The aim of this project was to study changes in cardiovascular responses brought about by long term oral treatment of Wistar rats with beta adrenoceptor antagonists. After chronic treatment with propranolol (12 or 60 mg/kg/day for up to 6 weeks) or timolol (1.2, 2.5, 5 or 25 mg/kg/day for up to 17 weeks), the log dose-response curves for mean rises in heart rate and mean arterial pressure on stimulation of the postsynaptic adrenoceptors of the pithed rat by I.V. noradrenaline or isoprenaline were not significantly changed. Chronic propranolol treatment significantly reduced the response of the heart to electrical stimulation of the whole sympathetic outflow but treatment with timolol failed to alter the cardiac chronotropic response. The rises in mean arterial pressure on stimulation of the whole sympathetic outflow were not altered by long term treatment with either propranolol or timolol. The high dose of propranolol significantly reduced the heart rate of conscious rats. However neither the lower dose of propranolol nor any dose of timolol affected heart rate. The systolic pressure of conscious rats was unaltered by treatment with the beta adrenoceptor blockers. The threshold for release of tritiated noradrenaline from the sympathetic nerves on stimulation of the whole spinal outflow was raised by chronic treatment with propranolol or timolol. Timolol significantly increased the concentration of 3H noradrenaline in the blood and decreased the heart content of tritium. Chronic propranolol treatment did not alter the blood or heart levels of 3H noradrenaline. Thus, although the plasma levels of the beta adrenoceptor blocking drugs were probably insufficient to ensure prolonged blockade of postsynaptic receptors, significant changes in presynaptic function were observed. It remains to be seen whether these changes play any significant part in cardiovascular responses to beta adrenoceptor antagonists in clinical practice.

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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.

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

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

Esta dissertação investigou os mecanismos envolvidos na vasodilatação focando no papel das cavéolas e na possível interação com a isoforma endotelial da sintase de óxido nítrico (eNOS)/ óxido nítrico e neuronal (nNOS)/ peróxido de hidrogênio (H2O2) em aorta de camundongos tratados com isoproterenol (ISO). Anéis de aorta foram montados em banho de órgãos para medida de tensão isométrica. A expressão das proteínas foi avaliada a partir da técnica de Western blot. Os resultados demonstraram que o tratamento com ISO: 1) não modifica nem a vasodilatação à acetilcolina (ACh) nem ao nitroprussiato de sódio; 2) aumenta a dependência das cavéolas na resposta vasodilatadora à ACh e ao ionóforo de cálcio e a expressão proteica da caveolina-1, mas não da caveolina-3; 3) aumenta a modulação das NOS, principalmente a nNOS, à ACh; 4) aumenta a participação do H2O2 na vasodilatação à ACh e 5) aumenta a expressão de proteínas da defesa antioxidante. Conclui-se que a hiperativação β-AR com ISO ativa mecanismos vasodilatadores compensatórios em resposta à ACh nas aortas de camundongos. / The aim of this thesis was to investigate the mechanisms involved on the endothelium-dependent vasodilation focusing on the role of caveolae and the possible interaction between endothelial nitric synthase (eNOS)/ nitric oxide and neuronal (nNOS)/ hydrogen peroxide (H2O2) in aorta of ISO-treated mice. Aortic rings were mounted in an organ bath for measurement of isometric tension. The expression of proteins was evaluated using Western blot. The results demonstrated that ISO treatment: 1) did not change acetylcholine (ACh) or NO donor-induced relaxation; 2) increases the caveolae participation in ACh and calcium ionophore-induced relaxation and caveolin-1 protein expression, while did not change caveolin-3; 3) increases the constitutive NOS modulation to ACh-induced relaxation, mainly through nNOS; 4) increases the H2O2 involvement on the vasodilation-induced to ACh and 5) increases the antioxidant proteins. It is concluded that β-AR hyperactivation ISO active vasodilators compensatory mechanisms in response to ACh in the aortas of mice.

