Made available in DSpace on 2015-07-22T20:49:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-04-27 / O sono é um importante evento fisiológico que diretamente influencia a saúde e está relacionado com o sistema imunológico no qual o cálcio age como um importante mensageiro. Neste estudo, nós realizamos as medidas de mobilização do cálcio citossólico em células vivas com o objetivo de compreender as mudanças na sinalização deste íon em células imunológicas do baço de camundongos após diferentes períodos de privação de sono. Esplenócitos de camundongos privados de sono por diferentes períodos (12 à 72 horas) mostraram uma progressiva perda da manutenção do cálcio intracelular proveniente do estoque do retículo endoplasmático e um comprometimento do tamponamento de cálcio transiente pela mitocôndria. Estes dados foram confirmados por mudanças no desempenho dos canais de cálcio SOCE e STIM1 e na integridade da fisiologia mitocondrial e lisossomal. Estes resultados foram corroborados pelo aumento na atividade de enzimas antioxidantes como a Superóxido Dismutase mitocondrial e citossólica, o que reforça que o rompimento na integridade da mitocôndria e lisossomos possivelmente ocorreu pela geração descontrolada de estresse oxidativo. A redução da atividade da Catalase sugere comprometimento na integridade celular, uma vez que o excesso de cálcio e radicais livres provavelmente danificaram o mecanismo de sinalização da célula. Ademais observamos resposta imunológica deficiente quando um grupo de camundongos pode dormir por 24 e por 48 horas após a privação de sono por 72 horas, seguido de infecção com parasita de malária (Plasmodium chabaudi). O envolvimento das células imunológicas foi confirmado pela redução na população de células totais do baço e, especificamente, na população de linfócitos B. Estes novos dados sugerem que a privação de sono prejudica a sinalização do cálcio provavelmente por um estresse no retículo endoplasmático, mitocôndria e lisossomos levando a um suprimento insuficiente de cálcio para eventos de sinalização com conseqüentes danos intracelulares. Estes dados confirmam mecanismos descritos previamente de efeitos imunossupressores da perda de sono. / Sleep is an important physiological event that directly influences health and is related to the immune system in which calcium acts as an important messenger. In this study, we performed measurements of cytosolic calcium mobilization in living cells in order to understand the changes in this ion signaling in immune cells from mice after different periods of sleep deprivation. Splenocytes of mice deprived of sleep for various periods (12 to 72 hours) showed a progressive loss of the intracellular calcium maintenance from the endoplasmic reticulum store and a commitment of transient calcium buffering by mitochondria. These data were confirmed by changes in the performance of calcium channels SOCE and STIM1 and in the integrity of lysosomal and mitochondrial physiology. These results were corroborated by the increase in activity of antioxidant enzymes such as mitochondrial and cytosolic superoxide dismutase, which reinforces the idea that the disruption in the integrity of mitochondria and lysosomes possibly occurred by uncontrolled generation of oxidative stress. The reduction of catalase activity suggests involvement in cellular integrity, since excess of calcium and free radicals probably compromise cell signaling. Moreover, deficient immune response was observed when a group of mice was allowed to sleep for 24 and 48 hours of sleep deprivation for 72 hours, followed by infection with malaria parasites (Plasmodium chabaudi). The involvement of the immune cells was confirmed by the reduction in the total population of cells in the spleen and, specifically, the population of B lymphocytes. These new data suggest that sleep deprivation affects calcium signaling probably by a stress in the endoplasmic reticulum, mitochondria and lysosomes, leading to an insufficient supply of calcium to signaling events and consequent intracellular damage. These data confirm previously described mechanisms of immunosuppressive effects of sleep loss. / TEDE

