Durante o desenvolvimento do sistema nervoso, as células têm a tarefa de proliferar, migrar, diferenciar, morrer ou amadurecer de modo altamente preciso para formar estruturas complexas. Tal precisão é alcançada em decorrência da interação perfeita entre as células que se comunicam por meio de mensageiros químicos no ambiente extracelular. Nesse contexto, nosso grupo tem reportado o envolvimento da bradicinina (BK) em processos do desenvolvimento neural. Recentemente, observou-se que a BK desempenha um papel importante na determinação do destino neural, favorecendo a neurogênese em detrimento da gliogênese em diversos modelos de diferenciação, além de potencializar a migração celular observada no modelo de neuroesferas de rato (Trujillo et al, 2012). Essas descobertas motivaram, como objetivo geral dessa tese, a investigação dos mecanismos subjacentes à BK que determinam seus efeitos. Dessa forma, o principal modelo de diferenciação utilizado foi as células precursoras neurais (CPNs) isoladas do telencéfalo de embriões de camundongos. Estas células proliferam na presença dos fatores de crescimento (GFs) EGF + FGF2, mantendo-se multipotentes e formando as neuroesferas, ao passo que migram e diferenciam em neurônios e glias pela remoção desses GFs, com boa proximidade aos eventos do desenvolvimento do cortex in vivo. Como resultados do presente trabalho, observou-se, inicialmente, que a BK também influencia efetivamente na diferenciação neural no modelo de CPNs murinas. Ao término da diferenciação, observou-se que esta cinina favoreceu a migração e promoveu o enriquecimento neuronal, evidenciado pelo aumento da expressão das proteínas β3-Tubulina e MAP2. Constatou-se também, que se observa uma baixa taxa de proliferação ao término da diferenciação na presença de BK (Trujillo et al, 2012), em consequência da grande proporção de neurônios em cultura estimulada por esta cinina. Esta relação causal foi evidenciada pelo ensaio de incorporação de EdU e concomitante imuno-detecção dos marcadores β3-Tubulina, GFAP e Nestina. Fatores que promovem a neurogênese podem promovê-la suprimindo a proliferação celular em CPNs indiferenciadas, mais especificamente, alongando a fase G1 do ciclo celular que resulta na divisão de diferenciação. Assim, investigou-se também se a BK influencia nesse processo. Análises por citometria de fluxo demonstraram que esta cinina suprimiu a proliferação estimulada pelos GFs, levando ao acúmulo de células na fase G1 do ciclo celular. Esse acúmulo não provém do bloqueio do ciclo, uma vez que se observam grandes proporções de células nas fases subsequentes à G1, indicando que essa fase foi apenas prolongada pela BK e, assim, corroboraria no favorecimento da neurogênese. Outra face dos mecanismos adjacentes à BK para seus efeitos na diferenciação neural se refere às vias de sinalização disparadas por esta cinina. Observou-se que a BK induz a produção de AMPc por intermédio de proteínas G sensíveis à toxina pertussis (TP) (provavelmente através da subunidade βγ de proteínas Gi) e promove a mobilização de cálcio dos estoques intracelulares, evidenciando o envolvimento da família de proteínas Gq. Esses resultados sugerem que o receptor B2 de cinina acopla-se tanto às proteínas Gi quanto às proteínas Gq em CPNs. A exposição dessas células à BK também ativou as vias da PI3K/Akt e da MAPK p38, mas não influenciou na ativação de STAT3 e JNK. Destaca-se o potencial da rota da MAPK ERK como uma das principais cascatas responsáveis por decodificar sinais de mensageiros externos em respostas celulares. O tratamento com BK em CPNs ativou a ERK por tempo prolongado e estimulou sua translocação para o núcleo. O efeito de BK na glio- e neurogênese de CPNs foi dependente da atividade de ERK, porque o bloqueio farmacológico dessa enzima impediu esse efeito de BK. Por outro lado, o favorecimento da migração induzido por esta cinina foi dependente da atividade da p38, enquanto, o seu efeito antiproliferativo foi condicionado à atividade das suas duas MAPKs, ERK e p38. Além disso, a via da PI3K/Akt ativada por BK não influenciou nos três eventos avaliados. Finalmente, utilizou-se nessa tese uma abordagem reducionista da diferenciação, porém amplamente utilizada por estudos mecanísticos de neurogênese, as células PC12. Assim, observou-se que a BK também