Os efeitos benéficos do exercício físico aeróbico (EFA) frente à prevenção e ao tratamento de doenças ocorrem por meio de adaptações agudas e crônicas em diversos tecidos e sistemas orgânicos, dentre os quais o tecido muscular esquelético assume papel de destaque. Considerando que a musculatura esquelética é rica em mitocôndrias, não surpreende o fato de que o aumento da capacidade e potência aeróbicas induzido pelo EFA ocorra principalmente em função das adaptações mitocondriais. Contudo, os mecanismos moleculares envolvidos nessas adaptações mitocondriais induzidas pelo EFA não são completamente compreendidos. Considerando-se que a atividade nervosa simpática aumenta consideravelmente durante a realização de uma sessão de EFA, parte das respostas mitocondriais ao EFA poderia ser mediada pela ativação dos receptores \'beta\'-adrenérgicos (\'beta\'-AR) na musculatura esquelética, representados predominantemente pelo subtipo \'beta\'2 (~ 90%). Com isso, na primeira parte do presente estudo, testou-se a hipótese de que a sinalização \'beta\'2-AR contribuiria para as respostas de função e dinâmica mitocondriais induzidas pela ativação adrenérgica aguda na musculatura esquelética. Para isso, o agonista \'beta\'-AR não seletivo isoproterenol foi administrado na presença ou ausência do antagonista \'beta\'2-AR específico, ICI 118,551, tanto em miotúbulos diferenciados a partir da linhagem de células C2C12 como em camundongos da cepa FVB. Os dados demonstraram que a ativação \'beta\'-AR aumentou significativamente a respiração mitocondrial em miotúbulos e em fibras musculares isoladas do músculo tibial anterior de camundongos, e que tal resposta foi dependente da ativação dos receptores \'beta\'2-AR via eixo proteína Gαs-AMPc-PKA. Em relação à dinâmica mitocondrial, os dados obtidos demonstraram que a ativação dos receptores \'beta\'2-AR aumentou a fusão mitocondrial na musculatura esquelética, e que esta resposta estava associada a um aumento da proteína de fusão Mfn1. Na segunda parte do presente estudo, testou-se a hipótese de que a ativação \'beta\'2-AR contribuiria para as respostas mitocondriais agudas induzidas pelo EFA na musculatura esquelética. Para isso, camundongos FVB foram submetidos a uma sessão de EFA em esteira rolante com e sem o bloqueio dos receptores \'beta\'2-AR com o antagonista ICI 118,551, e parâmetros de função e dinâmica mitocondriais foram avaliados. O EFA foi capaz de aumentar a respiração máxima mitocondrial em fibras musculares isoladas, e este efeito foi parcialmente prevenido pelo bloqueio dos receptores \'beta\'2-AR. Além disso, assim como observado com o tratamento com isoproterenol, o EFA promoveu aumento da fusão mitocondrial na musculatura esquelética, sendo que a ativação \'beta\'2-AR estava associada a essa resposta, porém em uma menor magnitude em relação aos efeitos observados com o estímulo farmacológico. Assim como observado no tratamento com isoproterenol, a expressão de Mfn1 foi aumentada pela sessão de EFA, a qual não foi atenuada pelo bloqueio dos receptores \'beta\'2-AR com ICI 118,551. Dessa forma, a partir dos resultados obtidos no presente estudo, pode-se concluir que os receptores \'beta\'2-AR têm papel importante no metabolismo muscular esquelético, modulando a função e a dinâmica mitocondriais. Essa