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Made available in DSpace on 2015-05-14T13:00:06Z (GMT). No. of bitstreams: 1 parte1.pdf: 1450137 bytes, checksum: 1838bf2efddf0ca147f276df88417bb6 (MD5) Previous issue date: 2009-09-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / It was been demonstrated that curine and reticuline, induced a vasodilator effect in the rat small mesenteric arteries through inhibition of voltage-gated Ca2+ channels (VGCC). These compounds, curine and reticuline were isolated from the root barks of Chondrondendron platyphyllum and Ocotea duckei Vattimo, respectively, therefore the aim of this work was to evaluate the vasodilator mechanism of curine and reticuline, bisbenzylisoquinoline alkaloids (BBA), isolated from the root barks of Chondrondendron platyphyllum and Ocotea duckei Vattimo, respectively, using functional and molecular approaches. Tension measurements in aorta rings, whole-cell patch-clamp and confocal techniques were employed to study the action of these alkaloids. The A7r5 smooth muscle derived cell line was used for Ca2+ currents measuring and the intracellular calcium concentration ([Ca2+]i) were evaluated using confocal microscopy. The main results are as follows: in aortic rings, curine (3 - 300 μM) antagonized KCl (60 mM) and Bay K8644 (3 x 10-7 M) induced contractions. In whole-cell configuration, curine reduced the voltage-activated peak amplitude of ICa,L in a concentration-dependent manner. However, the Ca+2 current density versus voltage relationship and maximal activation voltage of ICa,L were not changed. Moreover curine did not also affect the steady-state activation of ICa,L, but shifted the steady-state inactivation curve of ICa,L for more negative potentials, however this effect was not changed in the presence of IBMX, dbcAMP and 8-brcGMP, suggesting that cyclic mononucleotides, such as cAMP and cGMP, are not involved in curine effect. In confocal experiments, curine inhibited the rise on the [Ca2+]i induced by KCl (60 mM) in dispersed vascular smooth muscle cells. In reference to reticuline (3 300 μM) was verified that alkaloid agonized CaCl2 and KCl-induced contractions and elicited vasorelaxation in aortic rings. In whole-cell configuration, reticuline reduced the voltage-activated peak amplitude of ICa,L in a concentration-dependent manner, but did not change the characteristics of current density versus. voltage relationship. Reticuline shifted leftwards the steady-state inactivation curve of ICa,L, however this effect was not changed after application of dibutyryl cyclic adenosine monophosphate to the cell. In cells pretreated with forskolin, an adenylate cyclase activator, the addition of reticuline caused further inhibition of the Ca2+ currents suggesting an additive effect, indicating that cyclic mononucleotides were not involved. Taken together the results have shown that curine and reticuline elicits vasorelaxation due to the blockade of the L-type voltage-dependent Ca2+ current in rat aorta smooth muscle cells. The reported effect may contribute to the potential cardioprotective efficacy of curine and reticuline. / Curina e reticulina são alcalóides isolados das cascas do caule e raízes de Chondrondendron platyphyllum e de Ocotea duckei Vattimo, respectivamente. Estudos anteriores demonstraram que esses alcalóides são capazes de induzir efeito vasodilatador em artéria mesentérica e aorta de rato, respectivamente, devido possível inibição dos canais para Ca2+ dependentes de voltagem (VGCC). O objetivo deste trabalho foi investigar o mecanismo vasodilatador de curina e reticulina realizando experimentações funcionais e moleculares. Foram utilizadas medidas de tensão em anéis de aorta de rato, e empregadas técnicas de patch-clamp e de microscopia confocal para estudos da ação desses alcalóides. Também foram utilizadas células A7r5, uma linhagem de células musculares lisas embrionária derivada de aorta torácica de rato, que foram usadas para medir as correntes de Ca2+ macroscópicas e a concentração de cálcio intracelular ([Ca2+]i), que foram avaliadas usando a técnicas de patch-clamp e microscopia confocal, respectivamente. Os principais resultados são: em anéis de aorta, curina (3 - 300 μM) antagonizou as contrações induzidas por KCl (60 mM) e Bay K8644 (3 x 10-7 M). Na configuração whole-cell patch clamp , curina reduziu a amplitude da corrente de cálcio do tipo L (ICa,L) de maneira dependente de concentração. Porém, curina não alterou as características das correntes na relação corrente-voltagem. A