Contrairement aux HDL de sujets sains, les HDL de patients diabétiques ont perdu leur capacité à contrecarrer les effets inhibiteurs des LDL oxydées sur la vasorelaxation endothélium dépendante. Les mécanismes en cause ne sont pas connus. Or la glycation et l’oxydation sont deux phénomènes majeurs au cours du diabète. Nous avons donc étudié in vitro, le rôle de la glycation (associée ou non à une oxydation spontanée), et de l’oxydation d’HDL issues de sujets sains, sur leurs capacités à contrecarrer les effets inhibiteurs des LDL oxydées sur la vasorelaxation endothélium dépendante. Chaque condition a conduit au même constat: les HDL modifiées perdent leur pouvoir vasorelaxant en présence de LDL oxydées. En revanche, en l’absence de LDL oxydées, elles n’altèrent pas la vasorelaxation induite par l’acétylcholine. Ainsi les modifications structurelles des HDL (glycation, oxydation, ou les deux) induisent une perte de leur capacité à protéger l’endothélium du stress oxydatif, plutôt qu’un effet délétère direct sur l’endothélium. Un des mécanismes majeur impliqué dans ce phénomène est probablement l’absence de fixation de ces HDL modifiées à leur récepteur SR-BI. Elles ne pourraient plus alors s’opposer au niveau des cavéoles aux effets délétères des LDL oxydées, et ne favoriseraient plus la production de NO. Mais si aussi bien la glycation que l’oxydation des HDL entraînent ces effets néfastes, il semblerait qu’en condition physiopathologique (oxydation spontanée des HDL glyquées), l’oxydation ne majore pas cette perte de capacité des HDL à contrecarrer les effets inhibiteurs des LDL oxydées sur la vasorelaxation. / Contrary to HDL from normolipidaemic and normoglycaemic subjects, HDL from diabetic patients have lost their capacity to reverse the inhibition of vasorelaxation induced by oxidized LDL. Mechanisms involved are unknown. The glycation and oxidation of HDL are two major phenomena in diabetes mellitus. The aim of this work was to study in vitro the role of glycation (with or without spontaneous oxidation) and oxidation of HDL, on their capacity to counteract the inhibitory effect of oxidized LDL on endothelium-dependent vasorelaxation. Each state showed the same result, modified HDL lost their vasorelaxing power in stress conditions (with oxidized LDL). Nevertheless, modified HDL alone (without oxidized LDL) did not alter vasorelaxation induced by acetylcholine, after noradrenaline-induced vasoconstriction. Thus, modifications of HDL induce a loss of the ability to protect vessels from oxidative stress rather than have a direct deleterious effect on the vessel. One of the major mechanisms involved in this phenomenon is probably the loss of SR-BI binding of these modified HDL, that could lead to the inability of HDL to protect caveolae from deleterious effects induced by oxidized LDL and could not preserve NO production. However, though glycation, like oxidation of HDL, leads to these deleterious effects, it would seem that during physiopathological conditions, with the spontaneous oxidation of glycated HDL, oxidation does not aggravate the loss of the capacity of diabetic HDL to counteract the inhibitory effect of oxidized LDL on endothelium-dependent vasorelaxation.

Agelanthus dodoneifolius est une plante de la pharmacopée africaine utilisée en médecine traditionnelle pour le traitement de pathologies cardiovasculaires. Un fractionnement bioguidé a permis d'isoler une nouvelle dihydropyranone : baptisée Dodonéine (Ddn), elle possède des propriétés hypotensives et vasorelaxantes chez le rat.L'objectif de la thèse est de caractériser la ou les cible(s) moléculaire(s) de la Ddn dans le système cardiovasculaire et d'identifier un dérivé plus sélectif.Nos résultats montrent que la Ddn bloque le courant calcique de type L des cardiomyocytes ventriculaires de rat et des cellules musculaires lisses vasculaires, d'environ 30% avec une IC50 de l'ordre du µM. La Ddn apparaît comme un nouveau bloqueur des canaux calciques de type L avec des propriétés électrophysiologiques qui lui sont propres. Une étude biochimique a montré que l'anhydrase carbonique (CA) est également inhibée par la Ddn, et nous avons caractérisé l'expression de plusieurs isoformes au sein de muscle lisse vasculaire : CA II, III, XIII et XIV. Nos travaux précisent que leur inhibition augmente le pH intracellulaire, pouvant conduire à l'activation des canaux KCa. Ainsi, la Ddn induit un effet vasorelaxant en inhibant deux protéines: les canaux calciques et l'anhydrase carbonique. Nous avons ensuite démontré que les dérivés de la Ddn nouvellement synthétisés sont aussi vasorelaxants ; toutefois, le plus efficace, la Ddn-Bicyclique-OH, ne concurrence pas les bloqueurs calciques utilisés en clinique. Cependant, l'effet vasorelaxant de la Ddn et Ddn-Bc-OH caractérisé sur des artères systémiques humaines est en accord avec l'utilisation de la plante en médecine traditionnelle. / Agelanthus dodoneifolius is one of the medicinal plants used in African pharmacopeia and traditional medicine for the treatment of cardiovascular diseases. Bioguided fractionation has allowed isolating one of its main active principles named Dodoneine (Ddn). It is a new dihydropyranone which exerts hypotensive and vasorelaxant effects on rat.The aim of this work is to characterize the molecular(s) target(s) of Ddn on cardiovascular system and identify the most selective and effective Ddn's derivative.Electrophysiological studies revealed that Ddn blocks L-type calcium current density of about 30% with an IC50 value of about 2 µM on cardiac myocytes and on vascular smooth muscle cells. Ddn appears as a new natural calcium channel blocker which has its own electrophysiological properties. As it has been shown, by biochemical study, that carbonic anhydrase is a potential target for Ddn, we have demonstrated that isozymes II, III, XIII are present on vascular smooth muscle cells and inhibited by Ddn. This inhibition resulted in a rise in pHi of about 0.31, leading to KCa channel activation. Interestingly, Ddn induced vasorelaxation by targeting two proteins: calcium channel and carbonic anhydrase. Then, we demonstrated that Ddn's derivatives newly synthetized have also vasorelaxant properties via the inhibition of L-type calcium current. Among them, Ddn-Bicyclique-OH is the most effective but appears not to be a better pharmacological tool compared to the calcium channel blockers already use in clinical. However, in accordance to the use of the plant in traditional medicine, our results clearly show that Ddn and Ddn-Bc-OH induce a vasorelaxant effect on human systemic artery.

