In der vorliegenden Arbeit wurde erstmals mit Hilfe von drei unterschiedlichen experimentellen Modellen der Einfluß von Adrenomedullin (ADM) auf die myokardiale Kontraktilität des Rattenherzens untersucht. Die Methodik umfaßte die Verwendung des isolierten Herzen nach Langendorff, des isolierten Papillarmuskelpräparats und der konfokalen Laserscan Mikroskopie bei adulten Kardiomyozyten. In keinem der Ansätze konnte ein direkter Effekt von ADM auf die Kontraktilität nachgewiesen werden. An isolierten adulten Kardiomyozyten hatte ADM weder Einfluß auf die systolische Zellverkürzung noch auf den gleichzeitig registrierten zytosolischen Kalziumgehalt. Auch an isolierten Papillarmuskeln verursachte ADM keine Änderung der maximalen Kraftentwicklung oder Kraftanstiegsgeschwindigkeit. Endothelin-1 wurde sowohl am Papillarmuskelpräparat als auch an isolierten, adulten Kardiomyozyten als Positiv-Kontrolle verwendet und verursachte in beiden Versuchsaufbauten einen deutlichen Anstieg der kontraktilen Parameter. Als dritte Methode wurde das isoliert perfundierte Herz nach Langendorff verwendet. Unter Gabe von ADM zeigte sich ein Abfall des koronaren Perfusionsdrucks, entsprechend den bereits bekannten vasodilatierenden Eigenschaften des Peptids. Dieser Effekt war zwar begleitet von einer Abnahme der kontraktilen Parameter der Herzen, allerdings ist am isoliert und flußkonstant perfundierten Herz eine Abnahme des koronaren Perfusionsdrucks aufgrund des Gregg-Phänomes mit einer Abnahme der Kontraktilität vergesellschaftet. Aus den Ergebnissen dieser Arbeit kann daher der Schluß gezogen werden, das Adrenomedullin keinen Einfluß auf die Kontraktilität des Rattenherz hat. / The objective of this study was to investigate the influence of Adrenomedullin (ADM) on myocardial contractility of the rat heart in three different experimental models. First in the isolated rat heart preparation (Langendorf), second on the isolated papillary muscle preparation and third with confocal laserscan microscopy of isolated adult cardiomyocytes. None of these experimental models could show a direct effect of ADM on the contractility of the rat heart. In isolated adult cardiomyocytes, ADM had no influence on systolic cell shorting or on the simultaneously registered cytosolic calcium concentration. In isolated papillary muscle preparation ADM administration did not change maximal contractile force or time constants of contraction. Endothelin-1 was used as a positive control in both experimental setups and induced a marked increase in contraction parameters. In the isolated rat heart preparation (Langendorff), ADM induced a decrease of the coronary perfusion pressure, reflecting the already well described vasodilatatory properties of this peptide. This decrease was paralleled by a reduction of the contractile parameters of the heart, but in isolated rat heart preparations with constant coronary flow, a reduction of coronary perfusion pressure is always paralleled by a decrease of contractility due to the Gregg-Phenomenon. In conclusion the results of this study show that Adrenomedullin has no influence on myocardial contractility in the rat heart.

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No. of bitstreams: 2 Tese - Allancer Divino de Carvalho Nunes - 2018.pdf: 10933019 bytes, checksum: ec8f5a33649eddf1e5ff660120efcca9 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-04-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Despite Angiotensin-(1-7) [Ang-(1-7)] and Angiotensin II (Ang II) be considered important regulators of the cardiovascular system, its direct effects on cardiac contractility remain unclear and even controversial. Several studies, using different preparations, have showed that Ang-(1-7) or Ang II produce positive, negative or no inotropic effects. Thus, the aim of this study was to increase the knowledge about the effect of the low concentration of Ang-(1-7) and Ang II on cardiac contractility, coronary vascular function and the possible receptors and mechanisms of action involved in these effects. The in vivo effects of Ang-(1-7) and Ang II on cardiac contractility were evaluated in anesthetized rats. The Ang-(1-7) 4 nmol/L and Ang II 40 nmol/L caused negative inotropism in anesthetized rats. None of the peptides were able to change heart rate or arterial blood pressure in anesthetized rats. The direct effects of the Ang-(1-7) and Ang II on cardiac contractility were evaluated in isolated perfused rat hearts. After a basal period (30-40 minutes), the hearts were perfused for an additional 15 min with Ang-(1-7) and Ang II (20 pmol/L) in presence or absence of Mas receptor antagonist A-779 (2 nmol/L), AT2 receptor antagonist PD1233190 (2 nmol/L), AT1 receptor antagonist Losartan (1μmol/L), MrgD receptor antagonist D-PRO (2 nmol/L), ACE2 inhibitor DX600 (2nmol/L), Nitric Oxide synthase (NOS) inhibitor L-Name (10 nmol/L), Guanylyl cyclase (GC) inhibitor ODQ (200 nmol/L) and Adenylyl cyclase (AC) inhibitor MDL 12,330A (1μmol/L). Low concentrations of Ang-(1-7) and Ang II reduced the left ventricular end-systolic pressure (LVESP). The A-779 did not blocked the effect of Ang-(1-7) or Ang II. PD123319 and Losartan inhibited the Ang-(1-7) but not Ang II-induced negative inotropic effects. On the other hand, D-PRO was able to block the negative inotropic effect of the Ang II and Ang-(1-7). Furthermore, L-Name, ODQ and MDL 12,330A inhibited the Ang-(1-7) but not Ang II-induced negative inotropic effects. Similarly, low concentrations of Ang- (1-7) and Ang II-induced coronary vasodilatation. The A-779, D-PRO, L-Name, MDL 12,330A blocked the effect of Ang-(1-7) or Ang II. The DX600 blocked the vasodilatation induced by Ang II. PD123319, Losartan and ODQ inhibited the Ang-(1-7) but not Ang II-induced coronary vasodilatation. In addiction, the pharmacological blocked and