Esta dissertação investigou os mecanismos envolvidos na vasodilatação focando no papel das cavéolas e na possível interação com a isoforma endotelial da sintase de óxido nítrico (eNOS)/ óxido nítrico e neuronal (nNOS)/ peróxido de hidrogênio (H2O2) em aorta de camundongos tratados com isoproterenol (ISO). Anéis de aorta foram montados em banho de órgãos para medida de tensão isométrica. A expressão das proteínas foi avaliada a partir da técnica de Western blot. Os resultados demonstraram que o tratamento com ISO: 1) não modifica nem a vasodilatação à acetilcolina (ACh) nem ao nitroprussiato de sódio; 2) aumenta a dependência das cavéolas na resposta vasodilatadora à ACh e ao ionóforo de cálcio e a expressão proteica da caveolina-1, mas não da caveolina-3; 3) aumenta a modulação das NOS, principalmente a nNOS, à ACh; 4) aumenta a participação do H2O2 na vasodilatação à ACh e 5) aumenta a expressão de proteínas da defesa antioxidante. Conclui-se que a hiperativação β-AR com ISO ativa mecanismos vasodilatadores compensatórios em resposta à ACh nas aortas de camundongos. / The aim of this thesis was to investigate the mechanisms involved on the endothelium-dependent vasodilation focusing on the role of caveolae and the possible interaction between endothelial nitric synthase (eNOS)/ nitric oxide and neuronal (nNOS)/ hydrogen peroxide (H2O2) in aorta of ISO-treated mice. Aortic rings were mounted in an organ bath for measurement of isometric tension. The expression of proteins was evaluated using Western blot. The results demonstrated that ISO treatment: 1) did not change acetylcholine (ACh) or NO donor-induced relaxation; 2) increases the caveolae participation in ACh and calcium ionophore-induced relaxation and caveolin-1 protein expression, while did not change caveolin-3; 3) increases the constitutive NOS modulation to ACh-induced relaxation, mainly through nNOS; 4) increases the H2O2 involvement on the vasodilation-induced to ACh and 5) increases the antioxidant proteins. It is concluded that β-AR hyperactivation ISO active vasodilators compensatory mechanisms in response to ACh in the aortas of mice.

Beta adrenergic antagonists and antianginal drugs are used with the aim to ultimately decrease mortality and enable patients to lead an improved quality of life by avoidance of anginal episodes. Each class of medications used for this purpose has a variety of actual or potential side effects associated with their use. Side effects and drug interactions involving these medications are discussed in the following chapter. Evidence presented should be used in the context of the patient populations described and may aid clinical decision making through avoidance or identification of actual or potential side effects. This review includes literature published from January 2019 to January 2020 written in English.

Los receptores β-adrenérgicos (βAR) y de serotonina (5-HT4) median sus efectos en tejidos cardiacos a través de la ruta receptor-Gs-AC-AMPc. Las fosfodiesterasas (PDE) son una amplia familia de enzimas cuya función es la degradación del AMPc. Se desconocía que isoenzimas de PDEs son responsables de la hidrólisis de AMPc en las diferentes regiones cardiacas. El objetivo de esta tesis doctoral es investigar que isoenzimas de PDEs tienen actividad en el miocardio humano, porcino y de roedores. Se han realizado estudios cronotrópicos, inotrópicos, lusitrópicos, bioquímicos y electrofisiológicos. Los principales resultados son: Las PDEs se comportan de manera distinta en las diferentes regiones cardiacas y compartimentos celulares; y La frecuencia basal de nódulo sinusal está controlada por PDEs pero en ninguna especie estudiada las PDEs controlan la taquicardia causada por los βARs y los receptores 5-HT4. La extrapolación de la función de las PDEs al humano debe h acerse con cautela. / Myocardial β-adrenoceptors (βAR) and serotonin receptors (5-HT4) mediate their signals through the receptor-Gs-AC-cAMP pathway. Phosphodiesterases (PDEs) are a large enzyme family that degrade cAMP. It was unknown which PDE isoenzymes are responsible for the hydrolysis of the cAMP in different cardiac regions. The aim of this doctoral thesis is to investigate which isoenzymes have a role in human, porcine and rodent myocardium. We performed chronotropic, inotropic, lusitropic, biochemical and electrophysiological studies. The key results are: PDEs have different roles in different cardiac regions and cellular compartments; and the basal beating rate of the sinoatrial node is controlled by PDE3 and/or PDE4, but these PDEs do not limit the tachycardia mediated through the stimulation of β1AR, β2AR and 5-HT4. Given the diverse roles of PDE3 and PDE4 and their dependence on species, extrapolation to humans should be done cautiously because these animal models usually do not reflect the human myocardium.