Made available in DSpace on 2015-05-14T12:59:30Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3470014 bytes, checksum: ec4369a3d6de32a48a92ee347d505698 (MD5) Previous issue date: 2011-11-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Several epidemiological studies indicated that regular intake of red wine is associated with a decrease global mortality. The protective effect has been attributable, at least in part, to polyphenols. The cardiovascular effects induced by lyophilized red wine from Vale do São Francisco (LVTVSF) were evaluated in this study using in vivo and in vitro assays. The lyophilized red wine from Vale do São Francisco (LVTVSF) contains a series of flavonols, such, quercetin, myricetin and kaempferol. The red wine lyophilized was able to reduce mean arterial pressure (MAP) in L-NAME hypertensive rats compared to controls (143.7 ± 4.7 versus 172.5 ± 4.7 mmHg). In normotensive rats the administration of LVTVSF (10, 30, 90 mg/kg i.v.) randomly, induced hypotension (-24.0 ± 1.2; -32.4 ± 1.3; -51.4 ± 1.5%) and tachycardic effects (14.5 ± 2.3; 19.5 ± 1.7; 28.4 ± 2.1%). The pre-treatment with L-NAME (20 mg / kg i.v.), inhibitor of endothelial nitric oxide synthase, attenuated the hypotensive effect induced by LVTVSF. In isolated normotensive rat mesenteric artery rings LVTVSF elicited concentration-dependent relaxation of PHE-induced contraction (MR = 87.22 ± 3.59%). After removal of the vascular endothelial the vasorelaxant effect was significantly attenuated (MR = 32.07 ± 2.07%, p < 0.05), suggesting the involvement of endothelium-derived relaxing factor. After incubation with L-NAME (100 μM) and ODQ (10 mM), inhibitor of soluble guanylyl cyclase, the relaxation response was significantly attenuated (MR= 22.64 ± 3.74%, p < 0.05; MR = 37.08 ± 4.60%, p < 0.05, respectively). Similar results was also obtained rings incubed with KCl (20 mM), a modulator of potassium efflux; TEA (1 mM), nonselective blocker for potassium channels; (MR = 46.04 ± 9.87%, p < 0,05; MR = 44.40 ± 5.80%, p < 0,05; respectively). The LVTVSF was able to increase the NO levels in endothelial cells of the rabbit aorta. In the presence of charybdotoxin and apamin the relaxant response was not attenuated. Incubation with inhibitors of reactive oxygen species, N-acetylcysteine (NAC) (10 mM), tempol (100 μM) and apocynin (10 μM), reduces the vasorelaxation. After pre-treatment with atropine and indometacin, the vasorelaxant effect induced by LVTVSF was no attenuated. In endothelial cells of the rabbit aorta LVTVSF was able to increase production of ROS. These results together suggest that the hypotensive effect induced by LVTVSF in normotensive rats is probably due to a decrease in total peripheral resistance as a result of activation of the eNOS-NO-cGMP, involving the redox sensitive mechanism. These effects may be due to flavonoids found in LVTVSF. / Estudos epidemiológicos demonstram que o consumo regular de vinho está associado a um decréscimo da mortalidade global. Acredita-se que o efeito protetor seja decorrente, pelo menos em parte, da presença de polifenóis. A atividade biológica do Liofilizado do vinho tinto do Vale do São Francisco (LVTVSF) foi estudada no sistema cardiovascular usando técnicas combinadas in vivo e in vitro. A análise química do LVTVSF revelou a presença de vários polifenóis, como a quercetina, miricetina e caempferol. Em ratos com hipertensão induzida pelo L-NAME, o LVTVSF foi capaz de reduzir a pressão arterial média (PAM) quando comparado ao grupo controle (143,7 ± 4,7 versus 172,5 ± 4,7 mmHg), sem alterar a frequência cardíaca (FC). Ratos normotensos no qual foi administrado LVTVSF (10, 30, 90 mg/Kg i.v) aleatoriamente promoveu efeitos hipotensor (-24,0 ± 1,2; -32,4 ± 1,3; -51,4 ± 1,5 %),) e taquicárdico (14,5 ± 2,3; 19,5 ± 1,7; 28,4 ± 2,1%). A resposta hipotensora foi significantemente atenuada após a administração de NG-nitro- L-arginina-metil-éster (L-NAME) (20 mg/Kg i.v.), um inibidor da enzima sintase de óxido nítrico. Em preparações com anéis de artéria mesentérica superior isolada de ratos normotensos pré-contraídos com fenilefrina, o LVTVSF promoveu um efeito vasorelaxante (Emax= 87,22 ± 3,59%). Após a remoção do endotélio vascular o vasorelaxamento foi significativamente atenuado (Emax= 32,07 ± 2,07%, p < 0,05), sugerindo a participação de fatores relaxantes derivados do endotélio. Após a incubação com L-NAME (100 μM) e ODQ (10 μM), um inibidor da ciclase de guanilil solúvel, a resposta relaxante do composto foi significantemente atenuada (Emax.= 22,64 ± 3,74%, p < 0,05 e Emax= 37,08 ± 4,60%, p < 0,05, respectivamente). Resultados similares também foram obtidos em anéis com endotélio, na presença de KCl (20 mM), modulador do efluxo de K+; ou TEA ( 1mM), bloqueador não seletivo dos canais de K+; (Emax= 46,04 ± 9,87%, p < 0,05) e (Emax= 44,40 ± 5,80% p < 0,05). O LVTVSF aumentou os níveis de óxido nítrico em células endoteliais de aorta de coelho. Na presença de caribdotoxina (0,2 μM) e apamina (0,2 μM), bloqueadores dos canais de K+ sensiveis ao cálcio, o efeito relaxante induzido por LVTVSF não foi alterado. A incubação com os inibidores de espécies reativas de oxigênio, n-acetilcisteína (10 mM), tempol (100 μM) e apocinina (10 μM), diminuíram o vasorelaxamento. O efeito vasorelaxante não foi alterado na presença de atropina e indometacina, sugerindo que receptores muscarínicos, bem como metabólitos da via do ácido araquidônico não estão envolvidos neste efeito. Em células endoteliais de aorta de coelho o LVTVSF foi capaz de aumentar a produção de espécies reativas de oxigênio (ROS). Esses resultados em conjunto sugerem que o efeito hipotensor induzido por LVTVSF provavelmente ocorre devido à redução na resistência periférica total, resultado da ativação da via eNOS-NO-GMPc, envolvendo um mecanismo sensível a sinalização redox. Esses efeitos podem ser atribuídos aos flavonóides encontrados no LVTVSF.