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The purpose of this study was to verify the effect of dietary Ca2+ on growth and survival of silver catfish fingerlings (Rhamdia quelen) exposed to different water pH (5.5; 7.5 and 9.0). Silver catfish fingerlings were randomly placed in a termo-regulated water re-use system with twelve 250 L tanks, two 1000 L biofilters and a 2000 L reservoir with medium flow of 3.84 L min-1 tank. Stocking density was 0.16 fingerlings L-1. To prepare the treatment diets, the control diet (0.08% Ca2+) was supplemented with CaCO3 to yield experimental diets with 0.64; 0.95 and 2.39% Ca2+. There were three replicates/treatment. Survival was higher than 93.9% in all treatments. Exposure of silver catfish fingerlings to alkaline or acid water reduced growth, and this effect was not ameliorated by dietary Ca2+ supplementation. Moreover, best dietary Ca2+ range for silver catfish fingerling growth is 0.08-0.64%, mainly when fingerlings were maintained in water with neutral pH. / A proposta deste estudo foi verificar o efeito de diferentes níveis de Ca2+ na dieta no crescimento e na sobrevivência de alevinos de jundiá (Rhamdia quelen) expostos a diferentes faixas de pH da água (5,5; 7,5 e 9,0). Os alevinos de jundiá foram distribuídos aleatoriamente em densidade de estocagem de 0,16 alevinos/l, num sistema de recirculação de água termo-regulada, com 12 caixas de cimento amianto impermeabilizado de 250 l, 2 biofiltros de 1000 l e 1 reservatório de água elevado de 2000 l. Para o preparo dos diferentes tratamentos, a dieta controle (0,08% Ca2+) foi suplementada com CaCO3 para formulação das demais dietas, com 0,64; 0,95; e 2,39% Ca2+, com 3 repetições por tratamento. A sobrevivência foi superior a 93,9% em todos os tratamentos, não havendo diferença significativa. A exposição de alevinos de jundiá em águas ácidas ou alcalinas reduziu o crescimento, sendo que tais efeitos não foram compensados pela adição de Ca2+ na ração. Os resultados demonstram que dietas com níveis de Ca2+ em torno de 0,08-0,64% são as mais adequadas para o crescimento de alevinos de jundiá, principalmente quando os alevinos são mantidos em água com pH neutro.

Proteínas de membrana estão envolvidas em processos fisiológicos essenciais como, por exemplo, a manutenção do equilíbrio iônico e sinalização intracelular. No entanto, apesar do envolvimento em inúmeros processos fisiológicos e de grande interesse farmacêutico, o estudo estrutural de proteínas de membrana ainda é um processo custoso e muito mais complexo do que o estudo estrutural de proteínas solúveis. Os trocadores de Na+/Ca2+ são proteínas de membrana que atuam na manutenção da homeostase de Ca2+ intracelular e estão envolvidos em processos patológicos como doenças cardíacas. Estes trocadores estão presentes em diversas espécies de mamíferos (NCX) e insetos, por exemplo, na mosca Drosophila melanogaster (CALX). A topologia destas proteínas é constituída de dois domínios. O domínio transmembranar, que contém dois segmentos de 5 hélices transmembranares (TMH) e é responsável por promover o transporte específico de íons Ca2+ e Na+ através da membrana, e o domínio citoplasmático, responsável por regular a atividade do trocador. O domínio citoplasmático consiste de uma alça que contém dois domínios sensores de Ca2+ intracelular (CBD1 e CBD2). Trabalhos mostraram que o trocador CALX é inibido pela ligação de Ca em CBD1, enquanto que trocadores NCX são ativados. As regiões citosólicas que conectam CBD1 e CBD2 à TMH5 e TMH6 são conservadas e ainda não foram caracterizadas estruturalmente. Adjacente à TMH5 há um segmento anfipático, denominado exchanger inhibitory peptide (XIP), que está envolvido no mecanismo de regulação do trocador. Na ausência de dados estruturais do CALX completo, o estudo de TMH5-XIP poderá aumentar a compreensão sobre a estrutura e o funcionamento do trocador. A construção TMH5-XIP foi fusionada à MBP no N-terminal e a uma sequência de 8 histidinas no C-terminal. Apesar da expressão da proteína de fusão ter sido bem sucedida, problemas de precipitação e ineficiência durante a clivagem da conexão com a MBP impediram a conclusão dos estudos estruturais. Logo, uma construção menor, contendo apenas a região equivalente ao XIP, foi estudada por espectroscopia de RMN em solução e dicroísmo circular. XIP forma uma 310-hélice a baixa temperatura, 7 