gene silencing of AT1 receptor in endothelial human cells stimulated with Ang-(1-7) decreased AKT phosphorilation induced by Ang-(1-7). These data demonstrate that Ang-(1-7) and Ang II, at picomolar concentrations, induce significant and similar negative inotropic and coronary vasodilatation effects involving complex interaction mechanisms between many different receptors, altering intracellular signaling and their constitutive activity. / Embora a Angiotensina-(1-7) e Angiotensina II sejam considerados importantes peptídeos para regulação do sistema cardiovascular, seus efeitos sobre a contratilidade cardíaca ainda não estão totalmente elucidados. Diversos estudos, utilizando diferentes preparações, têm demonstrado que a Ang II e a Ang-(1-7) promovem efeito inotrópico positivo, negativo ou nenhum efeito inotrópico. Diante disso, o objetivo deste estudo foi ampliar os conhecimentos sobre os efeitos da Ang II e Ang-(1-7) no controle da contratilidade ventricular e função vascular coronariana. Além disso, avaliaremos o envolvimento dos receptores envolvidos nestes efeitos e os possíveis mecanismos de ação. Os efeitos in vivo da Ang-(1-7) e Ang II na contratilidade cardíaca foram avaliados em ratos anestesiados com uretana através da canulação intraventricular esquerda. A infusão de Ang-(1-7) 4 nmol/L e Ang II 40 nmol/L promoveram inotropismo negativo em corações de ratos anestesiados. Nenhum dos peptídeos foram capazes de alterar a pressão arterial e frequência cardíaca em ratos anestesiados. Os efeitos diretos da Ang- (1-7) e Ang II na contratilidade cardíaca foram avaliados em corações isolados de ratos. Após o período basal (30-40 minutos), os corações foram perfundidos durante 15 minutos com Ang-(1-7) ou Ang II na concentração de 20 pmol/L na presença ou ausência de antagonista do receptor Mas A-779 (2 nmol/L), antagonista do receptor AT2 PD123319 (2 nmol/L), antagonista do receptor AT1 Losartan (1μmol/L), antagonista do receptor MrgD D-PRO (2 nmol/L),inibidor da enzima ECA2 DX600 (2nmol/L), inibidor de Óxido nítrico sintase (NOS) L-Name (10 nmol/L), inibidor de Guanilato Ciclase (GC) ODQ (200 nmol/L) e inibidor de Adenilato Ciclase (AC) MDL 12,330A (1μmol/L). Concentrações picomolares de Ang-(1-7) e Ang II reduziram a pressão intraventricular sistólica (PIS). O A-779 não bloqueou os efeitos da Ang-(1-7) ou Ang II. PD123319 e Losartan inibiram somente o efeito inotrópico da Ang-(1-7). Por outro lado, D-PRO foi capaz de bloquear o efeito inotrópico de ambos os peptídeos angiotensinérgicos. Além disso, L-Name, ODQ e MDL 12,330A bloquearam apenas o efeito inotrópico da Ang-(1-7). Semelhantemente, baixas concentrações de Ang-(1-7) e Ang II induziram vasodilatação coronariana. O A- 779, D-PRO, L-Name, MDL 12,330A bloquearam a vasodilatação induzida por Ang-(1-7) e Ang II. O DX600 bloqueou a vasodilatação coronariana induzida por Ang II. PD 123319, Losartan e ODQ inibiram apenas a vasodilatação coronariana induzida por Ang-(1-7). Além disso, o bloqueio farmacológico e o silenciamento gênico do receptor AT1 em células endoteliais humanas estimuladas com Ang-(1-7) reduziram a fosforilação de AKT promovida por este peptídeo. Esses resultados demonstram que Ang-(1-7) e Ang II, em concentrações picomolares induzem significante e similar efeito inotrópico negativo e vasodilatação coronariana, envolvendo complexos mecanismos de interação entre os receptores.

Despite the development and use of specialised cardiac preservation solutions, the quality of the donor heart may still be compromised by its obligatory exposure to periods of ischaemia (both cold and warm) followed by reperfusion upon reintroduction of the recipient circulation. This is reflected in Transplant Registry data showing increased primary allograft failure as a function of increasing ischaemic time. The research described in this thesis is designed to further the understanding of the mechanisms by which the donor heart may be adapted to these prolonged periods of ischaemia and reperfusion by the activation of endogenous pro-survival signalling pathways by the addition of pharmacological agents to Celsior, a clinical preservation solution. Studies were conducted in an isolated working rat heart model of donor heart preservation. The first study investigated the cardioprotective effects of a novel inhibitor of poly(ADP-ribose) polymerase 1, INO-1153. Maximum protective effect (after a 6 hour storage period) was observed when the PARP inhibitor was administered prior to cardiac arrest and storage and when the agent was added to the Celsior cardioplegic / storage solution. This protective affect was associated with activation of the Akt signalling pathway and could be prevented by inhibition of Akt phosphorylation and activation. The second study examined functional protection and pro-survival signalling pathway activation in hearts arrested and stored for 6 hours in Celsior supplemented with glyceryl trinitrate (an exogenous source of nitric oxide) and Cariporide (an inhibitor of sodium hydrogen exchange). Here, cardiac protection was accompanied by activation of the ERK 1/2 pro-survival pathway as well as a decrease in apoptosis. The third study examined the cardioprotective effect of supplementation of Celsior with all three agents after an extended (10 hour) period of hypothermic storage. Significant recovery of function was only observed in the triply supplemented hearts, being accompanied by activation of both the Akt and ERK pathways. These studies demonstrate for the first time the feasibility of recruitment of endogenous pro-survival pathways as an approach to increasing the post-storage function of the donor heart. Importantly, for the logistics of clinical transplantation, these pathways can be recruited by addition of appropriate pharmacological agents to the arresting and storage solution.