voltagem de ativação máxima para ICa,L não foi diferente em relação ao controle. Além disso, curina também não afetou a ativação no estado estacionário das ICa,L, mas deslocou a curva da inativação estacionária para potenciais mais negativos. No entanto, esse efeito promovido por curina não foi alterado na presença de IBMX, dbcAMP e 8- brcGMP, sugerindo que os mononucleotídeos cíclicos, como APMc e GMPc, não estão envolvidos no efeito da curina. Em experimentos com microscopia confocal curina inibiu os transientes de cálcio intracelulares, e reduziu o aumento de [Ca2+]i induzidos por KCl (60 mM) em células de músculo liso vascular. Em relação à reticulina (3 300 μM), foi verificado que esse alcalóide antagonizou as contrações induzidas por CaCl2 e KCl, provocando vasorelaxamento em anéis de aorta. Na configuração whole-cell patch clamp , reticulina também reduziu a amplitude das ICa,L de maneira dependente de concentração, mas não mudou as características da corrente na relação corrente-voltagem. A reticulina deslocou para potenciais mais negativos a curva de inativação estacionária para as ICa,L. Porém, esse efeito não foi alterado após a aplicação de dbcAMP e 8-brcGMP. Em células pré-tradadas com forskolina, um ativador da adenilil ciclase, a adição da reticulina causou uma inibição adicional das correntes de Ca2+ que sugere um efeito aditivo da reticulina, indicando que os mononucleotídeos cíclicos não estão envolvidos. Dessa forma, curina e reticulina provocaram vasorelaxamento, devido ao bloqueio das correntes de Ca2+ dependentes de voltagem do tipo-L em células de músculo liso, em cultura e recémdispersas, de aorta de rato, revelando que esses alcalóides têm um importante potencial como modelo químico para a concepção e posterior desenvolvimento de novos fármacos com propriedade protetora cardiovascular.

Recent studies show that angiotensin II (AngII) can act from within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane AngII receptors. The role of this intracellular AngII (AngIIi) is unclear. Besides direct effects of AngIIi on cellular processes one could hypothesise a possible role of AngIIi in modulation of cellular responses induced after heterologous receptor stimulation. We therefore examined if AngIIi influences [Ca2+]i in A7r5 smooth muscle cells after serotonin (5HT) or UTP receptor stimulation. Application of AngIIi using liposomes, markedly inhibited 45Ca2+ influx after receptor stimulation with 5HT or UTP. This inhibition was reversible by intracellular administration of the AT1-antagonist losartan and not influenced by the AT2-antagonist PD123319. Similar results were obtained in single cell [Ca2+]i measurements, showing that AngIIi predominantly influences Ca2+ influx and not Ca2+ release via AT1-like receptors. It is concluded that AngIIi modulates signal transduction activated by heterologous receptor stimulation.

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Recent studies show that angiotensin II can act within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane angiotensin II receptors. The signal transduction of intracellular angiotensin II is unclear. Therefore, we investigated the effects of intracellular angiotensin II in cells devoid of physiological responses to extracellular angiotensin II (A7r5 vascular smooth muscle cells). Intracellular delivery of angiotensin II was obtained by using liposomes or cell permeabilisation. Intracellular angiotensin II stimulated Ca2+ influx, as measured by 45Ca2+ uptake and single-cell fluorimetry. This effect was insensitive to extracellular or intracellular addition of losartan (angiotensin AT1 receptor antagonist) or PD123319 ((s)-1-(4-[dimethylamino]-3-methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7- tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylate) (angiotensin AT2 receptor antagonist). Intracellular angiotensin II stimulated inositol-1,4,5-trisphosphate (Ins(1,4,5,)P3) production and increased the size of the Ins(1,4,5,)P3 releasable 45Ca2+ pool in permeabilised cells, independent of losartan and PD123319. Small G-proteins did not participate in this process, as assessed by using GDPβS. Intracellular delivery of angiotensin I was unable to elicit any of the effects elicited by intracellular angiotensin II. We conclude from our intracellular angiotensin application experiments that angiotensin II modulates Ca2+ homeostasis even in the absence of extracellular actions. Pharmacological properties suggest the involvement of putative angiotensin non-AT1-/non-AT2 receptors.