ativa a ERK por tempo prolongado e com translocação nuclear, sendo que tal forma de ativação dessa quinase é proposta na literatura como necessária e suficiente para induzir a neurogênese dessas células. Demonstrou-se ainda que o bloqueio apenas da ativação sustentada de ERK, pela inibição das atividades das PKCs clássicas, impede o favorecimento da neurogênese por BK em células PC12. Juntos, esses resultados contribuem para elucidação dos mecanismos de ação da BK na regulação da diferenciação neural, colaborando para melhor entender esse processo e prevendo possíveis aplicações em terapias de reparo neuronal em pacientes com doenças, por exemplo, de Parkinson, Alzheimer, Esclerose Múltipla e lesões isquêmicas. / During CNS development cells perform the task of proliferating, migrating, differentiating, dying or maturing in highly accurate patterns. Such accuracy is reached as a result of the perfect interaction among the cells that constantly communicate with each other through cell-cell contact or through chemical messengers present in the extracellular medium. In this context, our group has reported the involvement of bradykinin (BK) in neural differentiation of stem cell models (Trujillo et al, 2012). Recently, it has been observed that BK plays an important role in determining neural destination, favoring neurogenesis over gliogenesis in several models of differentiation, besides potentializing cell migration observed in the model of rat neurospheres. These discoveries have motivated, as the general objective of this thesis, the investigation of the mechanisms underlying BK-promoted effects on neural differentiation using neural precursor cells (NPCs) isolated from the telencephalon of mice embryos. These cells proliferate in the presence of growth factors (GFs) EGF + FGF2, remaining multipotent and forming neurospheres, while they migrate and differentiate in neurons and glias following removal of these GFs, resembling in simplified conditions events of the development of the cortex in vivo. As results of the present thesis, it was initially observed that BK also effectively influences neural differentiation fate of the mouse NPC model. This kinin favored migration and promoted neuronal enrichment, evidenced by increased expression of β3-Tubulin and MAP2 marker proteins. Moreover, proliferation rates were largely decreased following differentiation in the presence of BK (Trujillo et al, 2012), due to the large proportion of neurons in the culture stimulated by this kinin. This causal relation was evidenced by the EdU incorporation assay and the concomitant immunodetection rates of β3- Tubulin, GFAP and Nestin markers. Factors which promote neurogenesis can promote it by suppressing cell proliferation in undifferentiated NPCs, more specifically, prolonging the G1 phase of the cell cycle that result in the division of differentiation. Thus, it was further investigated whether BK influences this process. Flow cytometry analyses showed that this kinin suppressed the proliferation stimulated by GFs, resulting in the accumulation of cells in the G1 phase of the cell cycle. This accumulation is not caused by a cycle block, since wide proportions of cells are observed in phases subsequent to the G1, indicating that this phase was only prolonged by BK, thus corroborating for favoring neurogenesis. Another aspect of the mechanisms adjacent to BK for its effects on neural differentiation refers to the signaling pathways triggered by this kinin. Here, we show that the kinin B2 receptor couples to both Gi and Gq proteins in NPCs. BK induced the production of intracellular cAMP by activation of G proteins sensitive to pertussis toxin (PT) (probably through βγ subunit of Gi proteins) and promoted the mobilization of calcium from intracellular stocks, demonstrating the involvement of YM-254890-sensitive Gq proteins. Exposure of these cells to BK also activated PI3K/Akt and MAPK p38 pathways, but did not affect the activation of STAT3 and JNK. It is important to note the potential MAPK-ERK route as one of the main cascades responsible for decoding signals from external messengers into cellular responses. NPC treatment with BK activated ERK for prolonged time and stimulated its translocation into the nucleus. The effect of BK on glio- and neurogenesis