Made available in DSpace on 2015-05-14T13:00:12Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1253693 bytes, checksum: 473208aa372beaf58b2dc01deecdc9be (MD5) Previous issue date: 2010-03-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Organic nitrates are nitric oxide (NO) donors used in the treatment of cardiovascular diseases mimicking the role of endogenous NO. This study evaluated organic nitrates newly synthesized from glycerin, which cardiovascular actions had not yet been investigated. Therefore, the cardiovascular effects produced by organic nitrates derived from glycerin: 2-nitrate-1,3-dimethoxypropan (NDMP), 2-nitrate-1,3-diethoxypropan (NDEP), 2-nitrate-1 ,3-dipropoxypropan (NDPP) and 2-nitrate-1,3-dibutoxypropan (NDBP) in rats were investigated using in vitro and in vivo approaches. For in vitro studies, animals were euthanized and the superior mesenteric artery was isolated. Artery rings were kept in tanks with Tyrode at 37 ° C aerated with carbogen, then were attached to a force transducer (Fort 10, WPI, Sarasota, USA) coupled to a data acquisition system data (Miobath-4, WPI, Sarasota, USA) under a tension of 0.75 g for 1 hour. After this period, preparations were pre-contracted with phenylephrine (FEN) 10 &#956;M or KCl 80 mM and then increasing concentrations of organic nitrates were cumulatively added. The nitrate with the most promising effects was selected for further studies and concentration-response curves of the compound selected in the presence of HDX, a hijacker of NO; ODQ, inhibitor of soluble guanylyl cyclase, KCl 20 mM, a modulator of potassium efflux, and blockers for calcium-sensitive potassium channel (TEA, 1 mM), blockers for ATP-sensitive potassium channel (GLIB, 1 &#61549;M) and blockers for voltage-operated potassium channel (4-AP, 1 mM) were obtained. All compounds showed vasorelaxant activity endothelium-independent in superior mesenteric artery rings, being the NDBP was the most potent agent with Emax = 105.4 ± 2.7% in rings pre-contracted with FEN and Emax = 82, 7 ± 7.9% in KCl 80 mM induced contraction. The vasorelaxation was significantly attenuated in the presence of HDX and ODQ with Emax = 62.8 ± 14.9% and Emax = 15.2 ± 9.2%, respectively. In the presence of KCl 20 mM the vasorelaxat response was also reduced [Emax = 70.6 ± 15.02%] as well as in the presence of TEA [Emax = 87.97 ± 5.78%]; GLIB [Emax = 78, 2 ± 6.5%] and 4-AP, a lesser extent [Emax = 94.65 ± 6.6%]. For in vivo studies, we investigated the changes in blood pressure (BP) and heart rate (HR) in conscious rats treated acutely with NDBP. Intravenous administration of NDBP (2, 5, 10, 15 and 20 mg/kg, randomly) produced hypotension (-6 ± 1.7, -22 ± 6.8, -58 ± 3.7, -70 ± 5.5, -77 ± 5.4 mmHg) and bradycardia (-12 ± 5, -40 ± 19.7, -133 ± 18.6, -179 ± 23.5, and -266 ± 12.4 bpm) in a dose-dependent manner. Thus, the vasorrelaxant response produced by NDBP possibly involves the NO release and subsequent activation of the CGs/GMPc/PKG pathway and BKCa, KATP and KV channels. These mechanism of action may be contributing to hypotension and bradycardia showed in non-anesthetized normotensive rats. / Os nitratos orgânicos são doadores de óxido nítrico (NO) utilizados no tratamento de doenças cardiovasculares mimetizando o papel do NO endógeno. Neste estudo foram avaliados nitratos orgânicos recém-sintetizados a partir da glicerina cujas ações cardiovasculares ainda não haviam sido investigadas. Portanto, os efeitos cardiovasculares do 2-nitrato-1,3-dimetoxipropano (NDMP), 2-nitrato-1,3-dietoxipropano (NDEP), 2-nitrato-1,3-dipropoxipropano (NDPP) e 2-nitrato-1,3-dibutoxipropano (NDBP) em ratos normotensos foram observados, utilizando técnicas in vitro e in vivo. Para os estudos in vitro, os animais foram eutanasiados e a artéria mesentérica superior foi isolada. Anéis de artéria mesentérica superior isolada de rato foram mantidos em cubas contendo Tyrode a 37 ºC gaseificado com carbogênio, em seguida foram fixados a um transdutor de força (FORT 10, WPI, Sarasota, EUA), acoplado a um sistema de aquisição de dados (Miobath-4, WPI, Sarasota, EUA) sob tensão de 0,75 g, durante 1 hora. Após este período, as preparações foram pré-contraídas com fenilefrina (FEN) 10 &#956;M ou KCl 80 mM e, em seguida, concentrações crescentes dos nitratos orgânicos, foram adicionadas cumulativamente. O nitrato com efeito mais promissor foi selecionado para estudos subsequentes e foram obtidas curvas concentração-resposta do composto na presença de HDX, sequestrador de NO; ODQ, inibidor da ciclase de guanilil solúvel; KCl 20 mM, modulador do efluxo de potássio; e bloqueadores de canais para K+ sensíveis ao cálcio (TEA, 1 mM), ao ATP (GLIB, 1 &#61549;M) e operados por voltagem (4-AP, 1 mM). Todos os compostos apresentaram atividade vasorrelaxante em anéis de artéria mesentérica superior isolada de rato independente do