resposta é evidente mediante ativação direta via agonistas \'beta\'-AR no músculo esquelético, no entanto, os receptores \'beta\'2-AR modulam parcialmente as respostas mitocondriais em resposta à uma sessão de EFA / The beneficial effects of aerobic exercise (AE) for prevention and treatment of diseases are due to acute and chronic adaptations in several organ systems, including skeletal muscle, one of the main tissues involved in these adaptations. Since skeletal muscle contains high amounts of mitochondria, the increased aerobic capacity induced by AE occurs mainly due to mitochondrial adaptations. However, the molecular mechanisms underlying skeletal muscle mitochondrial adaptations induced by AE are not fully understood. One of the mechanisms by which these mitochondrial adaptations may occur is sympathetic activation mediated by \'beta\'2-adrenergic receptors (\'beta\'-AR) in skeletal muscle, predominantly represented by \'beta\'2 subtype (~90%). Considering that sympathetic activity increases during AE, \'beta\'2-AR signaling might be involved in skeletal muscle mitochondrial adaptations in response to AE. Thus, in the first part of this study, we tested the hypothesis that acute \'beta\'2-AR activation contributes to mitochondrial function and dynamics adaptations in skeletal muscle. For this, both differentiated C2C12 myotubes and FVB mice were treated with the nonselective \'beta\'-AR agonist isoproterenol in the presence or absence of the specific \'beta\'2-AR antagonist, ICI 118,551. The data showed that β-AR activation significantly increased mitochondrial respiration in myotubes and muscle fiber bundles isolated from mice tibialis anterior, and that this response was dependent on \'beta\'2-AR receptors activation via protein G\' alpha\'s-cAMP-PKA signaling cascade. Regarding mitochondrial dynamics, the data showed that \'beta\'2-AR receptor activation increased mitochondrial fusion in skeletal muscle and that this response was associated to an increase in Mfn1. In the second part of this study, we tested the hypothesis that \'beta\'2-AR activation would contribute to acute mitochondrial responses induced by AE in skeletal muscle. For this, FVB mice underwent one session of AE on a treadmill with and without \'beta\'2-AR receptor blockade with ICI 118,551, and parameters of mitochondrial function and dynamics were evaluated. AE was able to increase maximal mitochondrial respiration in isolated muscle fibers, and this effect was partially prevented by blocking \'beta\'2-AR receptors. Furthermore, as observed with isoproterenol treatment, AE increased mitochondrial fusion in skeletal muscle. \'beta\'2-AR activation was associated with this response, but in a smaller magnitude compared to the effects observed with pharmacological stimulation. As observed with the isoproterenol treatment, Mfn1 expression was increased by AE, which was not attenuated by blocking \'beta\'2-AR receptors with ICI 118,551. Therefore, one can conclude that \'beta\'2-AR receptors play an important role in skeletal muscle metabolism, modulating mitochondrial function and dynamics. This response is evident by direct activation through \'beta\'-AR agonists in skeletal muscle, however, \'beta\'2-AR receptors partially modulate mitochondrial responses induced by AE