of NPCs depended plainly on ERK activity, because the pharmacological blockade of this enzyme prevented the BK-exerted effects. On the other hand, the favoring of migration induced by this kinin was dependent on p38 activity, while its antiproliferative effect was conditioned to the activity of both the MAPKs ERK and p38. In addition, the PI3K/Akt pathway activated by BK did not affect any of the three evaluated events. Finally, we used in this thesis a reductionist approach of differentiation based on the use of PC12 cells, which has been widely used for mechanistic studies of neurogenesis. Thus, it was observed that BK also activated ERK for prolonged time and with nuclear translocation, considering that such form of kinase activation is proposed in the literature as necessary and sufficient to induce neurogenesis in these cells. This study also demonstrated that blockade only of the sustained ERK activation, through the inhibition of the activity of classic PKCs, prevents the favoring of neurogenesis by BK in PC12 cells. Together, these results compose novel mechanisms of action of BK on events of neural development in vitro, contributing to the better understanding of this process and foreseeing possible applications in the future for neuronal repair strategies

Using conventional microelectrodes, I studied the electrical basis for determining the resting V$\sb{\rm m}$ in intact EC's from hamsters. The resting V$\sb{\rm m}$ were found to be $-$40 mV for aortic EC's and $-$43 mV for vena caval EC's. The contributions of ions to the resting V$\sb{\rm m}$ of aortic EC's were compared in terms of the transference number (t$\sb{\rm ion}$). To develop a technique for in situ monitoring changes in V$\sb{\rm m}$ of postcapillary venular EC's in the hamster mesentery, a voltage-sensitive fluorescent probe, bisoxonol, was used to load the cells and the fluorescence signals were analyzed under an intravital microscope by recording the fluorescence intensity (I$\sb{\rm f}$) and processing fluorescent images of the bisoxonol-loaded cells. Calibrations were conducted by simultaneously measuring changes in V$\sb{\rm m}$ with microelectrodes and bisoxonol from aortic EC's and by varying extracellular Na$\sp{+}$ in microvessels. Both calibrations yielded the linear relationship between V$\sb{\rm m}$ and bisoxonol I$\sb{\rm f}$, showing the slope of 5.7%/mV for aortic EC's and 5.2%/mV for microvascular EC's. Altering extracellular K$\sp{+}$ to 25, 50, and 100 mM in the suffusate depolarized microvascular EC's by 5, 8, and 10 mV; whereas, the same alterations via both suffusion and perfusion induced the depolarization by 18, 30, and 42 mV, indicating that the K$\sp{+}$ conductance has an asymmetric distribution. Ba$\sp{2+}$ (1 mM) produced a depolarization by 70 mV, suggesting that the activity of K$\sp{+}$ channels dominates the resting V$\sb{\rm m}$. To correlate the bradykinin-induced increase in microvascular permeability to the changes in V$\sb{\rm m}$, the albumin flux (J$\sb{\rm A}$) was measured using TRITC-albumin along with monitoring V$\sb{\rm m}$. Bradykinin(l $\mu$M) induced a hyperpolarization of EC's by 8 mV and a biphasic increase in J$\sb{\rm A}$ from the basal level of 1.00 x 10$\sp{-6}$ to a transient peak of 9.17 x 10$\sp{-6}$ followed by a sustained level of 3.05 x 10$\sp{-6}$ cm/s. The linear correlations of net increases in both the peak and the sustained values of J$\sb{\rm A}$ to changes in V$\sb{\rm m}$ indicate that the hyperpolarization determines the peak in part and the sustained level in all. Under high K$\sp{+}$ (50 mM), bradykinin produced a repolarization from a depolarized V$\sb{\rm m}$ of -54 mV to -66 mV and a smaller increase in J$\sb{\rm A}$ from the basal level of 0.38 x 10$\sp{-6}$ to the peak of 5.51 x 10$\sp{-6}$ followed by a significantly lowered, sustained level of 1.11 x 10$\sp{-6}$ cm/s. The repolarization under high K$\sp{+}$ indicates that besides the activation of Ca$\sp{2+}$-dependent K$\sp{+}$ channels, other electrical events may be implicated. The correlation between the repolarization and the lowered value of J$\sb{\rm A}$ at the peak implies that this variation in V$\sb{\rm m}$ also mediates the bradykinin-induced increase in J$\sb{\rm A}$ under high K$\sp{+}$ condition. (Abstract shortened by UMI.)

