Músculos esqueléticos de ratos idosos apresentam uma reduzida capacidade de se regenerar após lesão. No presente estudo, nós lançamos a hipótese de que a estimulação farmacológica de adrenoceptores b2 em músculos de ratos idosos lesados poderia melhorar a regeneração destes. Ratos jovens e idosos foram tratados com injeção subcutânea do agonista b2-adrenérgico formoterol (2 mg/kg/dia) durante 10 e 21 dias após lesão do músculo sóleo. Os músculos de ratos idosos lesados e tratados com formoterol por 10 e 21 dias apresentaram menor processo inflamatório e fibras musculares em regeneração com maior calibre quando comparados aos músculos apenas lesados. O tratamento com formoterol preveniu a queda da força tetânica e aumentou a síntese de proteínas e a fosforilação de mTOR em músculos de ratos idosos lesados e avaliados após 10 dias. Nossos resultados sugerem que o formoterol melhora a capacidade regenerativa estrutural e funcional dos músculos esqueléticos de ratos idosos, e que esse efeito é mediado pelo aumento da síntese protéica através da ativação de mTOR. / Skeletal muscles from old rats fail to completely regenerate following injury. In the present work, we hypothesized that pharmacological stimulation of b2-adrenoceptors in aged muscles following injury could improve their regenerative capacity. Young and aged rats were treated with a subcutaneous injection of b2-adrenergic agonist formoterol (2 mg/kg/day) up to 10 and 21 days after soleus muscle injury. Formoterol-treated muscles from old rats evaluated at 10 and 21 days post-injury showed reduced inflammation and regenerating myofibers of greater caliber when compared to their injured controls. Formoterol minimized the decrease in tetanic force and increased protein synthesis and mTOR phosphorylation in old muscles at 10 days post-injury. Our results suggest that formoterol improves structural and functional regenerative capacity of regenerating skeletal muscles from aged rats by increasing protein synthesis via mTOR activation.

O controle neural da pressão arterial é essencial para a manutenção da homeostase do organismo. Este controle é realizado principalmente por núcleos bulbares e hipotalâmicos. Um dos principais sistemas de neurotransmissão envolvidos no controle da pressão arterial é o catecolaminérgico. Células noradrenérgicas e adrenérgicas estão presentes em todos os centros bulbares reguladores da pressão arterial, enquanto seus receptores, principalmente o receptor α2 adrenérgico (α2r), estão presentes nas mesmas regiões além de estarem presentes também nos núcleos hipotalâmicos. Estes receptores, quando ativados nestes núcleos, geram respostas cardiovasculares e atuam em conjunto com outros sistemas de neurotransmissão neste controle. Dentre estes sistemas de neurotransmissão, merecem destaque os sistemas angiotensinérgico e glutamatérgico, não apenas por estarem presentes nestes núcleos, mas também por atuarem no controle da pressão arterial. Contudo, pouco se sabe sobre como o sistema catecolaminérgico interage com estes sistemas. Desta forma, estudamos neste projeto a influência do sistema glutamatérgico e angiotensinérgico sobre o sistema catecolaminérgico no bulbo de ratos. Através de culturas de células bulbares, demonstramos que a ativação de receptores glutamatérgicos do tipo NMDA é capaz de elevar os níveis proteicos do α2R e que os receptores ionotrópicos do tipo não-NMDA precisam estar desbloqueados para tal. Em ratos adultos, microinjeções repetidas inibem a resposta bradicárdica induzida pela ativação dos α2rR no NTS. Contudo, o knockdown dos receptores AT1 de angiotensina restaura a resposta bradicárdica. A partir destes resultados, foi demonstrado que o sistema glutamatérgico é capaz de modular o sistema catecolaminérgico, enquanto o knockdown do receptor AT1 de angiotensina no NTS acentua a resposta bradicárdica dos α2R do NTS. Estes resultados sugerem que os sistemas de neurotransmissão bulbares interagem de diferentes formas e a compreensão deste controle pode vir a ser de grande valia para a compreensão de como se dá o controle da pressão arterial pelo sistema nervoso / The neural control of blood pressure is essential for the