(in English): The aim of the work was to perform in-depth analysis of M2KO mice both at baseline and upon a challenge with a cold stress and to explore the role of opposing receptors (i.e. adrenoceptors) in adaptation to lacking M2-receptors in the heart. We have performed receptor binding studies, study of receptor gene expression, echocardiography, telemetric monitoring of heart rate, body temperature and activity, heart rate variability and biorhythm analysis, analysis of heart rate response to the application of drugs (carbachol, atropine, isoprenaline, propranolol), assessment of adenylyl cyclase and NO synthase activity, measurement of catecholamine blood concentration and gene expression of catecholamine-synthesizing enzymes. We have found that the disruption of M2-receptor gene caused a compensatory decrease of cardiostimulatory β1-adrenoceptors and β2-adrenoceptors with corresponding down-regulation of the gene expression, M3-receptors were down-regulated as well. Missing M2-receptors were functionally replaced by the main cardioinhibitory β3-adrenoceptors that were up-regulated, not by cardioinhibitory M4-receptors. β3-adrenoceptors were found to signal through adenylyl cyclase instead of NO synthase. All these changes were found in the left ventricle only, so heterologous regulation is...

Summary: The effects of an alpha2 adrenoceptor blocker, yohimbine, and an alpha1 adrenoceptor blocker, phenoxybenzamine, and the central alpha2 adrenoceptor agonist, clonidine, on changes in arterial blood pressure and heart rate were studied during fatiguing muscular contractions to determine whether an adrenergic-opioidergic system might be involved in the mediation of cardiovascular function. Fatiguing contractions of the gastrocnemius and plantaris muscles of cats caused an increase in mean arterial blood pressure to 150-170 mmHg from resting values of 110-120 mmHg. Injection of clonidine into the cerebral aqueduct eliminated the increase in blood pressure; this effect was dose dependent. Naloxone antagonised the effects of the highest dose of clonidine (5 μg). Injections of yohimbine (1 μg) into the cerebral aqueduct had no significant effect on this pressor response. Yohimbine (1 μg) effectively counteracted the antipressor effects of clonidine when the two drugs were injected together until higher doses of clonidine (2-5 μg) were used. Phenoxybenzamine had no effect on the pressor response itself but unlike yohimbine was able to attenuate the effects of clonidine only when injected together. These data suggest that activation of muscle ergoreceptor afferent nerve fibres (group III and IV fibres) during muscular contractions may cause an increase in arterial blood pressure by interfering with an inhibitory adrenergic-endorphinergic pathway in the medullary region of the brainstem.

The role of protein kinase C (PKC) in mediating contractile responses in the rat vas deferens was studied. Phorbol-12,13-di-acetate (PDA) in the presence of 20 mM K+ elicited a concentration-dependent response with an EC50 of 190 nM. The non-PKC activator 4α-phorbol (2 μM) was unable to elicit contraction in 20 mM K+ buffer. Incubation of rat vas deferens with the PKC inhibitor iso-H7 (30 μM) attenuated the response to norepinephrine (NE) ane neurokinin A, with maximal effects depressed to 42 and 39% of control, respectively. Responses to 60 mM K+ and 2 μM PDA (20 mM K+) was also significantly inhibited by iso-H7. In the presence of 2 μM PDA and 20 mM K+, the NE concentration-effect curve was shifted 3,6-fold to the right of the control curve in a parallel manner. 4α-Phorbol (20 mM K+) at the same concentration did not produce this effect. These results suggest a significant role for PKC in the contractile response of the rat vas deferens.

Neste trabalho, mostramos que a extração de veneno promove aumento na ativação dos fatores de transcrição NF<font face=\"symbol\">kB e AP-1. Noradrenalina, liberada após extração de veneno, é responsável por esse aumento. A estimulação dos adrenoceptores <font face=\"symbol\">a e <font face=\"symbol\">b estão envolvidos nessa resposta, entretanto, o grau de ativação de AP-1 é alterado dependendo do tempo. Foi verificado dimorfismo sexual na ativação dos fatores de transcrição da glândula de veneno, sendo a ativação de NF<font face=\"symbol\">kB mais rápida nas fêmeas do que nos machos e a ativação de AP-1 maior nas fêmeas. Mostramos ainda que a extração de veneno ativa a glândula de veneno através da estimulação da inervação noradrenérgica. Noradrenalina atuando em adrenoceptores <font face=\"symbol\">a e <font face=\"symbol\">b regula a síntese de proteínas da glândula de veneno e não a síntese de toxinas do veneno. Em conclusão, a estimulação da inervação noradrenérgica desencadeia o ciclo de produção de veneno regulando a síntese de proteínas da glândula que provavelmente são essenciais para sua ativação e posterior produção de veneno, através da ativação de fatores de transcrição. Thesis: Noradrenergic Innervation in activation of venom gland of Bothrops jararaca snake. / In this work we showed that venom extraction increases the activation of transcription factors like NF<font face=\"symbol\">kB and AP-1. Noradrenaline released after venom extraction is responsible for NF<font face=\"symbol\">kB and AP-1 activation. Stimulation of both <font face=\"symbol\">a- and <font face=\"symbol\">b-adrenoceptors is involved in this response, however the increase of AP-1 activation is time dependent. A sexual dimorphism has been verified in NF<font face=\"symbol\">kB and AP-1 activation. Activation of NF<font face=\"symbol\">kB occurs earlier in female than in male snakes and activation of AP-1 is greater in female than in male snakes. We also showed that venom extraction activates venom gland by stimulating noradrenergic innervation. Noradrenaline acting at both <font face=\"symbol\">a- and <font face=\"symbol\">b-adrenoceptors regulates synthesis of protein of the venom gland, but not the toxin of the venom. In conclusion, stimulation of noradrenergic innervation triggers the venom production cycle by regulating synthesis of protein that probably is essential to activate venom gland to produce venom by activating transcription factors.