oC, que se desestabiliza a maior temperatura, 27 oC. Estes dados permitem a formulação de hipóteses sobre o papel de XIP no mecanismo de regulação do domínio transmembranar de CALX. / Membrane proteins are involved in essential physiological processes such as maintenance of the ionic balance and intracellular signaling. However, despite their role in numerous physiological processes of well-recognized pharmaceutical relevance, structural studies of membrane proteins remain being more complex than structural studies of globular proteins. Na+/Ca2+ exchangers (NCX) are membrane proteins that play essential roles in the maintenance of the intracellular Ca2+ homeostasis. Not surprisingly, the NCXs are involved in pathologies such as heart diseases. These exchangers are present in several species of mammals (NCX) and insects, for example, in the fly Drosophila melanogaster (CALX). The topology of these proteins consists of a transmembrane and a hydrophilic domain. The transmembrane domain corresponds to two segments of 5 transmembrane helices (TMH) forming a 10-helix bundle that is responsible for the specific transport of Ca2+ and Na+ across the cellular membrane. The hydrophilic domain is composed of a large cytoplasmic loop, which is associated with the regulation of the ion exchange activity of the transmembrane domain. The loop contains two Ca2+-sensors domains, CBD1 and CBD2, and uncharacterized regions. Studies showed that Ca2+ binding to CBD1 inhibits the CALX, whereas it activates the NCX. The juxtamembrane cytosolic regions linking the CBD1 and CBD2 domains to the TMH5 and TMH6, respectively, are highly conserved but have not yet been structurally characterized. The segment near TMH5 is amphipathic, and it is also called exchanger inhibitory peptide (XIP). In the absence of a three-dimensional structure of the complete CALX, the study of TMH5-XIP may contribute to our understanding of the structure and operation of the exchanger. In order to study TMH5-XIP, it was fused to an MBP tag at the N-terminus, and to a sequence of 8 histidines at the C-terminus. Although the expression of the fusion protein was successful, precipitation and inefficient MBP-tag cleavage prevented the isolation of pure TMH5-XIP for structural studies. Hence, a smaller construct, containing only the region equivalent to XIP, was studied by NMR spectroscopy in solution and circular dichroism. The structure assumed by XIP in solution is temperature dependent, being intrinsically disordered at 27 C or a 310-helix at 7 C, respectively. These findings allowed us to infer how XIP could participate in the CALX regulation mechanism.

Made available in DSpace on 2015-04-11T13:38:51Z (GMT). No. of bitstreams: 1 Thiago Mattos de Araujo Lima.pdf: 914896 bytes, checksum: be7fe7f16d2a312578b3d1011a681a23 (MD5) Previous issue date: 2010-03-30 / Fundação de Amparo à Pesquisa do Estado do Amazonas / Virola surinamensis (Myristicaceae), popularly known as ucuuba, ucuuba branca and sucuuba, is widely distributed in flooded areas of the Amazonian Forest. The bark resin is used in the folk medicine to treat erysipela and the tea of the leaves, to treat colics, dyspepsia, inflammations and malaria. The main constituents described for V. surinamensis are arylpropanoids, neolignans, lignans, lignoids, flavonoids, diarylpropanes, -butirolactones, alkaloids and acid benzoic derivatives. Among them, lignans and neolignans showed anti-parasitic and anti-fungal activities. Previous pharmacological studies described anti-parasitic effect of the essential oils and anti-leishmania, anti-inflammatory, anti-fungal, insecticide and gastroprotective actions. The aim of this work was to study the pharmacological activity of the standardized buthanolic fraction of Virola surinamensis and the mechanisms of the hypotensive effect and the potentiation of the skeletal muscle contraction. The chemical standardization was obtained after partition of the leaves aqueous extract (AE) in n-buthanol originating the buthanolic fraction (BuF). The BuF purification by high performance liquid chromatography (HPLC) originated 10 high purity fractions (F1 to F10) that are still in identification process. The pharmacological screening of AE and BuF showed low action in the CNS. In the