maintenance of the homeostasis of the organism. This control is performed mainly by nuclei in the medulla oblongata and in the hypothalamus. One of the main neurotransmitter system involved in this control is the catecholaminergic. Noradrenergic and adrenergic cells are present in all medullary nuclei involved in the arterial pressure regulation, while its receptors, especially the α2 adrenoceptor, are present in the same region plus hypothalamic nuclei. These receptors, upon activation in these nuclei, generate cardiovascular response, and act with other neurotransmission systems in this control. Among these systems, the glutamatergic and angiotensinergic deserve attention not only for also being present in the same nuclei, but for also acting in the control of the arterial pressure. Both angiotensinergic and glutamatergic systems interact with the catecholaminergic system throughout the nervous system. However, little is known about how the catecholaminergic system interacts with these systems in the modulation of blood pressure. Therefore, we studied in this project the influence of the glutamatergic and angiotensinergic systems over the catecholaminergic system in the medulla oblongata of rats. Through cell cultures of the medulla oblongata, we demonstrated that the activation of glutamatergic NMDA receptors is capable of elevating the proteic levels of α2-adrenoceptors, and that non-NMDA receptors need to be unblocked for such. In adult rats, repeated microinjections inhibit the bradycardic response elicited by the α2 adrenoceptors in the NTS. However, the angiotensin AT1 receptors knockdown restored the bradycardic response. Through the chronic knockout of angiotensinergic AT1 receptors in the NTS, we observed bradycardic response elicited by the activation of α2 adrenoceptors in the NTS of the knock-down rats. These results suggest that the medullary neurotransmission systems interact in different ways, and the comprehension of this control may be of great value for the comprehension of how the neural control of the blood pressure works

Orientador: Rosana Almada Bassani / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T18:24:30Z (GMT). No. of bitstreams: 1 Oliveira_ElizangelaSouto_M.pdf: 5397962 bytes, checksum: a6952b7daf58ed6c84d6fb7eecc0d06f (MD5) Previous issue date: 2015 / Resumo: Importantes ajustes cardiovasculares dependentes de estimulação adrenérgica do coração ocorrem no período perinatal. O objetivo deste trabalho foi estudar a resposta cronotrópica a catecolaminas em ratos imaturos, com foco em mecanismos intrínsecos e extrínsecos à via de transdução ?-adrenérgica. Foi determinada a resposta cronotrópica a tiramina (TIR), isoproterenol (ISO), noradrenalina (NA), forskolin (FSK) e 3-isobutil-1-metilxantina (IBMX) em átrios direitos de ratos de 0-23 dias de idade e adultos. No mesmo tecido, foram quantificados os níveis de mRNA de diversas proteínas pela técnica de reação em cadeia de polimerase em tempo real (qRT-PCR). Os principais resultados foram: a) a resposta cronotrópica máxima (Rmax) a TIR e NA exógena mostrou-se deprimida em átrios de ratos de 0-2 dias, enquanto a sensibilidade aos agonistas reduziu-se com o amadurecimento; b) o bloqueio do transportador neuronal de catecolaminas por desipramina foi menos efetivo em causar desvio à esquerda na curva concentração-efeito nos átrios de animais imaturos, e aboliu as diferenças de sensibilidade (mas não de Rmax) à NA; c) a inibição do transportador extraneuronal de monoaminas não modificou o pD2 do ISO em átrios de animais imaturos, os níveis de mRNA para este transportador foram ~30% daqueles em adultos; d) apesar da redução da Rmax à NA em neonatos, não houve diferença na Rmax a ISO, FSK e IBMX entre as diferentes idades; e) o bloqueio de adrenoceptores ?2 com ICI118,551 reduziu Rmax e pD2 do ISO apenas nos átrios de ratos imaturos, indicando contribuição deste subtipo de receptores apenas nos primeiros dias após o nascimento; f) a expressão de adrenoceptores ?2, Gi, Gs e adenilato ciclase (isoforma 1) foi maior em átrios de animais imaturos, enquanto aquela de adrenoceptores ?1 foi semelhante em todos os grupos etários. Conclui-se que os