acase@tulane.edu

Anhand einer umfassenden Falldarstellung einer jungen Patientin mit einem lebensbedrohlichen Gesichtsschmerzsyndrom, das nach septischer Thrombose der periorbitalen venösen und arteriellen Gefäße aufgetreten war, wurde die Bedeutung einer medikamentösen Antikoagulation für die erfolgreiche Schmerztherapie herausgearbeitet. An diesem Fallbeispiel konnte aber auch gezeigt werden, dass keine sicheren Parameter für die Indikation einer solchen Gerinnungstherapie vorlagen. Die Bedeutung dieses Falls lag unzweifelhaft in der Erkenntnis, dass in einer anhaltenden Aktivierung des Kontaktsystems der Gerinnung ein bislang unterschätztes Potential für die Entstehung und Unterhaltung ungeklärter Schmerzen liegen könnte und nicht zuletzt auch daran, dass sich diese ätiologische Komponente in der Komplexität der Erkrankung diagnostisch nicht eindeutig sichern ließ. Mit der Translokation von LPS aus der intestinalen Mukosa in endothelial vorgeschädigte Gefäßabschnitte wurde eine Hypothese vorgetragen, die neben einer schwer detektierbaren inflammatorischen Komponente auch das prokoagulatorische Potential der Schmerzentstehung erklären könnte. Die prokoagulatorische Komponente dieses hypothetischen Entstehungs-mechanismus chronischer Schmerzen müsste, so die Arbeitshypothese, umso dominanter sein, wenn prokoagulatorisch wirksame genetische Faktoren bei den Patienten hinzukommen. Unter der Annahme, dass eine solche zusätzliche Diathese nicht nur eine Schrittmacherfunktion haben, sondern auch einen diagnostischen Beitrag liefern könnte, wurde dieses diagnostische Pilotprojekt mit der empirisch begründeten Heparintherapie von 97 Schmerzpatienten verbunden. Alle Pa-tienten wurden mit dem niedermolekularen Heparin Enoxaparin behandelt und nach zehn Behandlungstagen in vier verschiedene Respondergruppen (Gruppe 1 bis 4) eingeteilt. Diese Gruppen wurden auf fünf prothrombotische Parameter untersucht. Dazu wurden die Allelprävalenzen des Plasminogen Aktivator Inhibitor-(PAI-1 4G/5G) Polymorphismus, der Faktor V-Leiden-Mutation, der Prothrombin (G20210A) Genmutation sowie die Prävalenzen der Hyperfibrinogenämie und des Protein S-Mangels ermittelt. Mit Hilfe des exakten Fisher Tests wurden jeweils die Allelprävalenzen und Parameter sowohl der Respondergruppen 1 bis 3 mit einem Kollektiv der Allgemeinbevölkerung als auch mit dem Kollektiv der Non-Responder (Gruppe 4) verglichen. Die Prävalenz des Allels A der Faktor V-Leiden-Mutation G1691A war im Enoxaparin-Kollektiv bei den Respondern der Gruppen 1 bis 3 im Vergleich zur Allgemeinbevölkerung und zur Non-Respondergruppe (Gruppe 4) signifikant erhöht. Die Allelprävalenzen und Parameter der übrigen prokoagulatorischen Faktoren unterschieden sich von denen der Kontrollgruppen nicht. Anhand des Kallikrein-Kinin-Systems als möglichem Effektor des Hämosta-sesystems konnten Hinweise auf die kausale Wirksamkeit des nieder-molekularen Heparins Enoxaparin bei der Behandlung chronischer Schmerzen gegeben werden. / We showed that low molecular heparins (enoxaparin) may help as a remedy in chronic pain syndromes. In our findings the inherited disorder Factor V Leiden was significantly higher in patients with chronic pain that had a benefit from enoxaparin in comparison to non-responders and to common population. The effect was proven by the Kallikrein-kinin-system.