arterial blood pressure of anesthetized rats, the intravenous injection of both AE and BuF produced hypotension apparently associated with the α1-adrenoceptors blockade. The BuF was effective to reduce the arterial blood pressure in the chronic treatment also. The absorbed substances did not modify the animal behavior in the Irwin Test, but produced reversible hypotension initiating in 30 min, time necessary to the gastric emptiness. The treatment during 7 days did not modify the animal behavior, but there was a residual accumulated effect of the BuF reducing the basal arterial blood pressure of the rats. In the isolated aortic rings without endothelium, the BuF relaxed the tonus induced by noradrenalin in a concentration-dependent manner, showing that BuF did not interfere with the NO system. This effect was indicative of a competitive antagonism and reinforced the evidences obtained in the arterial blood pressure that the mechanism of the hypotension produced by BuF was the α1 adrenoceptors blockade. The BuF potentiated the direct elicited twitches of the rat diaphragm. In microssomes isolated from the rabbit skeletal muscle, BuF inhibited the Ca2+-ATPase activity similarly to thapsigargin, indicating that the potentiation of the diaphragm contraction may be related to the decrease in the Ca2+ uptake by the sarcoplasmic reticulum. / A Virola surinamensis, conhecida popularmente como ucuúba, ucuúba branca, ucuúba de igapó, sucuba, é uma árvore que cresce em várzeas e em bancos de areia em rios na Floresta Amazônica. A resina da casca é utilizada na medicina popular para o tratamento de erisipelas e o chá das folhas é indicado em cólicas, dispepsia, processos inflamatórios e para o tratamento da malária. Dentre os constituintes de Virola surinamensis foram descritos arilpropanoides, neolignanas, lignanas, sesquilignóides, lignóides diméricos, flavonóides, diarilpropanos, -butirolactonas, alcalóides e derivados do ácido benzóico. Desses, destacam-se as lignanas e neolignanas por suas atividades anti-parasitária e anti-fúngica. Estudos farmacológicos anteriores descrevem atividade anti-parasitária dos óleos essenciais, atividade anti-leishmania, anti-inflamatória, antifúngica, inseticida e gastroprotetora. Este trabalho teve como objetivo o estudo das ações farmacológicas da fração butanólica padronizada de Virola surinamensis e a análise dos mecanismos do efeito hipotensor e da potenciação da contração do músculo esquelético. A padronização química foi obtida após partição do extrato aquoso das folhas (EA) em butanol dando origem à fração butanólica (FBut). A purificação da Fbut por cromatografia líquida de alta eficiência (CLAE) originou 10 frações de elevado grau de pureza (F1 a F10) que se encontra em processo de identificação. A triagem farmacológica do EA e da Fbut mostrou baixa ação no SNC. Na pressão arterial de ratos anestesiados a injeção endovenosa tanto do extrato aquoso como da fração butanólica produziu hipotensão aparentemente associada à uma ação bloqueadora de receptores α1-adrenérgicos. A Fbut mostrou-se eficaz na redução da pressão arterial durante o tratamento crônico. As substâncias absorvidas não modificaram o comportamento animal no teste de Irwin, mas produziram hipotensão reversível com início em torno de 30 minutos, tempo necessário para o esvaziamento gástrico. O tratamento repetido durante 7 dias também não modificou o comportamento animal, mas houve um efeito residual cumulativo da FBut que a cada dia reduziu o valor basal da pressão arterial dos ratos. Em anéis de aorta sem endotélio, a Fbut relaxou o tônus induzido pela noradrenalina de maneira concentração-dependente, mostrando que Fbut não interfere no sistema NOS. Este efeito foi indicativo de antagonismo competitivo e reforça as evidências obtidas na pressão arterial de que o mecanismo da hipotensão produzida pela Fbut é o bloqueio de adrenoceptores do tipo α1. A Fbut potenciou a contração obtida por estímulo direto da fibra muscular do diaframa de rato. Em microssomas isolados da musculatura esquelética do coelho, a Fbut inibiu a atividade da Ca2+-ATPase à semelhança da tapsigargina, indicando que a potenciação da contração do diafragma parece estar relacionada à diminuição da recaptação de Ca2+ pelo retículo sarcoplasmático.