componentes pós-receptor desta via de sinalização já estão presentes no neonato, embora tenha sido detectada aparente imaturidade no acoplamento de adrenoceptores ?1. Entretanto, foram identificados mecanismos compensatórios, tais como a participação de adrenoceptores ?2 e menor remoção neuronal e extraneuronal de catecolaminas. Palavras-chave: átrio direito, receptores adrenérgicos beta, catecolaminas, desenvolvimento pós-natal / Abstract: Important cardiovascular adjustments that depend on the adrenergic regulation of the cardiac function occur during the perinatal period. The goal of this work was to study the chronotropic responsiveness to catecholamines in immature rats, focusing on mechanisms intrinsic and extrinsic to the ?-adrenergic signaling pathway. The chronotropic response to tyramine (TYR), isoproterenol (ISO), norepinephrine (NE), forskolin (FSK), and 3-isobutyl-1-methylxanthine (IBMX) was determined in right atria isolated from 0-23 day-old and adults rats. In the same tissue, mRNA levels of relevant proteins were quantified by real-time polymerase chain reaction (qRT-PCR). The main results were: a) the maximum chronotropic response (Rmax) to TYR and to exogenous NE were lower in rigth atria from 0-2day-old rats, while the sensitivity to the agonists decreased during development; b) inhibition of neuronal norepinephrine transporter by desipramine was less effective at shifting the concentration-effect curve to the left in immature animals, and abolished the differences in sensitivity (but not in Rmax) to NE. c) inhibition of the extraneuronal monoamine transporter did not changed the ISO pD2 in atria from immature animals, in which the mRNA levels for this transporter were ~ 30% of those in adults; d) despite the lower Rmax to NE in neonates, the Rmax to ISO, FSK and IBMX was comparable among ages; e) ?2-adrenoceptor blockade with ICI118,551 reduced Rmax and ISO pD2 only in atria from immature rats, indicating contribution of this receptor subtype only in the first days after birth; f) ?2-adrenoceptor, Gi, Gs and adenylate cyclase (isoform 1) transcript levels were greater in the atria of immature animals, while that of ?1-adrenoceptor was similar in all age groups. In conclusion, post-receptor components of this signaling pathway seem to already be mature in newborns, although an apparent immaturity in ?1-adrenoceptor coupling was detected. However, compensatory mechanisms were identified, such as the participation of ?2-adrenoceptor and weaker neuronal and extraneuronal catecholamine removal. Keywords: right atria, ?-adrenoceptor, catecholamines, postnatal development / Mestrado / Farmacologia / Mestra em Farmacologia

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.

Dans le cœur, l’activation aiguë de la voie AMPc/PKA via la stimulation des récepteurs β-adrénergiques (β-ARs) permet de réguler la contraction cardiaque alors que l’activation chronique de cette voie est délétère, car elle est source de survenue d’arythmies cardiaques et de remodelage hypertrophique du cœur. Au niveau des cardiomyocytes, Il existe principalement deux sous-types de récepteurs β-ARs ; β1- et β2-ARs, qui exercent des effets différents sur la fonction cardiaque.Dans une première partie de ma thèse, je me suis intéressé à l’étude du rôle des récepteurs β1- et β2-ARs dans la régulation différentielle de l’activité PKA cytoplasmique et nucléaire. J’ai ainsi pu montrer que contrairement aux récepteurs β1-ARs qui ont la capacité d’activer la PKA au niveau du cytoplasme et aux noyaux, les récepteurs β2-ARs activent la PKA uniquement au niveau du cytoplasme, et ce indépendamment de la capacité des récepteurs β2-ARs à induire une augmentation des niveaux d’AMPc dans les noyaux. En accord avec ces résultats, les récepteurs β1- mais pas β2-ARs activent le facteur pro-apoptotique régulé par la PKA, ICER.Dans une seconde partie de ma thèse, je me suis intéressé aux différents mécanismes responsables de l’incapacité des récepteurs β2-ARs à activer la PKA au niveau des noyaux. Mes résultats soulignent le rôle de la localisation des récepteurs β2-ARs au niveau des cavéoles, leurs couplage aux protéines Gi, leurs désensibilisation par