No description available.

O adrenoceptor <font face=\"symbol\">b está presente na glândula de veneno de Bothrops jararaca, tanto no estado quiescente como no ativado por 4 dias, entretanto, quando estimulado, aumenta os níveis de AMPc somente em células secretoras no estado quiescente. Neste trabalho, estudamos funcionalmente o adrenoceptor <font face=\"symbol\">b durante o ciclo de produção de veneno e verificamos a sua função. Mostramos que a estimulação do adrenoceptor <font face=\"symbol\">b promove aumento na taxa de acidificação do meio extracelular em células quiescentes e ativadas por 4 dias, mas não por 15 dias. Em células quiescentes, a estimulação do adrenoceptor <font face=\"symbol\">b também promove o influxo de cálcio, via canais operados por voltagem e por receptor e a liberação de cálcio de estoques intracelulares sensíveis a tapsigargina. Em células ativadas por 4 dias, o aumento de cálcio citosólico é significativamente menor do que em células quiescentes, confirmando a dessensibilização deste receptor. PKA não participa desta sinalização. Dados in vivo mostram que o adrenoceptor <font face=\"symbol\">b é importante para desencadear o ciclo de produção de veneno. / <font face=\"symbol\">b adrenoceptor is present in quiescent and 4 days activated secretory cells of venom gland of Bothrops jararaca snake, however its stimulation increase cAMP production only in quiescent cells. In this work, we functionally characterized <font face=\"symbol\">b adrenoceptor during the venom production cycle and verified its function in venom production. We showed that the stimulation of <font face=\"symbol\">b adrenoceptor increases the rate of extracellular acidification in quiescent and 4 days activated cells, but not in 15 days activated cells. In quiescent cells, <font face=\"symbol\">b adrenoceptor stimulation also triggers calcium influx through voltage-operated and receptor-operated calcium channels, and calcium release from tapsigargin-sensitive stores. In 4 days activated cells, <font face=\"symbol\">b adrenoceptor stimulation promotes smaller increases in citosolic calcium concentration than in quiescent cells, confirming that this adrenoceptor undergoes to dessensitization. PKA does not participate in calcium signaling. In vivo experiments showed that <font face=\"symbol\">b adrenoceptor is important to trigger the cycle of venom production.

Während der letzten Jahre zeigten einige Studien, dass die Behandlung mit Knochenmark- Stammzellen (KMSZ) eine vielversprechende neue Therapieoption für den geschädigten Herzmuskel darstellen könnte. In dieser Arbeit wurde untersucht, ob es unter Behandlung mit Stammzellen zu einer zellulären Antwort in angezüchteten Kardiomyozyten (KMZ) kommt. Dafür wurden subkonfluente Kulturen aus Herzmuskelzellen von neonatalen Ratten für drei Tage mit Vybrant CM-DiI-markierten, sternalen humanen Knochenmarkstammzellen co-kultiviert. Im Anschluss wurden immunohistochemische Färbungen sowie eine quantitative Analyse mittels Western Blot für das Protoonkogen c-Myc durchgeführt. Des Weiteren wurde die Dichte der Beta-Adrenozeptoren unter Anwendung einer Histoautoradiographie mittels [125I]- iodocyanopindolol-Bindung analysiert. Die Auswertung der Immunohistochemie und der Western Blots zeigte eine signifikante Erhöhung der Expression von c-Myc in den Kardiomyozyten, welche in naher Umgebung der Stammzellen lagen. Dieser Effekt war direkt abhängig von der Entfernung der KMZ zur SZ. Die Histoautoradiographie zeigte eine signifikant höhere Beta-Rezeptor-Dichte in Kardiomyozyten in direkter Nähe zur Stammzelle. Mit steigender Entfernung von der Stammzelle verringerte sich die Rezeptordichte. Somit konnte gezeigt werden, dass eine kleine Anzahl von Knochenmark-Stammzellen ausreicht, um eine große Zahl von Kardiomyozyten zu beeinflussen, indem eine intrazelluläre Signalkaskade über c-Myc aktiviert und die Anzahl der Beta-Adrenozeptoren erhöht wird.