Orientador: José Ricardo de Arruda Miranda / Resumo: Inflammatory bowel disease (IBD) is an idiopathic gastrintestinal tract disease that affects a large part of the world population and has its symptoms related to changes in colon motility. However, the evolution of these changes is not completely understood, and may be related to symptoms that appear in stages when the disease is apparently controlled. The objective of this work was to perform a long-term evaluation of the mechanical and electrical aspects of colonic contractility during the model of inflammation induced by trinitrobenzene sulfonic acid (TNBS) in rats. This work applied an efficient and low invasive methodology associating mechanical, electrical, chemical and histological information. The contractility and inflammatory parameters were acquired in the same animal at six different times: before induction of TNBS (control) and at 2, 5, 8, 11 and 14 days thereafter. The mechanical activities were acquired by Alternating Current Biosusceptometry (ACB) and subdivided in Rhythmic Propagation Ripples (RPR) and Rhythmic Propulsive Motor Complex (RPMC). We recorded the electrical activity by electromyography (EMG) and evaluated the inflammation processes determining the myeloperoxidase activity (MPO) in the feces. Additionally, we compared the thickness of colon layers throughout the inflammation by histopathological analyzes. Our results showed transient changes in MPO activity levels and frequency of RPMC contraction, while RPR and electrical activity underwent perma... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Estudamos o efeito do citrato de sildenafil (ViagraÂ), vasodilatador largamente utilizado na terapÃutica da disfunÃÃo erÃtil, sobre o comportamento motor do trato gastrintestinal (TGI) de ratos Wistar. Para tanto, utilizamos 175 animais machos, pesando entre 200 a 350g, distribuÃdos nos quatro seguintes grupos de estudo: efeitos do citrato de sildenafil sobre o i) esvaziamento gÃstrico (EG) e os trÃnsitos gastrintestinal (GI) e ii) intestinal de lÃquido em ratos acordados; iii) a complacÃncia gÃstrica de ratos anestesiados e iv) a contratilidade de tiras isoladas do duodeno de ratos ex vivo. i) Avaliamos, em 64 ratos acordados sob jejum e livre acesso à Ãgua por 24h, o efeito da injeÃÃo (0,2mL; e.v.) de sildenafil (4mg/Kg) ou veÃculo (HCl 0,01N) sobre o EG e o trÃnsito GI de lÃquido, bem como sobre a pressÃo arterial (PA). Mediante gavagem, 1,5mL da refeiÃÃo-teste (vermelho de fenol - 0,5mg/mL em glicose a 5%) foi injetada no estÃmago. Depois de 10, 20 ou 30min, sacrificamos os animais e, apÃs laparotomia, obstruÃmos o piloro, o cÃrdia e o Ãleo terminal. Removemos e dividimos o TGI em: estÃmago e segmentos consecutivos do intestino delgado (40% iniciais; 30% mediais e 30% terminais). ApÃs o processamento destas porÃÃes viscerais, determinamos as absorbÃncias das amostras a 560nm. A retenÃÃo fracional de vermelho fenol em cada segmento permitiu o cÃlculo do EG e trÃnsito GI. Em um grupo separado de animais, a PA foi monitorada continuamente por meio de um sistema digital de aquisiÃÃo de dados durante 20min antes e 30min apÃs o tratamento com sildenafil ou diluente. Comparado ao grupo controle, houve aumento significativo da retenÃÃo gÃstrica (44,2Â2,0 vs 53,2Â2,1; 25,4Â1,3 vs 37,3Â1,6; 20,9Â2,5 vs 32,5Â2,9%) nos animais tratados com sildenafil e sacrificados aos 10, 20, ou 30min, respectivamente, bem como retarde significativo no trÃnsito GI. Embora o sildenafil tenha provocado hipotensÃo, a PA retoma nÃveis basais logo apÃs 10min. O prÃ-tratamento com omeprazol (bloqueador da secreÃÃo Ãcida estomacal) nÃo modificou o efeito do sildenafil sobre os valores de retenÃÃo gÃstrica e intestinal nem nos nÃveis de PA. ii) Noutros animais (n=44), sob jejum de 24h e dotados previamente (3d) de uma cÃnula crÃnica no bulbo duodenal, estudamos o efeito do sildenafil sobre a progressÃo ao longo do intestino delgado de uma refeiÃÃo teste (10MBq de TecnÃcio ligado a fitato e diluÃdo em 1mL de salina 0,9%). Decorridos 20, 30 ou 40min da injeÃÃo (0,2mL e.v.) de sildenafil (4mg/Kg) ou diluente (HCL 0,01N), sacrificamos os animais e, apÃs laparotomia e remoÃÃo do TGI, dividimo-o em: estÃmago, cinco segmentos congruentes e consecutivos de intestino delgado e o intestino grosso. A contagem da radiatividade foi determinada num colimador de gama-cÃmara. O sildenafil promoveu retarde (p<0,05) do TI, indicado pelos retardes dos centros geomÃtricos da refeiÃÃo de 2,8&#61617; 0,2 vs 3,3&#61617; 0,1; 3,0&#61617; 0,2 vs 3,7&#61617; 0,1 e 3,4&#61617; 0,1 vs 4,2&#61617; 0,2 em relaÃÃo ao grupo controle, aos 20, 30 ou 40min. iii) Os estudos de complacÃncia gÃstrica foram conduzidos em 39 ratos anestesiados, sob jejum de 24h. As variaÃÃes do volume gÃstrico (VG), foram medidas por pletismografia, enquanto a PA foi monitorada