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors. The beta-arrestins, adaptor proteins involved in GPCR desensitization, may also act as scaffolds for signaling pathways such as the mitogen-activated protein kinase (MAPK) cascade. The MAPK family, which includes the extracellular-signal regulated kinases (ERK) 1 and 2, promotes cellular differentiation and proliferation. Herein, the activation of ERK1/2 upon stimulation of the GPCR bradykinin type 2 receptor (B2R) with bradykinin was examined. Various B2R mutants with modified C-termini were employed to examine the temporal kinetics of ERK1/2. One of these receptor mutants displayed a loss of beta-arrestin binding as well as greatly enhanced ERK1/2 activation, compared to the wild-type receptor, when a cluster of serine/threonine residues important for B2R internalization was mutated. The other receptor mutants exhibited a correlation between their affinity for beta-arrestin and the intensity of ERK1/2 activation. Data from a mouse embryonic fibroblast cell line null for beta-arrestin suggested that beta-arrestin is involved in late-phase ERK1/2 activation by the B2R. These data point to the involvement of beta-arrestin in the activation of the ERK1/2 MAPKs through the B2R.

Introduction: Plasma kallikrein (PK) is a cofactor in blood coagulation and modulates inflammation through the release of bradykinin (BK). Previously it was believed that plasma prekallikrein (PPK), the precursor of PK and a member of the serine protease superfamily, was synthesised exclusively by hepatocytes and secreted into circulation. However, recent studies show that various human tissues contain PPK mRNA. In this study we sought to determine in which human tissues PK is expressed. Methods: Following approval by the Ethics Committee at the University of Natal, tissue samples from the spinal cord, 13 different regions of the brain, 7 different blood vessels and various other organs were collected at autopsy within 24h of death (n =10). Sections were probed using polyclonal antibodies specific for PK. PK concentrations in extracts of these tissues were measured by competitive EllSA. Results: A Western blot analysis demonstrated the monospecificity of the antibody for the PK protein. The presence of immunoreactive PK in cells of the pancreatic islets of Langerhans served as a positive control for each immunolabeling experiment. The hepatocytes, renal distal convoluted tubules and epithelial cells lining the bronchiole and pulmonary alveoli labeled positively for PK. In the gastrointestinal tract tissue, immunoreactive PK was visualised in the acinar cells of the salivary gland, in stromal and glandular duct cells of the oesophagus, and in some chief and glandular cells in the stomach. Some of the above-mentioned tissues contained a few inflammatory cells which stained intensely for PK. Immunoreactive PK was visualised in the endothelial cells and smooth muscle cells of the all the blood vessels examined, except the renal vein. Increased immunolabeling for PK in the endothelial cells, foam cells and macrophages was observed in arteries with atheromatous plaques. In neural tissue immunoreactive PK was observed in neurons, ependymal cells, fibre tracts, and in secretory cells of the anterior pituitary gland. Immunolabeling for PK was visualised in some neurons of the spinal cord and in different brain regions viz. hypothalamus, cerebral cortex, thalamus, brain stem and hippocampus. In sections of the hypothalamus and spinal cord, we observed immunolabeling for PK in ependymal cells lining the third ventricle and central canal respectively. Positive labeling for PK was evident in fibre tracts of the pons, medulla and hippocampus. No immunoreactive PK was visualised in the choroid plexus or cerebellum. High amounts of PK were measured by competitive ELlSA in extracts of the pancreas (12.94 ± 2.04 /-lg/ml), the pons (1.67 ± 1.46 /-lg/ml) and aorta (0.44 ± 0.14 /-lg/ml). The basilar artery (0.09 ± 0.07 /-lg/ml) and spinal cord (0.09 ± 0.04 /-lg/ml) had the least PK concentrations. Discussion and Conclusions: We have shown that the PPK mRNA demonstrated in various human tissues is most likely translated into protein by the immunolocalisation of PK within specific cells in the different tissues examined. The actions of PK within these tissues may be two fold, firstly by its kininogenase activity it may release BK from high molecular weight kininogen, or alternatively, PK may act as a proteolytic enzyme on other proteins. With respect to the latter) PK may