<p>Oscillations in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) is the key signal in glucose-stimulated β-cells governing pulsatile insulin release. The glucose response of mouse β-cells is often manifested as slow oscillations and rapid transients of [Ca²⁺]_i. In the present study, microfluorometric technique was used to evaluate the role of amino acids, glucagon, ryanodine and caffeine on the generation and maintenance of [Ca²⁺]_i oscillations and transients in individual murine β-cells and isolated mouse pancreatic islets. The amino acids glycine, alanine and arginine, at around their physiological concentrations, transformed the glucose-induced slow oscillations of [Ca²⁺]_i in isolated mouse β-cells into sustained elevation. Increased Ca²⁺ entry promoted the reappearance of the slow [Ca²⁺]_i oscillations. The [Ca²⁺]_i oscillations were more resistant to amino acid transformation in intact islets, supporting the idea that cellular interactions are important for maintaining the oscillatory activity. Individual rat β-cells responded to glucose stimulation with slow [Ca²⁺]_i oscillations due to periodic entry of Ca²⁺ as well as with transients evoked by mobilization of intracellular stores. The [Ca²⁺]_i oscillations in rat β-cells had a slightly lower frequency than those in mouse β-cells and were more easily transformed into sustained elevation in the presence of glucagon or caffeine. The transients of [Ca²⁺]_i were more common in rat than in mouse β-cells and often appeared in synchrony also in cells lacking physical contact. Depolarization enhanced the generation of [Ca²⁺]_i transients. In accordance with the idea that β-cells have functionally active ryanodine receptors, it was found that ryanodine sometimes restored oscillatory activity abolished by caffeine. However, the IP3 receptors are the major Ca²⁺ release channels both in β-cells from rats and mice. Single β-cells from ob/ob mice did not differ from those of lean controls with regard to frequency, amplitudes and half-widths of the slow [Ca²⁺]_i oscillations. Nevertheless, there was an excessive firing of [Ca²⁺]_i transients in the β-cells from the ob/ob mice, which was suppressed by leptin at close to physiological concentrations. The enhanced firing of [Ca²⁺]_i transients in ob/ob mouse β-cells may be due to the absence of leptin and mediated by activation of the phospholipase C signaling pathway.</p>

Oscillations in cytoplasmic Ca2+ concentration ([Ca2+]i) is the key signal in glucose-stimulated β-cells governing pulsatile insulin release. The glucose response of mouse β-cells is often manifested as slow oscillations and rapid transients of [Ca2+] i. In the present study, microfluorometric technique was used to evaluate the role of amino acids, glucagon, ryanodine and caffeine on the generation and maintenance of [Ca2+] i oscillations and transients in individual murine β-cells and isolated mouse pancreatic islets. The amino acids glycine, alanine and arginine, at around their physiological concentrations, transformed the glucose-induced slow oscillations of [Ca2+] i in isolated mouse β-cells into sustained elevation. Increased Ca2+ entry promoted the reappearance of the slow [Ca2+] i oscillations. The [Ca2+] i oscillations were more resistant to amino acid transformation in intact islets, supporting the idea that cellular interactions are important for maintaining the oscillatory activity. Individual rat β-cells responded to glucose stimulation with slow [Ca2+] i oscillations due to periodic entry of Ca2+ as well as with transients evoked by mobilization of intracellular stores. The [Ca2+] i oscillations in rat β-cells had a slightly lower frequency than those in mouse β-cells and were more easily transformed into sustained elevation in the presence of glucagon or caffeine. The transients of [Ca2+] i were more common in rat than in mouse β-cells and often appeared in synchrony also in cells lacking physical contact. Depolarization enhanced the generation of [Ca2+] i transients. In accordance with the idea that β-cells have functionally active ryanodine receptors, it was found that ryanodine sometimes restored oscillatory activity abolished by caffeine. However, the IP3 receptors are the major Ca2+ release channels both in β-cells from rats and mice. Single β-cells from ob/ob mice did not differ from those of lean controls with regard to frequency, amplitudes and half-widths of the slow [Ca2+] i oscillations. Nevertheless, there was an excessive firing of [Ca2+] i transients in the β-cells from the ob/ob mice, which was suppressed by leptin at close to physiological concentrations. The enhanced firing of [Ca2+] i transients in ob/ob mouse β-cells may be due to the absence of leptin and mediated by activation of the phospholipase C signaling pathway.