continuamente por um sistema digital de aquisiÃÃo de dados. Em relaÃÃo aos valores basais (2,91Â0,19mL) o sildenafil (3mg/Kg â e.v.) aumentou (p<0,05) o VG apÃs 10, 20 e 30min (3,08Â0,18; 3,10Â0,17 e 3,09Â0,17mL). A PA basal (105,8Â2,28mmHg) caiu significativamente com o sildenafil (59,8Â3,2; 64,8Â3,7 e 59,3Â4,6mmHg) enquanto o diluente (HCl 0,01N) nÃo modificou seja o VG ou a PA. O prÃ-tratamento mediante esplancnotomia ou injeÃÃo e.v. com azul de metileno (3mg/Kg-bloqueador da guanilato ciclase), L-NNA (3mg/Kg-bloqueador da NO sintetase) ou propranolol (2mg/Kg-Ã-bloqueador) preveniram o aumento do VG pelo sildenafil; jà o pÃs-tratamento com nitroprussiato de sÃdio (1mg/Kg - e.v.) o ampliou significativamente. iv) Avaliamos ainda o efeito do sildenafil sobre a contratilidade de tiras isoladas do duodeno de ratos ex vivo (n=28), sacrificados por deslocamento cervical. Tiras dissecadas do duodeno foram suspensas longitudinalmente em cuba de vidro (10mL), plena de soluÃÃo de Tyrode (37oC e pH 7,4), e submetidas a uma tensÃo inicial de 1g. ApÃs 1h de estabilizaÃÃo, a contratilidade espontÃnea ou induzida das tiras foi registrada continuamente por um sistema digital de aquisiÃÃo de dados. O sildenafil em doses crescentes e cumulativas (0,1 a 300Âmol/L) relaxou (EC50 de 9,6Âmol/L) o duodeno, mais atà que o zaprinaste ou a papaverina (bloqueadores de FDEs) (EC50 91,6 e 78,5Âmol/L, nesta ordem). Observamos ademais que o sildenafil inibiu as contraÃÃes induzidas por acetilcolina ou carbacol (IC50 26,7 e 16,2Âmol/L, respectivamente). Jà o prÃ-tratamento com azul de metileno, ODQ (bloqueador da guanilato ciclase) ou L-NAME (bloquedor da NO sintetase), mas nÃo o D-NAME (isÃmero inativo da NO sintetase) preveniram o efeito do sildenafil. O efeito mio-relaxante do sildenafil foi ampliado pela L-arginina (substrato do NO sintetase) ou nitroprussiato de sÃdio (doador de NO). O prÃ-tratamento com forskolina (estimulador da adenilato ciclase) tambÃm aumentou o efeito mio-relaxante do sildenafil. Em resumo, observamos que o sildenafil diminui a motilidade gastrintestinal, retardando o EG, os trÃnsitos GI e intestinal de lÃquido em ratos acordados; aumenta a complacÃncia gÃstrica em ratos anestesiados alÃm de apresentar efeitos antiespasmÃdico e mio-relaxante sobre tiras isoladas de duodeno de ratos ex vivo; por estimulaÃÃo do sistema nervoso simpÃtico e tendo como provÃvel mecanismo de aÃÃo ao nÃvel do miÃcito gastrintestinal a via do NO/GMP cÃclico. / We evaluated the effect of sildenafil citrate (ViagraÂ) a vasodilator largely used for the treatment of male erectile dysfunction, on the gastrointestinal motility in rats. Experiments were performed on 175 male, Wistar rats, weighing 200-350g. Four groups of study were done: the sildenafil effects on the: i) Gastric emptying (GE) and gastrointestinal (GI) transit and ii) Intestinal transit (IT) of liquid in awake rats; iii) Gastric compliance in anesthetized rats and iv) Contractility of rat duodenal isolated strips. i) In 64 rats fasted for 24h with previous vascular access (right jugular vein and left carotid artery), we studied the effect of an i.v. injection (0.2mL) of sildenafil (4mg/Kg) or vehicle (0.01N HCl) on GE and GI transit of a liquid meal, as well as on arterial pressure (AP) in a separated group of rats. Animals were gavage-fed with 1.5mL of a test meal (0.5mg/mL of phenol red in 5% glucose). After 10, 20 or 30min, animals were sacrificed and submitted to a laparotomy to obstruct the pylorus, cardia and terminal ileus. The gut was removed and then divided into: stomach and consecutive three small intestine segments (40% proximal; 30% medial and 30% terminal). After processing these segments, the dye retention was determined at 560nm. The percentage of dye retention in each segment permitted to evaluate GE and GI transit. Arterial pressure was continuously monitored by a digital acquisition system during 20min before and 30min after sildenafil injection. We observed a significant increase of gastric retention in sildenafil treated rats at 10, 20, or 30min after the test meal (44,2Â2,0 vs 53,2Â2,1; 25,4Â1,3 vs 37,3Â1,6; 20,9Â2,5 vs 32,5Â2,9%, respectively), as well as a significant GI transit delay. Despite of sildenafil inducing hypotension, AP returned to basal levels 10min afterwards. Acid gastic secretion blocking pre-treatment with omeprazol did not modify the sildenafil effect on gastric retention, GI transit or AP. ii) In another group we evaluated the sildenafil (4mg/Kg) or diluente (0.01N HCl, 0.2mL) effects on the IT in awake rats, fasted for 24h. Animals were studied 3d after the insertion of a silastic cannula (0.6cm ID) into the duodenal bulb. We evaluated the progression of a radioactive liquid test meal fed (10MBq of 99mTc â 1mL of saline 0,9%) administered through the inserted cannula into the small intestine. After 