la GRK2 ainsi que la dégradation de l’AMPc généré par ces récepteurs par la PDE3 et 4 dans la régulation de la signalisation PKA cytoplasmique et pointent vers la PDE4 comme un régulateur central permettant de limiter l’activation de la PKA holoenzyme responsable des réponses PKA nucléaires. Mes résultats montrent également que la mAKAP est un élément clé dans la transduction de la signalisation PKA nucléaire induite par les récepteurs β2-ARs et à un moindre degré, les récepteurs β1-ARs. Dans la dernière partie de ma thèse, j’ai étudié le remodelage de la signalisation PKA nucléaire induite par les récepteurs β1- et β2-ARs au cours de l’insuffisance cardiaque. J’ai ainsi pu montrer qu’en plus de la diminution de la signalisation PKA nucléaire induite par les récepteurs β1-ARs, il existe une signalisation PKA nucléaire de novo induite par les récepteurs β2-ARs dans les cardiomyocytes de rat adulte insuffisants.En conclusion, ce travail a mis à jour une nouvelle différence entre les récepteurs β1- et β2-ARs dans la signalisation PKA au niveau des noyaux des cardiomyocytes de rat adultes, et souligne le rôle important de la PDE4 et de la mAKAP dans la régulation de la signalisation PKA nucléaire induite par les récepteurs β2-ARs. / In the heart, acute activation of the cAMP/PKA pathway upon stimulation of β-adrenoceptors (β-ARs), plays a fundamental role in the regulation of cardiac function, whereas chronic activation of this pathway is deleterious, as it is responsible for cardiac arrhythmias and hypertrophic remodeling of the heart. In cardiac myocytes, there are mainly two subtypes of β-ARs: β1- and β2-ARs, which exert different effects on cardiac function.In the first part of my thesis, my work was focused on understanding the role of β1- and β2-ARs in the differential regulation of cytoplasmic and nuclear PKA activity. Hence, I have showed that unlike β1-ARs which have the capacity to induce the activation of PKA in the cytoplasm and the nucleus, β2-ARs induce the activation of PKA only in the cytoplasmic compartment, regardless of their ability to induce an increase in cAMP in the nuclei. Consistently, β1- but not β2-ARs were able to induce the activation of the pro-apoptotic factor regulated by PKA, ICER.The second aim of my thesis was to decipher the different mechanisms involved in the inability of β2-ARs to activate PKA in the nucleus. I concentrated my efforts on investigating the role of the localization of β2-ARs in caveolae, their coupling to Gi proteins, their desensitization by GRK2 as well as the hydrolysis of cAMP by PDE3 and 4 in the regulation of β2-AR-induced cytoplasmic PKA activity. My results point to PDE4 as a central regulator which limits the activation of the PKA holoenzyme pool involved in the nuclear PKA responses. My results also show that mAKAP is a key component of nuclear PKA signaling induced by β2-ARs and to a lesser extent by β1-ARs. In the last part of my thesis, I have studied the remodeling of nuclear PKA signaling induced by β1- and β2-ARs that occurs during heart failure. I showed that, besides a decrease in β1-AR-induced nuclear PKA signaling, there is a de novo β2-AR-induced nuclear PKA signaling in cardiomyocytes from rat with heart failure.In conclusion, this work uncovers a new difference in PKA signaling between β1- and β2-ARs at the nuclear compartment of adult rat cardiomyocytes and underlines the importance of PDE4 and mAKAP in the regulation of β2-AR-induced nuclear PKA signaling.

In der vorliegenden Arbeit wurden die Effekte einer frühzeitigen Zelltherapie im Langzeit in-vivo Infarktmodell studiert. Erstmals wurden dabei auch Veränderungen der kardialen -Adrenozeptoren untersucht und Zelltherapie mit einer reversiblen präinfarziösen Ischämie kombiniert. Initial wurden dafür bei 38 männlichen weißen Neuseeländer Kaninchen Knochenmarkspunktionen durchgeführt, MSC durch Kultur isoliert und 60 Minuten nach induziertem Infarkt und ohne Reperfusion in den Randbereich des Infarktgebietes injiziert. Zur Untersuchung möglicher Interaktionen zwischen Zelltherapie und Präinfarktgeschehen wurde bei einigen Tieren das Myokard durch eine kurzzeitige Präinfarktischämie