloqueadores seletivos: em traqueia o efeito de FGAL não foi alterado na presença de TEA+ 1 mM, bloqueador dos canais K+ de grande condutância ativados pelo Ca2+ (BKCa), glibenclamida, bloqueador dos canais de K+ sensíveis ao ATP (KATP), BaCl2, bloqueador dos canais de K+ retificadores de entrada (Kir) ou de 4-AP, bloqueador dos canais de K+ sensíveis à voltagem (KV), porém foi reduzido na presença de apamina, bloqueador dos canais de K+ de pequena condutância ativados pelo Ca2+ (SKCa). Em aorta, o efeito relaxante de FGAL não foi alterado na presença de TEA+ 1 mM, por outro lado foi reduzido na presença de apamina, glibenclamida, BaCl2 e 4-AP, sugerindo a participação dos SKCa, KATP, Kir e KV na ação vosorrelaxante do flavonoide. O fato de FGAL deslocar para direita com redução do Emax as contrações induzidas por CaCl2 em meio despolarizante, e por CaCl2 na presença de FEN e verapamil, bloqueador de CaV, sugere o envolvimento dos CaV e dos ROCs, respectivamente. Ainda em aorta, FGAL inibiu as contrações induzidas por FEN em meio livre de Ca2+, sugerindo inibição da liberação de Ca2+ do retículo sarcoplasmático (RS). Avaliou-se ainda a participação dos nucleotídios cíclicos, e observou-se que o relaxamento induzido pela aminofilina, inibidor não seletivo de fosfodiesterases (PDEs) em traqueia e aorta foi potencializado com FGAL, sugerindo a participação de AMPc e/ou GMPc. Em aorta foi avaliado o efeito de FGAL sobre o relaxamento induzido com inibidores seletivos de PDE-3 (milrinona, seletiva para AMPc) e PDE-5 (sildenafila, seletiva para GMPc), sendo que FGAL só potencializou o relaxamento induzido por sildenafila, sugerindo a participação do GMPc. Como os canais de K+ são modulados negativamente pela PKC, investigou-se uma possível inibição da PKC por FGAL, que relaxou a aorta pré-contraída com o ativador de PKC (PMA), sugerindo inibição dessa enzima. Em conclusão, o mecanismo de ação espasmolítica de FGAL em traqueia envolve modulação positiva dos SKCa e dos nucleotídios cíclicos, e em aorta modulação positiva dos KATP, SKCa, Kir, KV; inibição dos CaV e dos ROCs, da liberação de Ca2+ do RS, da PDE-5 e PKC.

A atividade nervosa simpática está aumentada na insuficiência cardíaca (IC) e relacionada com a gravidade e o prognóstico da doença. Camundongos com deleção dos receptores adrenérgicos α2A e α2C (α2A/α2CARKO) desenvolvem IC induzida pela hiperestimulação simpática e apresentam 50% de mortalidade aos sete meses de idade. A diminuição na contratilidade cardíaca, a perda de cardiomiócitos e a intolerância à realização de esforço físico sugerem esse modelo genético para o estudo de possíveis terapias farmacológicas e não-farmacológicas para o tratamento da IC. O presente estudo avalia o possível efeito terapêutico do treinamento físico (TF) na cardiomiopatia induzida pela hiperatividade simpática. Os benefícios sobre a tolerância ao esforço e a função sistólica observados nos camundongos α2A/α2CARKO com o TF foram acompanhados pelo aumento do pico do transiente de Ca2+ intracelular e da expressão de proteínas cardíacas que regulam o transiente de Ca2+ SERCA2 (20 %), fosfo-PLB-Ser16 (92 %), fosfo-PLB-Tre17 (285 %), bem como pela redução na expressão do NCX e da PP1. Portanto, esse estudo fornece evidências de alterações na sinalização intracelular de Ca2+ nos camundongos α2A/α2CARKO que contribuem para o agravamento da IC nesse modelo, e que o TF melhora a função ventricular associada ao aumento no transiente de Ca2+ intracelular no cardiomiócito. / The sympathetic nervous activity is increased in heart failure (HF) and is associated with the severity and prognosis of disease. Mice lacking both α2A and α2C adrenergic receptors (α2A/α2CARKO) develop sympathetic hyperactivity- induced HF and present 50% mortality rate by seven mo of age. The decreased cardiac contractility, cardiomyocytes degradation and exercise intolerance suggest that these mice are a good genetic model to unravel molecular mechanisms involved in the improvements of ventricular function by different pharmacological and non-pharmacological therapies for HF. The present study was underlined to test the possible therapeutic effect of exercise training in sympathetic hyperactivity- induced HF. The improved exercise tolerance and systolic function after exercise training in α2A/α2CARKO was accompanied by increased intracellular Ca2+ transient and the expression of cardiac proteins which regulate Ca2+ transients, such as expression SERCA2 (20%), phospho-PLB-Ser16 (92%), phospho-PLB-Tre17 (285%), paralleled by reduction in NCX and PP1 expression. Therefore, this study provide direct evidence for the altered intracellular Ca2+ signaling in α2A/α2CARKO mice and that exercise training improves the ventricular function associated with an increase in intracellular Ca2+ transient in cardiomyocyte.