20, 30 or 40min, animals were sacrificed by anesthetic overdose. After laparotomy, we removed and divided the gut in: stomach, five congruent and consecutive segments of the small intestine and the large intestine. Radioactivity counting was obtained in a gamma-chamber collimator. Sildenafil promoted an IT delay (p<0.05), indicated by shifting the center of mass to the proximal portions of the TGI (2.8Â0.2 vs 3.3Â0.1; 3.0Â0.2 vs 3.7Â0.1 and 3.4Â0.1 vs 4.2Â0.2) in relation to control group. iii) Gastric compliance study was performed on 39 anesthetized rats after 24h of fasting. Gastric volume (GV) variations were measured by plethysmography while AP was continuously monitored. We have also observed that GV increased (p<0.05) after sildenafil treatment (3mg/Kg - e.v) (3.08Â0.18; 3.10Â0.17 and 3.09Â0.17mL vs 2.91Â0.19mL) at 10, 20 and 30min after drug administation, respectively. Basal AP (105.8Â2.28mmHg) dropped by the sildenafil injection (59.8Â3.2; 64.8Â3.7 and 59.3Â4.6mmHg-p<0.05) while vehicule (0.01N HCl) did not change either GV or AP. After splanchnotomy or pre-treatments (e.v.) with methylene blue (3mg/Kg-guanilate cyclase blocker), L-NNA (3mg/Kg - NO synthase blocker) or propranolol (2mg/Kg - Ã-blocker) prevented GV increase due to sildenafil; while post-treatment with sodium nitroprusside (1mg/Kg - NO donor) raised it. iv) The in vitro contractility studies were performed on isolated duodenal strips obtained from rats (n=28) killed by cervical dislocation. Duodenal strips were suspended longitudinally in a glass chamber (10mL), filled with Tyrode solution (37oC and pH 7.4). After 1h of stabilization under 1g of initial tension, the spontaneous or induced contractility were continuously recorded by a digital acquisition system. Increasing and cummulative doses of sildenafil (0.1 to 300Âmol/L) relaxed (9.6Âmol/L of EC50) the duodenal strips. This effect was more intense than those displayed by zaprinast or papaverine (PDEs blockers) (91.6 and 78.5Âmol/L of EC50, in this order). Sildenafil showed significant antispasmodic and myorelaxant effects on the duodenal contractions induced by acetylcholine or carbamylcholine (IC50 26.7 and 16.2Âmol/L, respectively). Pre-treatment with methylene blue, ODQ (guanilato cyclase blocker) or L-NAME (NO synthase blocker) also prevented these sildenafil effects, but D-NAME (an inactive substrate for NO synthase) did not. Myorelaxant sildenafil effect was reverted by L-arginine (substrate for NO synthase) and contrarily it was largely increased by sodium nitroprusside. Forskolin adenylate cyclase activation pre-treatment also increased the myorelaxant effect of sildenafil. In summary, we have observed that sildenafil slowed down the gastrointestinal motility, delaying GE, GI and intestinal transits of a liquid meal in awake rats; Gastric compliance was also increased in anesthetized rats treated with sildenafil. Sildenafil also exhibited both antispasmodic and myorelaxant effects on isolated strips of duodenum of ex vivo rats. Besides central or peripheral sympathetic nervous system activation, sildenafil possibly acts at the gastrointestinal myocite level by activating the NO/GMPc system.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Quercetin is a flavonoid widely distributed in plants, and it is shown to have several biological activities. This study aims to describe the effects of quercetin on the contracting and electrophysiological properties of the cardiac muscle as well as the homeostasis of intracellular calcium. This study evaluated the inotropic effect of quercetin in guinea pigs’ left atria, its effects on the adrenergic receptors, and on the electrocardiographic parameters. In mice ventricular cardiomyocytes the L-type Ca2+ current (ICa,L), the intracellular Ca2+ global transient and the calcium sparks were evaluated. The results revealed a positive inotropic effect from quercetin (EC50 3.64 x 10-6 M, n = 4) that was significantly reduced by 1 M propranolol. Furthermore, quercetin caused diastolic relaxation (~7%). In ventricular cardiomyocytes, 30 μM quercetin increased the density of ICa,L at 0 mV of −0.95 ± 0.01 A/F from −1.21 ± 0.08 A/F (n = 5, p < 0.05) and membrane potential at which 50% of the channels are activated (V0.5) diminished to -13.06 ± 1.52 mV from -19.26 ± 1.72 mV (n = 5, p < 0.001) not altering the slope factor. Furthermore, cardiomyocytes exposure at 30 μM quercetin presented [Ca2+]i transient of 4.61± 0.22 (n = 91 cells, p < 0.001) that was 28% higher compared with control situation (3.60 ± 0.14, n = 40 cells). Quercetin also accelerated the [Ca2+]i transient decay kinetics at 90% (t90) from 875.8 ± 13.44 ms to 740.0 ± 26.74 ms (p < 0.001) and at 50% (t50) from 253.3 ± 7.75 ms to 181.4 ± 12.67 ms (p < 0.001). In cardiomyocytes not electrically stimulated, quercetin did not increase the