endotélio funcional, sendo o NDBP o mais potente com Emáx = 105,4 ± 2,7 % em anéis pré-contraídos com FEN e Emáx = 82,7 ± 7,9 % na contração induzida por KCL 80 mM. O vasorrelaxamento foi significativamente atenuado na presença de HDX e ODQ, com Emáx = 62,8 ± 14,9 % e Emáx = 15,2 ± 9,2 %, respectivamente. Na presença de KCl 20 mM a resposta vasorrelaxante também foi reduzida [Emáx = 70,6 ± 15,02 %], bem como na presença do TEA [Emáx = 87,97 ± 5,78 %]; GLIB [Emáx = 78,2 ± 6,5 %] e 4-AP, em menor proporção [Emáx = 94,65 ± 6,6 %]. Para os estudos in vivo, foram investigadas as alterações na pressão arterial (PA) e frequência cardíaca (FC) em ratos não-anestesiados tratados agudamente com o NDBP. A administração aleatória do NDBP (2, 5, 10, 15 e 20 mg/kg, i. v) produziu hipotensão (-6 ± 1,7; -22 ±6,8; -58 ± 3,7; -70 ± 5,5; -77 ± 5,4 mmHg) e bradicardia (-12 ± 5; -40 ± 19,7; -133 ± 18,6; -179 ± 23,5; e -266 ± 12,4 bpm) de maneira dose-dependente. Deste modo, a resposta vasorrelaxante promovida pelo NDBP possivelmente envolve a liberação de NO e posterior ativação da via CGs/GMPc/PKG e canais para K+ do tipo BKCa; KATP e KV. Este mecanismo de ação pode estar contribuindo para a hipotensão e bradicardia observadas em animais normotensos não-anestesiados.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21973号 / 農博第2363号 / 新制||農||1071(附属図書館) / 学位論文||R1||N5224(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 井上 和生, 教授 谷 史人, 准教授 大日向 耕作 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

No description available.

L'objectif de notre travail a consisté en l'étude des mécanismes enzymatiques impliqués dans la libération de l'oxyde nitrique à partir des S-nitrosothiols (RSNO) et dans leurs effets vasorelaxants. Notre intérêt porte sur deux enzymes : la gamma-glutamyltransférase (GGT) et la protéine disulfure isomérase (PDI) car elles jouent un rôle important dans la dénitrosation des RSNO. Nous avons choisi d'étudier la dénitrosation de deux RSNO : le S-nitrosoglutathion (GSNO), un mononitrosothiol endogène et la S,S'-dinitrosobucillamine (BUC(NO)2), un nouveau dinitrosothiol. Nous avons synthétisé ces RSNO et nous avons vérifié la nature du produit obtenu par une caractérisation physico-chimique complète. Les analyses ont montré que ces RSNO présentent une pureté élevée (>97%) avec un niveau faible d'impuretés permettant leur utilisation dans des expérimentations biologiques. Les effets vasorelaxants des RSNO ainsi que l'implication des enzymes ont été évalués. Nos résultats montrent que la GGT et la PDI sont capables de dénitroser in vitro le GSNO. Le modèle ex vivo d'anneau aortique isolé de rat Wistar nous a permis de démontrer que l'effet vasorelaxant de GSNO (CE50=3,2±0,5.10-7 M) est dépendant de l'endothélium et de l'activité de la GGT et de la PDI. Concernant la BUC(NO)2, ce dinitrosothiol est catabolisé in vitro par la PDI, est un vasorelaxant plus puissant que la plupart des RSNO (CE50=2,2±0,2.10-8 M) et met en jeu l'activité de la PDI vasculaire. Nos travaux ont conduit à une meilleure compréhension des mécanismes enzymatiques impliqués dans les effets vasculaires des RSNO, ce qui permettra d'optimiser le choix de la meilleure RSNO à utiliser dans une finalité thérapeutique / The aim of our work was to evaluate the enzymatic pathways involved in the release of nitric oxide and in the vasorelaxant effect of S-nitrosothiols (RSNO). We were interested in two enzymes: the gamma-glutamyltransferase (GGT) and the protein disulfide isomerase (PDI), because they play an important role in RSNO denitrosation. Two RSNO were studied: S-nitrosoglutathione (GSNO), an endogenous mononitrosothiol, and S,S'-dinitrosobucillamine (BUC(NO)2), a new dinitrosothiol. We synthesized RSNO and we structurally characterized these products. The resulting data are consistent with the expected structure. Our products have a high purity (>97%) and a limited amount of impurities allowing their suitable use in biological experiments. The vasorelaxant effects of RSNO and the involvement of GGT and PDI were evaluated. The results indicate that purified GGT and PDI denitrosate GSNO in vitro. Furthermore, we demonstrated by using an ex vivo model consisting in an aortic ring isolated from Wistar rat that the vasorelaxant effect of GSNO (EC50=3,2±0,5.10-7 M) was dependent on the endothelium and GGT and PDI activities. As concerns BUC(NO)2, this dinitrosothiol catabolized in vitro by PDI, is more potent (EC50=2,2±0,2.10-8 M) than the most of nitrosothiols described in the literature. This vasorelaxation effect was dependent on PDI activity. In conclusion, our data led to a better understanding of the enzymatic mechanisms involved in the vascular effects of RSNO, which will permit, in physiopathological context, to optimize the choice of the best RSNO for use in a therapeutic purpose