präkonditioniert. Die Ergebnisse der vorliegenden Arbeit zeigen, dass auch die frühzeitige Zellinjektion ohne Reperfusion mit signifikanten Effekten auf die Kontraktilität und spezifischen sympathoadrenergen Veränderungen verbunden ist.:Abkürzungsverzeichnis vii Literaturübersicht 1 Myokardinfarkt und postischämische Herzinsuffizienz 1 sympathoadrenerge Veränderungen bei Myokardinfarkt und Herzinsuffizienz 1 akuter Myokardinfarkt 1 Herzinsuffizienz 2 Veränderungen der -Adrenozeptoren bei Herzinsuffizienz 3 Stammzellen und Zelltherapien 4 Zelltherapien bei Myokardinfarkt 5 Knochenmark 5 hämatopoetische Stammzellen 5 mesenchymale Stammzellen 6 endotheliale Progenitorzellen 8 klinische Studien 8 Effekte adulter Stammzellen und anderer Zelltypen 10 Angiogenese 11 Zytoprotektion, Apoptosehemmung und antiinflammatorische Effekte 12 Unterstützung endogener Reperaturmechanismen 12 Stabilisierung der extrazellulären Matrix 13 Optimierung der Stammzelltherapien 13 Injektionszeitpunkt 14 Stammzellmigration 15 Ziel der Arbeit, Thesen und Fragestellung 17 Methoden 18 Versuchsaufbau 18 Tierhaltung 19 Tiermodell und Versuchsprotokoll 19 Knochenmarksgewinnung 19 Zellaufbereitung 19 Induktion des Myokardinfarktes und Zellapplikation 20 Sakrifizierung und Probenentnahme 21 Auswertung 22 transthorakale Echokardiografie 23 Durchführung 23 Radioligandenbindungsstudien 25 Rezeptortheorie 25 -Adrenozeptoren 25 Grundlagen der Radioligandenbindungsstudien 27 Auswertung der Radioligandenbindungsstudien 28 -Adrenozeptorbestimmung durch Radioligandenbindungsstudien 30 Durchführung 30 Radioligand 31 Probenvorbereitung 31 Proteinbestimmung 32 Bindungsstudien 32 Auswertung 34 Noradrenalinbestimmung durch HPLC 36 Flüssigkeitschromatografie und HPLC 36 Prinzipien der Auftrennung 36 Aufbau eines HPLC Systems 37 chromatografische Kenngrössen 39 Probenvorbereitung 42 Durchführung 42 Katecholaminextraktion 43 Chromatografie 43 Auswertung 44 Immunhistochemie 45 c-Kit 45 Durchführung 46 Gewebeproben 46 Entparaffinierung und Gewebevorbehandlung 46 immunhistochemische Färbung 47 Auswertung der histologischen Schnitte 47 statistische Auswertung 48 Ergebnisse 49 allgemeine Daten 49 Echokardiografie 50 linksventrikuläre Funktion 50 Infarktausdehnung und Wandstärke im Infarktbereich 54 sympathoadrenerge Veränderungen 55 Radioligandenbindungsstudien 55 -Adrenozeptordichte im LV, S und RV 57 Noradrenalin Plasmakonzentration 64 c-Kit positive Zellen im Infarktbereich und infarktfernen Myokard 66 Diskussion 68 Ziel und Fragestellung 68 Tiermodell 68 Echokardiografie 70 Radioligandenbindungsstudien 73 Veränderungen der -Adrenozeptoren nach Myokardinfarkt 74 Therapiebedingte Veränderungen 76 plasmatisches Noradrenalin 79 Immunhistochemie 82 Zusammenfassung 84 Literaturverzeichnis I Anhang a Abbildungsverzeichnis a Tabellenverzeichnis b Tabellen c Materialien und Geräte f Tierversuche und Laborgeräte f Apperaturen und Geräte zur Durchführung der RLBS f HPLC System f statistische Auswertung und grafische Darstellung g Verbauchsmaterialien g allgemeine Laborchemikalien g Verbrauchsmateralien zur Durchführung der HPLC h Verbrauchsmateralien zur Durchführung der RLBS h Verbrauchsmateralien zur Durchführung der Immunhistochemie h Erklärung über die eigenständige Abfassung der Arbeit i Danksagung j Lebenslauf k Publikationsverzeichnis l

The expression of the α1C-adrenoceptor subtype in human and rabbit blood vessels has been analyzed using the reverse transcriptase/polymerase chain reaction technique (RT/PCR). The 20 bp primers employed were designed from the bovine α1C-adrenoceptor and flank a least conserved region - the putative third cytoplasmic loop. RT/PCR products generated from rabbit and human brain mRNA both had 93% homology to the bovine α1C-adrenoceptor and were used as species and subtype specific probes in Southern blot analysis of vascular RT/PCR products. Poly A+ RNA was purified from the human saphenous vein and rabbit aorta, renal, pulmonary and central ear arteries and amplified by RT/PCR. Size analysis by agarose gel electrophoresis, together with Southern hybridization of the resulting cDNA products confirm the expression of the α1C-adrenoceptor in these vessels.