No description available.

No description available.

This diploma thesis focuses on study of the expression of purinergic receptors P2X and P2Y in the cells of neonatal adenohypofysis. Purinergic receptors are activated by binding of extracellular ATP and their activation leads to an increase in intracellular concentrations of calcium ions. The aim of this thesis was to determine by microfluorimetry method whether neonatal pituitary cells contain purinergic receptors. In addition, the influence of various agonists and antagonists of purinergic receptors on intracellular concentrations of ions Ca2+ in neonatal pituitary cells was monitored. The influence of extracellular ATP on secretion of a luteinizing hormone was observed by radioimmunoassay and it was compared with the effect of a hormone GnRH. It was found that neonatal pituitary cells are less sensitive to extracellular ATP than adult pituitary cells, but the relative sensitivity to other agonists of purinergic receptors is similar to the one of adult pituitary cells. In the mixed population of neonatal pituitary cells there were identified ATP- and 2 MeSATP- sensitive purinergic receptors P2X, as well as ADP-sensitive receptors P2Y. Further, the BzATP-sensitive receptors P2X7, PPADS-sensitive receptors P2X2 and BDBD-sensitive receptors P2X4 were identified. Finally, it was proved that...

Os receptores a2-adrenérgicos pré-sinápticos (a2 AR) regulam a função cardiovascular através da inibição da liberação do neurotransmissor no terminal nervoso simpático. Recentemente, foi publicado que a ablação dos subtipos a2A e a2C -AR em camundongos (KO) leva a hiperatividade simpática com evidências de disfunção cardíaca aos 4 meses de idade. Esses camundongos constituem um modelo experimental para o estudo dos tratamentos farmacológicos e não farmacológicos para a prevenção e o tratamento da insuficiência cardíaca. Nós estudamos se o treinamento físico melhoraria a função cardíaca e a expressão de proteínas cardíacas envolvidas no controle do Ca2+ intracelular em camundongos KO. Métodos e Resultados: Camundongos controle e KO foram estudados dos 3 aos 5 meses, onde a cardiomiopatia está em estágio inicial, e divididos aleatoriamente em treinados e sedentários. O treinamento físico (TF) foi realizado com natação, 1 hora por dia, 5 vezes por semana, por 8 semanas. A pressão arterial e a freqüência cardíaca de repouso (FC) foram medidas por pletismografia de cauda. A fração de encurtamento (FS) por ecocardiograma. A tolerância ao esforço físico por teste progressivo em esteira rolante. O tônus simpático cardíaco foi estimado por bloqueio farmacológico dos receptores muscarínicos e adrenérgicos com atropina na presença ou ausência do antagonista b-adrenérgico propranolol. O diâmetro dos cardiomiócitos e a fração de colágeno cardíaco por microscopia óptica. A expressão das proteínas cardíacas: bomba de Ca2+ do retículo sarcoplasmático (SERCA2), fosfolambam (PLB), fosfo-Ser16-PLB, fosfo-Thr17-PLB, trocador sódio-cálcio (NCX), canais de rianodina (RYR), fosfo-Ser2809-RYR, e as fosfatases 1 e 2A foram analisadas por Western Blot. Aos 3 meses de idade não foram observadas diferenças significantes na pressão arterial, FS e tolerância ao esforço físico entre os animais controle e KO, no entanto, KO apresentou taquicardia basal. Aos 5 meses de idade, quando a disfunção cardíaca está em estágio inicial, KO apresentou intolerância ao esforço, taquicardia basal, com aumento significante do tônus simpático cardíaco (34%), aumento do diâmetro dos cardiomiócitos (15%) e da fração de colágeno cardíaco (32%) quando comparados os animais controle. Além disso, a FS estava diminuída no KO vs. controle (16 ± 0,2 vs. 20 ± 0,9%, p<0,05). A diminuição na FS no grupo KO estava associada à