frequency of calcium sparks. In isolated guinea pig heart, quercetin increased the heart rate from 133.1 ± 5.49 bpm to 146.2 bpm ± 5.28 (n = 5, p <0.0001); decreased the PR interval from 107.3 ± 4.69 ms to 100.3 ± 1.79 ms (n = 5, p < 0.05) and decreased the QTc from 10.49 ± 0.048 ms to 10.23 ± 0.06 ms. The duration of the QRS complex was not significantly changed, and there was also no evidence of the appearance of cardiac arrhythmias. Thus, we showed that quercetin activates β-adrenergic receptors, leading to increased L-type calcium current and intracellular calcium transient, not inducing the increase of calcium sparks or arrhythmogenic effects. / A quercetina é um flavonoide, amplamente distribuído nas plantas e apresenta várias atividades biológicas. Esse trabalho tem como objetivo descrever os efeitos da quercetina sobre as propriedades contráteis e eletrofisiológicas do músculo cardíaco, assim como a homeostase do cálcio intracelular. Nesse estudo, foi avaliado o efeito inotrópico da quercetina em átrio esquerdo de cobaia, sua ação nos receptores adrenérgicos e sobre os parâmetros eletrocardiográficos. Em cardiomiócito ventricular de camundongo, foram estudadas as correntes de Ca+2 tipo-L (ICa,L), o transiente intracelular global de cálcio e as sparks de cálcio. Os resultados revelaram que a quercetina promoveu efeito inotrópico positivo (EC50 3,64 x 10-6 M, n = 4) que foi significativamente reduzido pelo propranolol a 1 M. Além disso, a quercetina induziu relaxamento diastólico (~7%). Em cardiomiócito ventricular, 30 μM de quercetina promoveu aumento da densidade da ICa,L em 0 mV de -0,95 ± 0,01 A/F para -1,21 ± 0,08 A/F (n = 5, p < 0,05) e o potencial de membrana, onde 50% dos canais estão ativados (V0,5) diminuiu de -13,06 ± 1,52 mV para -19,26 ± 1,72 mV (n = 5, p < 0,001) sem alterar a inclinação da curva. Além disso, os cardiomiócitos expostos a 30 μM de quercetina apresentaram um transiente intracelular de cálcio de 4,61 ± 0,22 (n = 91 células, p < 0,001) que foi 28% maior comparado com cardiomiócito na situação controle (3,60 ± 0,14, n = 40 células). A quercetina também acelerou a cinética do decaimento do transiente da Ca+2, em 90% (t90) foi reduzido de 875,8 ± 13,44 ms para 740,0 ± 26,74 ms (p < 0,001) e em 50% (t50) de 253,3 ± 7,7 ms para 181,4 ± 12,67 ms (p < 0,001). Em cardiomiócito não estimulados eletricamente, a quercetina não aumentou a frequência de sparks de cálcio. Em coração isolado de cobaia, a quercetina aumentou a frequência cardíaca de 133,1 ± 5,49 bpm para 146,2 ± 5,28 bpm (n = 5, p < 0,0001); diminuiu o intervalo PR de 107,3 ± 4,69 ms para 100,3 ± 1,79 ms (n = 5, p < 0,05); diminuiu o QTc de 10,49 ± 0,048 ms para 10,23 ± 0,06 ms. A duração do complexo QRS não apresentou alteração significativa, assim como não foi evidenciado o aparecimento de arritmias cardíacas. Assim, evidenciamos que a quercetina ativa receptores β-adrenérgicos, levando ao aumento da corrente de cálcio tipo-L e do transiente intracelular de cálcio, sem induzir o aumento de sparks de cálcio ou efeitos arritmogênicos.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The farnesol (C15H26O) is a sesquiterpene alcohol found in herbs and essential oils. Research show some beneficial properties of farnesol as: antioxidant, anti-inflammatory and chemopreventive properties. However, the farnesol futher inhibition of activity of L-type calcium channels in smooth muscle. This study sought to analyze the involvement of calcium channels L-type in the cardiodepressant farnesol response in the rat heart. To this, contractile studies were conducted on left atria drawn to rest tension 5mN (0,5gf) and subjected to field stimulation with above-threshold current pulses of the 1 Hz, kept in isolated organ vessel, submerged in Krebs-Henseleit solution (8 mL) and aerated with carbogênica mixture (95% O2 and 5% CO2). The atrial contraction force was capture by an isometric transducer. Electrocardiographic recordings were performe on isolated heart, aortic perfusion under a constant flow (10 ml / min) Langendorff type system. The hearts were keep spontaneously beating, to determine the heart rate, and in heart stimulated were determined PR interval (PRI), QT (QTi) and complex QRS. Left ventricular pressure was determined in the heart stimulated electrically by means of a balloon inflated with water at a pressure of 15 cmHg. In rat left atrium, the farnesol response to the contraction strength showed a negative inotropic effect, reducing the contraction force at 41.63% at the maximum concentration used, with an EC50 of 2.84 ± 0.19 mM. To evaluate the effect of farnesol of the positive inotropic response of CaCl2 and (±)-Bay K8644 was observed that the farnesol shifted to the right CaCl2 concentration-response curve and decreased maximum efficiency (100% to 23%) and abolished curve of (±)-Bay