The circulatory system is one of the first systems that develops during embryogenesis. Angiogenesis describes the formation of blood vessels as a part of the circulatory system and is essential for organ growth in embryogenesis as well as repair in adulthood. A dysregulation of vessel growth contributes to the pathogenesis of many disorders. Thus, an imbalance between pro- and antiangiogenic factors could be observed in infantile hemangioma (IH). IH is the most common benign tumor during infancy, which appears during the first month of life. These vascular tumors are characterized by rapid proliferation and subsequently slower involution. Most IHs regress spontaneously, but in some cases they cause disfigurement and systemic complications, which requires immediate treatment. Recently, a therapeutic effect of propranolol on IH has been demonstrated. Hence, this non-selective β-blocker became the first-line therapy for IH. Over the last years, our understanding of the underlying mechanisms of IH has been improved and possible mechanisms of action of propranolol in IH have postulated. Previous studies revealed that gap junction proteins, the connexins (Cx), might also play a role in the pathogenesis of IH. Therefore, affecting gap junctional intercellular communication is suggested as a novel therapeutic target of propranolol in IH. In this review we summarize the current knowledge of the molecular processes, leading to IH and provide new insights of how Cxs might be involved in the development of these vascular tumors

O Controle de qualidade de proteína (CQP) consiste na supervisão e no processamento de proteínas danificadas por meio de processos catalíticos (proteassoma e autofagia). Nesse estudo, caracterizamos o CQP, bem como os benefícios da ativação β2-adrenérgica (β2-AR) modulador positivo do CQP, em modelo animal de disfunção/atrofia muscular induzida por constrição crônica do nervo isquiático (CCI). Observamos que, apesar de um aumento na atividade catalítica, a atrofia está associada à um CQP insuficiente, detectado por um acúmulo de proteínas citotóxicas nessa musculatura. O tratamento com Formoterol (agonista β2-AR) aumentou a atividade proteassomal e restaurou o fluxo de degradação via autofagia, resultando na melhora do CQP e da miopatia esquelética. A inibição da autofagia, mas não do proteassoma, foi capaz de abolir os efeitos do Formoterol na CCI. Nossos resultados sugerem uma nova contribuição da sinalização β2-AR no quadro de miopatia esquelética, no qual sua ativação foi capaz de restaurar o CQP, contribuindo para a melhora do trofismo e função muscular. / The protein quality control (PQC) detects, repairs and disposes cytotoxic proteins through different proteolytic systems (proteasome and autophagy). Here, we characterized the PQC profile as well as the benefits of sustained β2-adrenoceptor activation (β2-AR) a positive PQC modulator, during skeletal muscle atrophy in a rat model of sciatic nerve constriction (SNC). PQC is disrupted in SNC rats, demonstrated by elevated proteasomal and lysosomal activities along with accumulation of cytotoxic proteins and pro-apoptotic factors. The β2-AR activation (Formoterol) promotes a further increase in proteasomal activity, along with autophagic flux reestablishment. Of interest, sustained autophagy inhibition, but not proteasomal inhibition, is sufficient to abolish Formoterol effects on skeletal muscle PQC, mass and strength. These findings suggest a new contribution of β2-AR signalling pathway to the pathophysiology of skeletal muscle where β2-AR restores the impaired PQC, therefore contributing to a better skeletal muscle morphology and function.

Dados da literatura mostram que a inervação simpática possui papel apenas na síntese e secreção das proteínas da saliva. Neste trabalho mostramos que após o tratamento crônico com reserpina, uma droga simpatolítica, houve alteração na síntese de diferentes proteínas em glândulas submandibulares e parótidas relacionadas com vários processos biológicos e a posterior administração de agonistas de adrenoceptores reverteu esses efeitos. Além disso, na glândula parótida, a estimulação da inervação simpática é importante para o processo de exocitose das proteínas secretadas. Esses resultados sugerem que a inervação simpática possui um importante papel para a funcionalidade das glândulas salivares de camundongos, isto é, mantendo estas glândulas em constante estado de ativação, regulando a síntese de diferentes proteínas dessas glândulas ou promovendo a exocitose de proteínas da saliva. Além disso, os resultados mostram que a inervação simpática atua de maneira diferente nas glândulas submandibulares e parótidas de camundongos. / Data in literature show that sympathetic outflow has a role only in stimulating synthesis and secretion of the saliva proteins in mammals. We show that after chronic treatment with reserpine, a sympatholytic drug, there were changes in the synthesis of different proteins in submandibular and parotid glands associated with various biological processes and subsequent administration of adrenoceptor agonists reversed these effects. Furthermore, in the parotid gland, stimulation of the sympathetic outflow is important to the process of exocytosis of secretory proteins. These results suggest that sympathetic outflow plays an important role for the functionality of the mouse salivary glands, namely, keeping these glands in a constant activated stage, regulating the synthesis of different proteins in these glands or promoting the exocytosis of saliva proteins. Furthermore, these results show that the sympathetic outflow acts differently in the mouse.