redução na expressão de SERCA 2 (26%) e NCX (34%). Por outro lado, a expressão de fosfo-Ser16-PLB e fosfo-Ser2809-RYR estavam 56% e 42% aumentadas no grupo KO quando comparado ao grupo controle, respectivamente. O TF nos camundongos KO preveniu a intolerância ao esforço físico e a disfunção sistólica, normalizou a FC de repouso e o tônus simpático cardíaco, mas não alterou a fração de colágeno cardíaco. A melhora na função ventricular estava associada à restauração da expressão de SERCA2 e fosfo-Ser2809-RYR. Além disso, o TF aumentou a razão SERCA2/PLB no KO e a expressão de fosfo-Ser16-PLB no grupo KO com 5 meses de idade. Conclusão: Os dados apresentados no presente trabalho fornecem evidências de mecanismos moleculares que confirmam o benefício do treinamento físico como medida preventiva da insuficiência cardíaca. / Presynaptic a2-adrenoceptors (a2 AR) regulate the cardiovascular function by inhibiting neurotransmitter release on the sympathetic nerve terminals. We have recently reported that disruption for both a2A and a2C AR subtypes in mice (KO) leads to sympathetic hyperactivity with evidence of cardiac dysfunction by 4 mo of age. These mice provide a model system for evaluating non-pharmacological and pharmacological approaches for the prevention and treatment of heart failure. We investigated whether exercise training would improve the cardiac function and expression of myocardial Ca2+ handling proteins in KO. Methods and Results: We studied a cohort of congenic KO and their wild type (WT) controls over a period of 2 months (from 3-5 mo of age). Mice from both groups were randomly assigned into sedentary and trained. Exercise training (ET) consisted of 8-wk swimming session of 60 minutes, 5 days/wk. Blood pressure and heart rate (HR) were determined non-invasively by tail cuff. Cardiac contractility was evaluated by echocardiography. Exercise capacity was measured using a graded treadmill protocol. Cardiac sympathetic tone was estimated by pharmacological blockade of muscarinic receptors with atropine in the presence or absence of the b-adrenergic receptor antagonist propranolol. Cardiomyocyte cross-sectional diameter and cardiac collagen fraction were evaluated by optical microscopy. The protein expression of cardiac sarcoplasmic reticulum Ca2+ pump (SERCA2), phospholamban (PLB), phospho-Ser16-PLB, phospho-Thr17-PLB, sodium-calcium exchanger (NCX), ryanodine receptor (RYR), phospho-Ser2809-RYR2, and phosphatases 1 and 2A were analyzed using Western blotting technique. At 3 mo of age, no significant difference in blood pressure, FS and exercise capacity was observed between WT and KO mice, although KO mice had significantly higher HR. At 5 mo of age, when cardiac dysfunction is in an early stage, KO presented exercise intolerance and higher HR with significantly increased cardiac sympathetic tone (34%), cardiomyocyte cross-sectional diameter (15%) and cardiac collagen fraction (32%) when compared with WT mice. In addition, KO presented lower FS than WT mice (16± 0.2 vs. 20± 0.9%, p£0.05). The impaired FS in KO was associated with a reduction of SERCA2 (26%) and NCX (34%) expression. Conversely, phospho-Ser16-PLB and phospho-Ser2809-RYR2 was 56% and 42% increased in KO when compared with WT mice, respectively. ET prevented exercise intolerance and systolic dysfunction, normalized baseline HR and cardiac sympathetic tone, while it did not change cardiac collagen fraction. The improved ventricular function was associated with restored SERCA2 and phospho-Ser2809-RYR2 expression levels. Indeed, ET increased SERCA/PLB ratio and phospho-Ser16-PLB expression in 5 mo-old KO mice. Conclusion: Our data provide evidence of molecular mechanisms by which ET is a successful adjuvant to pharmacological therapy of HF.