Submitted by Clebson Anjos (clebson.leandro54@gmail.com) on 2016-03-30T18:39:51Z No. of bitstreams: 1 arquivototal.pdf: 1445770 bytes, checksum: 93635c33711265255604ec58e14b9042 (MD5) / Made available in DSpace on 2016-03-30T18:39:51Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1445770 bytes, checksum: 93635c33711265255604ec58e14b9042 (MD5) Previous issue date: 2015-08-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The process of discovering new drugs that act on the smooth muscle layer causing vascular relaxation is of fundamental importance in the treatment of cardiovascular diseases. In this context highlights the isatin, a versatile substance found in various tissues of mammals and plants which can easily undergo structural changes to yield new substances. The pharmacological effects of four novel drugs derived from isatin (IS-AK1, IS-BK1, B-001 and D-001) on the cardiovascular system using techniques in vivo and in vitro. In in vitro studies, cranial mesenteric rings from rat were kept in vats containing Tyrode at 37 ° C gassed with carbogen then they were fixed to a force transducer (PowerLab™, ADInstruments, MA, EUA) and coupled to a data acquisition system (WinDaq/XL, DI 148-U, Insight, Brasil) under tension 0.75 g, for 1 hour. After this period, the preparations were pre-contracted with phenylephrine (PHE, 10 μM) and KCl (60 mM) and then increasing concentrations of isatin derivatives were added cumulatively. All tested compounds showed vasorelaxant effect and IS-AK1 the most promising compound for presenting higher power when both in presence and in absence of endothelium compared to others (pD2 = 7.99 ± 0.11 and 7.95 ± 0.15, respectively), but these vasorelaxant responses were not different (Emax = 85.69 ± 5.18% vs. 74.62 ± 5.33%) in presence or in absence of endothelium. Therefore, IS-AK1 was then was selected for further studies in order to investigate its mechanism of action of the compound by concentration-response curves in the presence of blockers ODQ (10 μM), cyclase blocker soluble guanylyl; TEA 3 (mM) nonspecific blocker of K+ channels; TEA (1 mM) BKCa blocker; GLIB (1 μM), KATP blocker; 4-AP (1 mM) KV blocker and BaCl2 (30 μM), KIR blocker. In the presence of ODQ vasorelaxation was significantly attenuated (Emax = 39.68 ± 6.56%; pD2 = 5.07 ± 0.2, n = 6). In addition, in the presence of TEA 3 mM (Emax = 43.53 ± 8.16%, 6.72 pD2 ± 0.2, n = 6) and presence of TEA 1 mM (Emax = 39 72 ± 7.86%; pD2 = 5.63 ± 0.2, n = 6) vasorelaxant response were reduced. There was reduction in the power of the IS-AK1 when blocked by GLIB (pD2 = 7.23 ± 0.17, n = 5), 4-AP (pD2 = 7.1 ± 0.17, n = 5) and BaCl2 (pD2 = 6.23 ± 0.16, n = 5), suggesting the participation of different types of K+ channels in this response. For in vivo studies, changes in blood pressure and heart rate were investigated in non-anesthetized rats treated acutely with the IS-AK1. The IS-AK1 administration (10 mg/kg) produced blood pressure and bradycardia both in normotensive (-50 ± 12 mmHg -258 ± 40 bpm, n = 7) and hypertensive rats (-99 ± 7 mmHg, -278 ± 40 bpm, n = 6) with effects more pronounced in hypotensor effect in hypertensive rats. Thus, the vasorelaxant response promoted by IS-AK1 possibly involves the participation of sGC enzyme, thereby leading to the activation of PKG causes activation of K+ channels, KATP, KV, KIR and especially BKCa types. This mechanism of action may be contributing to hypotension and bradycardia observed in non-anesthetized rats. / O processo de descoberta de novas drogas que atuam na camada muscular lisa causando relaxamento vascular tem importância fundamental no tratamento de doenças cardiovasculares. Neste contexto, destaca-se a isatina, uma substância bastante versátil encontrada em diversos tecidos de mamíferos e de plantas que pode facilmente sofrer modificações estruturais para dar origem a novas substâncias. Neste contexto foram estudados os efeitos farmacológicos de quatro novas drogas derivadas da isatina (IS-AK1, IS-BK1, B-001 e D-001) sobre o sistema cardiovascular, utilizando-se técnicas in vivo e in vitro. Nos estudos in vitro, anéis de mesentérica cranial isolada de rato foram mantidos em cubas contendo Tyrode a 37 ºC gaseificado com carbogênio, em seguida foram fixados a um transdutor de força (PowerLab™, ADInstruments, MA, EUA) e acoplado a um sistema de aquisição de dados (WinDaq/XL, DI 148-U, Insight, Brasil) sob tensão de 0,75 g, durante 1 hora. Após este período, as preparações foram pré-contraídas com fenilefrina (FEN, 10 μM) ou KCl (60 mM) e em seguida, concentrações crescentes dos derivados da isatina foram adicionadas cumulativamente. Todos os compostos testados apresentaram efeito vasorrelaxante, sendo o IS-AK1 o composto mais promissor por apresentar maior potência tanto na presença como na ausência do endotélio (pD2 = 7,99 ± 0,11 e 7,95 ± 0,15, respectivamente). Entretanto, as respostas vasorrelaxantes