The aim of this thesis was to clarify the influence of the stress on the adrenergic and muscarinic receptors in the heart and in the lungs. Research was perform on rat hearts and lungs and on the hearts and lungs of the CRH KO mice. First, we assessed mRNA levels of all α- and β-adrenergic receptor and muscarinic receptor subtypes. Subsequently, we performed the radioligand-binding studies to determine densities of these receptors. We identified all three α1-adrenergic receptor subtypes in the rat lungs. In the lungs of WT mice, we found that the amount of α1-adrenergic and muscarinic receptors was sex-dependent. Densities of the former were higher in females and those of the latter were higher in males. There was no difference between males and females in β-adrenergic receptor density. As for CRH KO mice, the basal densities of studied receptors were lower than in CRH WT mice (except β1-adrenergic receptors in females). The main purpose of the thesis was to detect the immobilization-induced changes in the studied receptors in the kontrol (WT) and CRH KO mice. Short-term and long-term immobilization caused decrease in all α1-adrenergic receptor subtypes in females, whereas only α1A-adrenergic receptors decreased in males. The amount of β1-adrenergic receptors decreased in males and remained without...

Neste trabalho, mostramos que a extração de veneno promove aumento na ativação dos fatores de transcrição NF<font face=\"symbol\">kB e AP-1. Noradrenalina, liberada após extração de veneno, é responsável por esse aumento. A estimulação dos adrenoceptores <font face=\"symbol\">a e <font face=\"symbol\">b estão envolvidos nessa resposta, entretanto, o grau de ativação de AP-1 é alterado dependendo do tempo. Foi verificado dimorfismo sexual na ativação dos fatores de transcrição da glândula de veneno, sendo a ativação de NF<font face=\"symbol\">kB mais rápida nas fêmeas do que nos machos e a ativação de AP-1 maior nas fêmeas. Mostramos ainda que a extração de veneno ativa a glândula de veneno através da estimulação da inervação noradrenérgica. Noradrenalina atuando em adrenoceptores <font face=\"symbol\">a e <font face=\"symbol\">b regula a síntese de proteínas da glândula de veneno e não a síntese de toxinas do veneno. Em conclusão, a estimulação da inervação noradrenérgica desencadeia o ciclo de produção de veneno regulando a síntese de proteínas da glândula que provavelmente são essenciais para sua ativação e posterior produção de veneno, através da ativação de fatores de transcrição. Thesis: Noradrenergic Innervation in activation of venom gland of Bothrops jararaca snake. / In this work we showed that venom extraction increases the activation of transcription factors like NF<font face=\"symbol\">kB and AP-1. Noradrenaline released after venom extraction is responsible for NF<font face=\"symbol\">kB and AP-1 activation. Stimulation of both <font face=\"symbol\">a- and <font face=\"symbol\">b-adrenoceptors is involved in this response, however the increase of AP-1 activation is time dependent. A sexual dimorphism has been verified in NF<font face=\"symbol\">kB and AP-1 activation. Activation of NF<font face=\"symbol\">kB occurs earlier in female than in male snakes and activation of AP-1 is greater in female than in male snakes. We also showed that venom extraction activates venom gland by stimulating noradrenergic innervation. Noradrenaline acting at both <font face=\"symbol\">a- and <font face=\"symbol\">b-adrenoceptors regulates synthesis of protein of the venom gland, but not the toxin of the venom. In conclusion, stimulation of noradrenergic innervation triggers the venom production cycle by regulating synthesis of protein that probably is essential to activate venom gland to produce venom by activating transcription factors.