K8644. TEA was use to evaluate the role of K+ channels in the negative inotropic response and the maximum effect by farnesol (41.63 to 63.02%) was increased. In isolated heart, there was an increase of PRI, QTi and QRS complex, and reduced left ventricular pressure (37, 38%) and heart rate (25.22%). Thus, farnesol exerts inotropic and negative chronotropic responses in the heart by reducing current to the L-type Ca2+. / O farnesol (C15H26O) é um álcool sesquiterpênico encontrado em óleos essenciais e ervas aromáticas. Pesquisas evidenciam algumas propriedades benéficas do farnesol como: propriedades antioxidantes, anti-inflamatórias e quimiopreventivas. Entretanto, o farnesol promoveu inibição dos canais de cálcio tipo-L em músculo liso. Neste trabalho procurou-se analisar os efeitos do farnesol sobre os mecanismos contráteis e eletrofisiológicos sobre o coração de rato. Para esse fim, os estudos contráteis foram realizados em átrios esquerdo estirados a para uma tensão de repouso de 5mN (0,5gf) e submetidos a estimulação de campo com pulsos de corrente supralimiares de 1 Hz, mantido em cuba para órgão isolado, submerso em solução de Krebs-Henseleit (8 mL) e aerado com mistura carbogênica (95 % O2 e 5 % CO2). A força de contração atrial foi captada por um transdutor isométrico. Os registros eletrocardiográficos foram obtidos em coração isolado, sob perfusão aórtica de fluxo constante (10 mL/min), em sistema de Langendorff. Os corações foram mantidos com batimento espontâneo, para determinar a frequência cardíaca, e em corações estimulados foram determinados os intervalos PR (PRi), QT (QTi) e complexo QRS. A pressão ventricular esquerda foi determinada, em coração estimulado eletricamente, por meio de um balonete insuflado com água até uma pressão de 15 cmHg. Em átrio esquerdo de rato, a resposta do farnesol sobre a força de contração apresentou efeito inotrópico negativo, diminuindo a força de contração em 41,63% na concentração máxima usada, apresentando uma CE50 de 2,843 ± 0,19 mM. Ao avaliar o efeito do farnesol sobre a resposta inotrópica positiva do CaCl2 e (±)-BAY K8644 foi observado que o farnesol deslocou para direita a curva concentração-resposta do CaCl2 e diminuiu a eficácia máxima (100% para 23%) e aboliu a curva do (±)-BAY K8644. Para avaliar a participação dos canais para potássio na resposta inotrópica negativa foi utilizado TEA tendo aumento da eficácia máxima do farnesol de 41,63 para 63,02%. Em coração isolado, foi observado aumento do PRi, QTi e complexo QRS, e redução da pressão ventricular esquerda (37,38%) e frequência cardíaca (25,22%). Assim sendo, o farnesol exerce respostas inotrópicas e cronotrópicas negativas no coração, por redução das correntes para Ca2+ tipo-L.

Zymosan-induced peritonitis is a model commonly used to study systemic inflammatory response syndrome and multiple organ dysfunction syndrome. However, effects of zymosan on cardiac function have not been reported. We evaluated cardiac responses to zymosan in mice and the role of β-Glucan and dectin-1 in mediating these responses. Temperature and cardiac function were evaluated before and after intraperitoneal (i.p.) injection of zymosan (100 or 500 mg/kg) or saline. Chronotropic and dromotropic functions were measured using electrocardiograms (ECGs) collected from conscious mice. Cardiac inotropic function was determined by echocardiography. High-dose zymosan caused a rapid and maintained hypothermia along with visual signs of illness. Baseline heart rate (HR) was unaffected but HR variability (HRV) increased, and there was a modest slowing of ventricular conduction. High-dose zymosan also caused prominent decreases in cardiac contractility at 4 and 24 h. Because zymosan is known to cause gastrointestinal tract pathology, peritoneal wash and blood samples were evaluated for bacteria at 24 h after zymosan or saline injection. Translocation of bacterial occurred in all zymosan-treated mice (n=3), and two had bacteremia. Purified β-Glucan (50 and 125 mg/kg, i.p.) had no effect on temperature or ECG parameters. However, deletion of dectin-1 modified the ECG responses to high-dose zymosan; slowing of ventricular conduction and the increase in HRV were eliminated but a marked bradycardia appeared at 24 h after zymosan treatment. Zymosan-treated dectin-1 knockout mice also showed hypothermia and visual signs of illness. Fecal samples from dectin-1 knockout mice contained more bacteria than wild types, but zymosan caused less translocation of bacteria. Collectively, these findings demonstrate that zymosan-induced systemic inflammation causes cardiac dysfunction in mice. The data suggest that dectin-1-dependent and -independent mechanisms are involved. Although zymosan treatment causes translocation of bacteria, this effect does not have a major role in the overall systemic response to zymosan.