máximas não foram diferentes (Emax = 85,69 ± 5,18 % vs. 74,62 ± 5,33 %) na presença e na ausência de endotélio. Portanto, o IS-AK1 foi selecionado para estudos subsequentes afim de investigar seu mecanismo de ação por meio de curvas concentração-resposta ao composto na presença dos bloqueadores ODQ (10 μM), bloqueador da ciclase de guanilil solúvel; TEA (3 mM), bloqueador inespecífico de canais para K+; TEA (1 mM), bloqueador de BKCa; GLIB (1 μM), bloqueador de KATP; 4-AP (1 mM), bloqueador de Kv e BaCl2 (30 μM), bloqueador de KIR. Na presença do ODQ, o vasorrelaxamento foi significativamente atenuado (Emax = 39,68 ± 6,56 %; pD2 = 5,07 ± 0,2, n = 6). Além disso, a resposta vasorrelaxante foi reduzida na presença de TEA (3 mM), (Emax = 43,53 ± 8,16 %; pD2 6,72 ± 0,2, n = 6) e na presença de TEA (1 mM) (Emax = 39,72 ± 7,86 %; pD2 = 5,63 ± 0,2, n = 6). Ocorreu redução na potência do IS-AK1 quando bloqueado por GLIB (pD2 = 7,23 ± 0,17, n = 5), 4-AP (pD2 = 7,1 ± 0,17, n = 5) e BaCl2 (pD2 = 6,23 ± 0,16, n = 5), sugerindo a participação de diferentes canais para K+ nesta resposta. Para os estudos in vivo, foram investigadas as alterações na pressão arterial e frequência cardíaca em ratos não-anestesiados tratados agudamente com o IS-AK1. A administração de IS-AK1 (10 mg/kg) produziu hipotensão e bradicardia tanto em ratos normotensos (-50 ± 12 mmHg, -258 ± 40 bpm, n = 7) quanto em ratos hipertensos (-99 ± 7 mmHg, -278 ± 40 bpm, n = 6) com efeito mais pronunciado no efeito hipotensor em ratos hipertensos. Deste modo, a resposta vasorrelaxante promovida pelo IS-AK1 parece envolver a participação da enzima sGC, consequentemente levando a ativação da PKG, ocasionando a ativação de canais para K+ do tipo KATP, KV, KIR e, principalmente, BKCa. Este mecanismo de ação pode estar contribuindo para a hipotensão e bradicardia observadas em ratos não-anestesiados.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-02-02T13:02:03Z No. of bitstreams: 1 arquivototal.pdf: 2110108 bytes, checksum: 608a6dd3b7ef97ff56ce7a532de666a1 (MD5) / Made available in DSpace on 2017-02-02T13:02:03Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2110108 bytes, checksum: 608a6dd3b7ef97ff56ce7a532de666a1 (MD5) Previous issue date: 2016-04-26 / This study aimed to quantify the content of phenolic compounds of the bark extracts of the stem and pulp of the fruit of Hymenaea rubriflora Ducke and its effect on vascular reactivity to contractile and relaxing agents thoracic aorta of rats, as well as oxidative stress and biochemical parameters. extraction was performed phenolic compounds of the shell, the stem and the pulp using the ratio of water and alcohol solvents (1: 1). The presence of total phenolic compounds was performed by the Folin-Ciocalteu method and the antioxidant capacity for the kidnapping of free radical DPPH of hydroalcoholic extracts. The vasorelaxant effect of the hydroalcoholic extract of the stem bark (HR-HAc), as well as acute toxicity for the realization of rats supplemented rats with HR-HACwas studied at doses of 25 (G25), 50 (G50) and 150 mg / kg (G100) for four weeks (8 animals / group). Biochemical analysis was used to quantify nitrite (NO), malondialdehyde (MDA), antioxidant activity, lipid profile, and glucose. It was identified that the HR-HAcshowed higher concentration of phenolic compounds (274, 63 mg AC Gál / g) and antioxidant activity (5076.50 micromol Tx / g), the fruit pulp extract (HR-HAp) ( Ac Ga 3.49 mg / g and 29.26 micromol Tx / g, respectively). In addition, the HR-HAcrelaxed the aorta pre-contracted with mouse FEN concentration dependent manner in both, presence and absence of functional endothelium (EC50 = 5.0 ± 1.1 vs. 8.7 ± 1.8 ug / mL), and the attenuated relaxation in the presence of L-NAME (EC50 = 142.4 ± 21.6 mg / mL), however, was not observed change in the relaxation induced by the extract in the presence of indometHAcin (EC50 = 2.9 ± 0.3 ug / mL). Acute supplementation HR-HAcshowed low toxicity, while the chronic supplementation showed increased relaxation potency of ACh compared to G50 G100 (pD2 = 7.8 ± 0.1 vs. 7.6 ± 0.1). The cumulative curve with sodium nitroprusside (SNP) showed greater potency in relaxing G100 compared to G50 (pD2 = 12.1 ± 0.04 vs 11.3 ± 0.3). The contractile efficacy without endothelium in rat aorta was reduced G100 (Emax = 40.4 ± 5.1%). The cumulative curve FEN in the presence of L-NAME showed reduction in contractile potency in rat aorta with endothelium G25 and G50 in comparison GC in the presence and absence of L-NAME (pD2 = 6.4 ± 0.1, 6.4 ± 0.1 and 6.9 ± 0.1 respectively) as well as contractile efficiency of FEN was reduced in the groups G25, G50 and G100 when compared to the control group in the presence and absence of L-NAME (Emax = 61.4 ± 5.2, 74.4 ± 5.3, 76.9 ± 3.7, 100 and 100%, respectively). It was found that there was no significant