Résumé : La contraction cellulaire est une activité centrale dans plusieurs processus physiologiques. Entre autre, elle joue un rôle dans la régulation de la perméabilité vasculaire et de la pression artérielle. Il est également établi qu’une contraction anormale des cellules du muscle lisse vasculaire (VSMC) est souvent associée à l’hypertension et ses complications. Il est donc suggéré que l’étude des mécanismes impliqués dans la transduction de signaux menant à la contraction cellulaire et les mécanismes impliqués dans la régulation de celle-ci pourrait permettre d’identifier de nouvelles cibles potentielles afin d’envisager de nouveaux traitements pour cette maladie. Étant donné le rôle connu du système angiotensinergique dans l’activité contractile des VSMC et les nombreux indices suggérant une dérégulation de ce système dans de développement de l’hypertension; la présente thèse a été consacrée, dans un premier temps, à identifier de nouveaux mécanismes de signalisation impliqués dans la contraction cellulaire induite par l’angiotensine II. Ainsi, il est montré, pour la première fois, que les β-arrestines; des protéines adaptatrices impliquées dans la désensibilisation et la signalisation des récepteurs couplés aux protéines G, participent à la contraction cellulaire associée à l’activation du récepteur de l’angiotensine de type 1, et ce, en exerçant des effets opposés sur la phosphorylation de la chaîne légère de la myosine. Dans un second temps, étant donné qu’en condition diabétique une dysfonction vasculaire est observée et que cette dernière serait reliée à des changements dans le phénotype fonctionnel des VSMC; la deuxième partie de cette thèse traite des effets de l’exposition aux produits de glycation avancée (AGE) sur le phénotype et l’activité contractile des VSMC. Effet, les AGE, issus de la réaction entre les protéines et le glucose et dont la formation est particulièrement favorisée en contexte diabétique, pourraient être impliqués dans la dysfonction vasculaire observée. Ainsi, il est montré, pour la première fois, que l’exposition des VSMC aux AGE inhibe l’expression de leur phénotype contractile en affectant leurs propriétés mécaniques et diverses fonctions cellulaires telles que la signalisation, la contraction et l’organisation du cytosquelette. Dans un contexte plus large, cette thèse confirme également l’importance d’intégrer les approches biophysiques à la biologie cellulaire. En effet, les résultats obtenus par microscopie à force atomique sont uniques puisqu’ils offrent une nouvelle perspective concernant l’activité cellulaire via la caractérisation du phénotype mécanique de la cellule et la mesure de la contraction au niveau d’une seule cellule. || Abstract : Cell contraction plays a key role in a variety of physiological processes, among those; it regulates vascular permeability and blood pressure. It is known that abnormal contraction of vascular smooth muscle cells (VSMC) is often associated with hypertension and its complications. Therefore, it is suggested that studying mechanisms involved in signal transduction leading to cell contraction and mechanisms regulating the cell ability to contract may allow identifying new therapeutic targets in order to suggest new treatments for hypertension. Knowing the role of the angiotensin system in VSMC contractility and the numerous evidences suggesting a dysregulation of this system in the development of hypertension; this thesis first sought to identify new signaling mechanisms involved in cell contraction induced by angiotensin II. Therefore, it is shown here, for the first time, that β- arrestins; which are scaffolding proteins involved in the desensitisation and the signalling of protein G-coupled receptors, participate in cell contractile activity induced by angiotensin receptor type 1 activation by reciprocally regulating myosin activity through its light chain phosphorylation. Secondly, knowing that a vascular dysfunction is observed in diabetes which could be attributed to changes in VSCM functional phenotype; this thesis also investigated the effect of advanced glycation end-products (AGE) exposure on the contractile phenotype and function of VSMC. Indeed, AGE, which are the product of a reaction between proteins and glucose, are increased significantly in diabetes and are suspected to be involved in the vascular dysfunction observed in this metabolic disease. Therefore, it shown, for the first time, that AGE stimulation of the VSMC A7r5 interfere with the expression of their contractile phenotype by changing their mechanical properties and various cellular functions such as signal transduction, contraction and cytoskeletal organisation. Finally, this thesis also underlines the utility of integrating biophysical tools into studying cellular biology. Indeed, the various results obtained using atomic force microscopy are unique in a way that they provide new insights into studying cellular activity trough the measurement of single cell contraction and characterisation its mechanical phenotype

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