Le syndrome d'apnée obstructive du sommeil (SAOS), est une maladie chronique qui se caractérise par des interruptions répétées de la respiration durant le sommeil en raison de la fermeture temporaire des voies aériennes supérieures. L'hypoxie intermittente chronique (HI) résultante de cette fermeture transitoire des voies aériennes supérieures, constitue l’une des conséquences majeures du SAOS, et elle est la responsable de la plupart des complications liées à cette pathologie, dont nous citons: l’hypertension artérielle, l’infarctus de myocarde et plus généralement le remodelage cardiovasculaire.D’autre part, l’entrainement physique intensif(EI)est bien connu d’avoir des bénéfices sur le système cardiovasculaire, d’où nous avons poser l’hypothèse que l’EI peut inverser les effets délétères de l’HI sur la réactivité et le remodelage vasculaire ainsi que sur la signalisation calcique intracellulaire dans les cellules musculaires.Pour répondre à cette question, nous avons choisi le rat comme modèle animal, pour étudier l’effet potentiel de l'EI dans la prévention et l’inversion des effets délétères de (HI) en termes de réactivité et signalisation calcique dans les tissues musculaires.Des rats ont été exposés durant 21 jours à l’hypoxie intermittente dans des cages spécialement équipées pour maintenir un flux d’air alternant entre 21% et 5% de PO2 dans les cages contenant les rats hypoxique et a 21% de PO2 dans les cages contenant les rats contrôles. Durant les deux dernières semaines d’exposition à l’HI, un groupe des rats hypoxiques et un des rats normoxiques ont subi des sessions d'EI en courant sur un tapis roulant avec une vitesse allant de 16m/min jusqu'à 30 m/min.Les paramètres physiologiques ont été mesurés (Pression artérielle, fréquence cardiaque, hématocrites), l’aorte a été prélevé pour étudier la réactivité vasculaire, les cellules musculaires lisses de l’aorte ont été ensuite prélevés et cultivées pour étudier la signalisation calcique par microscopie à EPIfluorescence. Finalement les gènes codant pour les médiateurs de la signalisation calcique : RyR1, RyR2 RyR3, (ryanodine receptors), TRPV4 (transient receptor potential channel), SERCA1, SERCA2 (Sarco/Endoplasmic Reticulum Ca2+ -ATPase) et IP3R1 (Inositol 1,4,5-Trisphosphate Receptor) dans différentes tissues vasculaires et squelettiques ont été étudiés au niveau moléculaire par Q-PCR et Western Blot.Nos résultats montrent que l'HI induit une augmentation significative de pression artérielle et de l’hématocrite et une diminution dans la relaxation de l'aorte induite par l'acétylcholine pré contractée par la phénylnephrine. Ceci est conforme à notre observation selon laquelle HI augmente le niveau de calcium intracellulaire dans le muscle lisse aortique cultivé. D'autre part, l'EI induit une diminution significative de l’hématocrite et de la vasoconstriction aortique induite par la phénylnephrine et l'endothélie-1, conformément à l'observation que l'EI réduit la différence HI-N dans la réponse calcique. A l’échelle moléculaire, HI induit une augmentation significative de l'expression de RyR1, RyR2, RyR3, SERCA1, SERCA2, TRPV4 et IP3R1 au niveau de l'ARNm dans les tissus de tous les groupes, avec une plus grande quantité de RyR1,RyR2,et RyR3 dans les tissus HI des muscles lisses (principalement dans l'aorte thoracique et abdominale) et le SERCA1 (9 fois plus haut dans les tissus IH) et le SERCA2 (10 fois plus élevé dans les tissus HI) dans les muscles squelette (Gastrocnemius, plantaris et soléus). De plus, HI induit une augmentation significative de RYR1, RYR2 et TRPV4 au niveau protéique dans l'aorte thoracique et abdominale; et l'EI réduit la différence d'expression entre les animaux N et IH.Nos résultats suggèrent que l'EI représente un traitement prometteur non pharmacologique ou complémentaire pour limiter les complications cardio-vasculaires induites par l’HI et le remodelage musculaire chez les patients atteints de SAOS. / Obstructive sleep apnea syndrome (OSAS) is a chronic disease characterized by repeated interruptions of breathing during sleep due to the temporary closure of the upper airway. Its prevalence increases with the increasing in prevalence of obesity, especially in developed countries.Chronic intermittent hypoxia (IH) resulting from this transient closure of the upper airway is one of the major consequences of OSAS and is responsible of most of the complications related to this pathology, including hypertension, myocardial infarction, atherosclerosis and more generally cardiovascular remodeling.On the other hand, intensive physical training(IT) is well known to have benefits on cardiovascular system, thus we hypothesize that physical training can reverse the deleterious effects of IH on reactivity and vascular remodeling as well as intracellular calcium signaling in muscle cells.To answer this question, we chose the rat as an animal model to study the potential effect of IT in the prevention and reversal of deleterious (IH) effects in terms of reactivity and calcium signaling in muscle tissue.Rats were exposed for 21 days to intermittent hypoxia and housed in cages specially equipped to maintain an airflow alternating between 21% and 5% PO2 in cages containing hypoxic rats and 21% PO2 in cages containing the control rats. During the last two weeks of exposure to IH, a group of hypoxic rats and one of the normoxic rats underwent IT sessions on a treadmill at a speed of 16m / min to 30m / min.Physiological parameters were measured (blood pressure, heart rate, hematocrit), the aorta was removed to study the vascular reactivity, then vascular smooth muscle cells were removed and cultured to study calcium signaling by EPIfluorescence microscopy. Finally, the genes coding for the key mediators of the calcium signaling: RyR1, RyR2 RyR3, (ryanodine receptors), TRPV4 (transient receptor potential channel), SERCA1, SERCA2 (Sarco / Endoplasmic Reticulum Ca2 + -ATPase) and IP3R1 , 5-Trisphosphate Receptor) in various vascular and skeletal tissues were studied at the molecular level as mRNA by Q-PCR or as protein by Western Blot.Our results show that IH induces a significant increase in blood pressure and hematocrit and a decrease in acetylcholine-induced aortic relaxation pre-contracted with phenylnephrine. This was consistent with our observation that HI increases the level of intracellular calcium in cultured aortic smooth muscle. On the other hand, IT induced a significant decrease in hematocrit and aortic vasoconstriction induced by phenylnephrine and endothelial-1, consistant with the observation that IT reduces the IH-N difference in the calcium response. On the molecular scale, IH induces a significant increase in the expression of RyR1, RyR2, RyR3, SERCA1, SERCA2, TRPV4 and IP3R1 at the mRNA level in the tissues of all groups with a greater amount of RyR1,RyR2,& RyR3 higher in IH tissue of smooth muscles (mainly in the thoracic and abdominal aorta) and SERCA1 (9-fold higher in IH tissues) and SERCA2 (10-fold higher in IH tissues) in the skeletal muscles (Gastrocnemius, plantaris and soléus). In addition, IH induces a significant increase in RYR1, RYR2 and TRPV4 at the protein level in the thoracic and abdominal aorta; And IT reduces the difference in expression between animals N and IH.Our results suggest that IT is a promising, non-pharmacological or complementary treatment for limiting cardiovascular complications induced by IH and muscle remodeling in patients with OSAS.

No description available.