difference in the values of antioxidant capacity, NO, lipid profile and glucose in plasma of the treated animals and the control group. Thus, it is concluded that this product may be indicated for prophylactic benefits to pathologies related to changes in vascular reactivity. / O presente estudo teve como objetivo quantificar o conteúdo de compostos fenólicos de extratos da casca do caule e da polpa do fruto da Hymenaea rubriflora Ducke e sua ação na reatividade vascular a agentes contráteis e relaxantes de aorta torácica de ratos, assim como extresse oxidativo e parâmetros bioquímicos. Foi realizada extração dos compostos fenólicos da casca do caule e da polpa utilizando como solventes água e álcool etílico (1:1). A presença de compostos fenólicos totais foi realizada pelo método de Folin-Ciocalteau e a capacidade antioxidante pelo sequestro do radical livre DPPH dos extratos hidroalcóolicos. O efeito vasorrelaxante do extrato hidroalcóolico da casca do caule (HR-HAc), assim como a toxicidade aguda para a realização da suplementação de ratos Wistar com o HR-HAc foram investigados nas doses de 25 (G25), 50 (G50) e 150 mg/kg (G100) por quatro semanas (8 animais/grupo). A análise bioquímica foi utilizada para quantificar nitrito (NO), malondialdeído (MDA), atividade antioxidante, perfil lipídico e glicose. Identificou-se que o HR-HAc apresentou maior concentração de compostos fenólicos (274, 63 mg ác gál/g) e atividade antioxidante (5076,50 μmol Tx/g), que o extrato da polpa do fruto (HR-HAp) (3,49 mg ác gál/g e 29,26 μmol Tx/g, respectivamente). Além disso, o HR-HAc relaxou a aorta de rato pré-contraída com FEN de maneira dependente de concentração em ambos, presença e ausência do endotélio funcional (CE50 = 5,0 ± 1,1 vs 8,7 ± 1,8 μg/mL), sendo o relaxamento atenuado na presença do L-NAME (CE50 =142,4 ± 21,6 μg/mL), entretanto, não foi observado alteração no relaxamento induzido pelo extrato na presença da indometacina (CE50 = 2,9 ± 0,3 μg/mL). A suplementação aguda do HR-HAc apresentou baixa toxicidade, enquanto que a suplementação crônica mostrou aumento da potência relaxante da ACh em G50, quando comparado ao G100 (pD2 = 7,8 ± 0,1 vs 7,6± 0,1). A curva cumulativa com nitroprussiato de sódio (NPS) apresentou maior potencia relaxante em G100 quando comparado a G50 (pD2= 12,1 ± 0,04 vs 11,3 ± 0,3). A eficácia contrátil em aorta de rato sem endotélio foi reduzida em G100 (Emax = 40,4 ± 5,1%). A curva cumulativa a FEN na presença de L-NAME mostrou diminuição na potência contrátil em aorta de rato com endotélio em G25 e G50 quando comparado GC na presença e ausência L-NAME (pD2 = 6,4 ± 0,1, 6,4± 0,1 e 6,9 ± 0,1, respectivamente), assim como a eficácia contrátil da FEN foi reduzida nos grupos G25, G50 e G100 quando comparado ao GC na presença e ausência de L-NAME (Emax = 61,4 ± 5,2; 74,4 ± 5,3; 76,9 ± 3,7; 100 e 100%, respectivamente). Verificou-se que não ocorreu diferença significativa para os valores da capacidade antioxidante, NO, perfil lipídico e glicose no plasma dos animais tratados e do grupo controle. Desse modo, conclui-se que este produto poderá ser indicado para benefícios profiláticos a patologias relacionadas à alterações na reatividade vascular.

Made available in DSpace on 2015-05-14T12:59:49Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1974119 bytes, checksum: 87d088fb9d6973822abebb19f69d61d5 (MD5) Previous issue date: 2013-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The organic nitrates are classified as drugs donors of nitric oxide (NO) that might potentially be useful in the treatment of cardiovascular diseases, principally to mimick endogenous NO. A new-found organic nitrate, the nitrate-12- cis- 9- ethyl octadecanoate (NCOE), synthesized from ricinoleic acid from castor oil, was studied in order to evaluate its cardiovascular effects, using in vivo and in vitro approaches. In normotensive rats, NCOE (10, 20, 30, 40 and 60 mg /kg, iv) induced hypotension (2.5 ± 0.9, -3.9 ± 0.7, -31.0 ± 6.6, -40.6 ± 3.9 and -50.4 ± 3.5%) and bradycardia (-5.6 ± 0.9, -8.9 ± 1.0, -57.2 ± 8.9; -70.9 ± 5.3 and -77.9 ± 2.7%) transients, both dose-dependent effects. In isolated rat superior mesenteric artery rings pre-contracted with phenylephrine (Phe) (1 mM), NCOE (10-10-10-3 M) induced a concentration-dependent vasorelaxation in presence (MR = 107.3 ± 4.43%; pD2 = 5.59 ± 0.06) or absence (MR = 118.0 ± 3.5%; pD2 = 5.90 ± 0.05) of endothelium, suggesting a NCOE effect independent of endothelium-derived factor. All subsequent experiments were performed in the absence of endothelium. The NCOE effect was attenuated after a contraction induced by depolarizing solution with high K+ (MR = 92.0 ± 4.1%) compared to the nitrate effect on Phe-contractions. The pre-incubation with PTIO (300 mM), a free radical form of NO (NO&#9679;) scavenger, attenuated the NCOE vasorelaxation potency (pD2 = 5.10 ± 0.05), suggesting NO&#9679; involvement in the nitrate effect. However, in the presence of L-cysteine (3 mM), a reduced form of NO (NO-) scavenger, NCOE response was potentiated (pD2 = 6.34 ± 0.03). Similar effect was observed in the presence of N-acetylcysteine (NAC) (3 mM), a free radicals intracellular scavenger (pD2 = 6.56 ± 0.05). The NCOE effect was not altered in the presence of proadifeno (10 mM), an inhibitor of cytochrome P450 (pD2 = 5,99 ± 0,07%). However, the vasodilation was reduced in the presence of cyanamide (1 mM), inhibitor of mitochondrial aldehyde dehydrogenase (mtALDH) (MR = 94.3 ± 6.26%), and of ODQ (10 &#956;M), inhibitor of soluble guanylyl cyclase (sGC) (MR = 55.2 ± 3.60%), suggesting these enzymes involvement in NCOE response. After preincubation with TEA (3 mM), a K+ channels non-selective blocker, the nitrate vasorelaxation was reduced (MR = 107.1 ± 7.09), demonstrating the participation of these channels in nitrate effect. When using iberiotoxin (100 nM) and glibenclamide (10 &#956;M), KATP and BKCa selective blockers, respectively, the vasodilatory effect was reduced (MR = 106.2 ± 1.49%; pD2 = 5.61 ± 0.04, respectively), although the effect was not modified in the presence of 4-aminopyridine (1 mM), KV blocker (pD2 = 5,70 ± 0,04%). Furthermore, NCOE increased NO levels in rat aorta smooth muscle cells, detected by NO-sensitive dye DAF-2T. These results together suggest that NCOE induces hypotension and bradycardia transients, and promotes vasorelaxation due NO&#9679; release through the compound metabolism via mtALDH and consequent sGC, KATP and KBCa activation. / Os nitratos orgânicos são classificados como drogas doadoras de óxido nítrico (NO), utilizadas no tratamento de doenças cardiovasculares, mimetizando as ações do NO endógeno. Um nitrato orgânico inédito, o 12-nitrato-cis-9-octadecanoato de etila (NCOE), sintetizado a partir do ácido ricinoléico do óleo da mamona, foi estudado com o objetivo de avaliar seus efeitos cardiovasculares, utilizando abordagens in vivo e in vitro. Em ratos normotensos, o NCOE (10; 20; 30; 40 e 60 mg/kg, i.v.) promoveu hipotensão (- 2,5 ± 0,9; -3,9 ± 0,7; -31,0 ± 6,6; -40,6 ± 3,9 e -50,4 ± 3,5%) e bradicardia (-5,6 ± 0,9; -8,9 ± 1,0; -57,2 ± 8,9; -70,9 ± 5,3 e -77,9 ± 2,7%) transientes, ambas dependente de dose. Em anéis de artéria mesentérica superior isolada de rato, pré-contraídos com fenilefrina (FEN) (1 &#956;M), o NCOE (10-10 - 10-3 M) induziu vasorrelaxamento, dependente de concentração, na presença (Emáx = 107,3 ± 4,43%; pD2 = 5,59 ± 0,06) e ausência (Emáx = 118,0 ± 3,53%; pD2 = 5,90 ± 0,05) do endotélio, sugerindo que o efeito do NCOE parece ser independente dos fatores liberados pelo endotélio. Todos os experimentos subsequentes foram realizados na ausência do endotélio. O efeito do NCOE foi atenuado após contração induzida por solução despolarizante de 60 mM de KCl (Emáx = 92,0 ± 4,1%), quando comparado com o efeito do NCOE frente à FEN. A pré-incubação com PTIO (300 &#956;M), sequestrador do NO na forma radicalar (NO&#9679;), atenuou a potência do vasorrelaxamento do NCOE (pD2 = 5,10 ± 0,05), sugerindo a participação do NO&#9679; no efeito deste nitrato. Entretanto, na presença de L-cisteína (3 mM), um sequestrador do NO na forma reduzida (NO-), o vasorrelaxamento do NCOE foi potencializado (pD2 = 6,34 ± 0,03). Efeito semelhante foi observado na presença da N-acetilcisteína (NAC) (3 mM), um sequestrador de radicais livres intracelulares (pD2= 6,56 ± 0,05). O efeito do NCOE não foi alterado na presença do proadifeno (10 &#956;M), um inibidor do citocromo P450 (pD2 = 5,99 ± 0,07%). No entanto, o efeito vasorrelaxante foi reduzido na presença da cianamida (1 mM), inibidor da aldeído desidrogenase mitocondrial (mtALDH) (Emàx = 94,3 ± 6,26%); e do ODQ 10 &#956;M, inibidor da ciclase de guanilil solúvel (sGC) (Emáx = 55,2± 3,60%), sugerindo o envolvimento destas enzimas no efeito do NCOE. Após pré-incubação com TEA (3 mM), concentração que bloqueia de forma não seletivo os canais para K+, o vasorrelaxamento do nitrato foi atenuado (Emáx = 107,1 ± 7,09%), demonstrando o envolvimento destes canais neste efeito. Ao utilizar a iberiotoxina (100 nM) e a glibenclamida (10 &#956;M), bloqueadores seletivos dos BKCa e KATP, respectivamente, o efeito vasodilatador foi diminuído (Emàx = 106,2 ± 1,49%; pD2 = 5,61 ± 0,04, respectivamente); entretanto, o efeito não foi modificado na presença de 4-aminopiridina (1 mM), bloqueador dos KV (pD2 = 5,70 ± 0,04%). Além disso, NCOE aumentou os níveis de NO em células musculares lisas de aorta, detectado pela fluorescência emitida por DAF-2T. Estes resultados, em conjunto, sugerem que NCOE induz hipotensão e bradicardia transientes, e promove vasorrelaxamento, devido a liberação de NO&#9679;, por meio da metabolização do composto via mtALDH, e consequente ativação da sGC e dos canais KATP e KBCa.