be involved in the processing of protein precursors, for example precursors of the digestive enzymes found in saliva and in gastric secretion, insulin precursors in the pancreas, and hormonal precursors in the pituitary gland. The localisation of PK and B1 and B2 kinin receptors in the kidney, lung, stomach, blood vessels and brain suggests that the effects of PK in these tissues are mediated by BK-receptor interaction. These may include the regulation of glucose uptake in the pancreas, water and ion transport in the kidney, and local and systemic blood pressure in the cardiovascular system. The presence of immunoreactive PK in neurons suggests that BK-receptor mediated interaction may regulate neurophysiological processes such as synaptic transmission. Immunolabeling for PK in polymorphonuclear leukocytes observed in some of these tissue sections suggests the potential to mediate the inflammatory process. / Thesis (M.Med.Sc.)-University of Natal, Durban, 2000

Bradykinin (BK) acts through eNOS to reduce MAPK-mediated feedback inhibition of insulin signalling. Preliminary data suggest that the sGC-cGMP-PKG pathway, a prominent NO target, is involved. Our present study aimed to support the role of this pathway with atrial natriuretic peptide (ANP), which uses a receptor associated GC (NPR-A) to generate cGMP. We found that treating adipocytes with ANP mimicked BK effects on insulin-stimulated glucose uptake, Tyr-IRS-1 and Akt/PKB phosphorylation, as well as JNK and ERK1/2 inhibition. These outcomes depended on GC-cGMP-PKG signalling since A71915 (NPR-A antagonist), and KT-5823 (PKG inhibitor), completely abrogated them, while zaprinast (phosphodiesterase inhibitor), prolonged ANP actions. Furthermore, decreased MAPK phosphorylation was independent of upstream kinase activity, suggesting that MAPK phosphatases may be involved. These data indicate that BK and ANP act through the GC-cGMP-PKG pathway to potentiate insulin signalling via attenuated feedback inhibition. Stimulating the GC-cGMP-PKG pathway may, therefore, be a promising therapy for T2DM.

La posture, composante statique du contrôle moteur permettant une position érigée du corps, repose sur une décharge tonique des motoneurones innervant nos muscles antigravitaires. La décharge prend la forme de « potentiel de plateau » au niveau de motoneurones matures chez de nombreux vertébrés. Pour déterminer une éventuelle concordance entre l'émergence des propriétés de plateau et le développement postural, notre travail a eu pour but d'étudier la maturation et la nature ionique des potentiels de plateau des motoneurones innervant le muscle triceps surae (extenseur de la cheville) chez le rat nouveau-né.La réalisation de ces travaux de thèse nous a permis de dégager un mécanisme fondamental dans la genèse des propriétés de plateau des motoneurones lombaires. Ce mécanisme dont le fondement repose sur l'activation d'un « ménage à trois » jouerait un rôle majeur dans le développement moteur chez le rat. Dans la mesure où les potentiels de plateau des motoneurones sont fortement perturbés à la suite d'une lésion médullaire, cette avancée scientifique permettra éventuellement de mieux comprendre l'origine de certains déficits sensori-moteurs (spasticité, hyperalgésie...) et le développement de nouvelles stratégies thérapeutiques. / Posture allowing an erect posture of the body relies on spiking activity of motoneurons innervating antigravitary muscle. Discharge could take the form of plateau potential on mature motoneurons of numerous vertebrates. To determine a possible concordance between the emergence of plateau potential and postural control development, we studied the maturation and ionic nature of plateau potential of motoneurons innervating triceps surae muscle of neonatal rat.The conclusion of our work allows us to propose a fundamental mechanism in the genesis of plateau potential on lumbar motoneurons. This mechanism based on a "ménage a trois" seems to play an important role in the neonatal motor development. This scientific advance could eventually lead to a better understanding of the origin of some sensori-motor impairments (spasticity, hyperalgesia...) and development of therapeutic strategies.

Durante o desenvolvimento do sistema nervoso, as células têm a tarefa de proliferar, migrar, diferenciar, morrer ou amadurecer de modo altamente preciso para formar estruturas complexas. Tal precisão é alcançada em decorrência da interação perfeita entre as células que se comunicam por meio de mensageiros químicos no ambiente extracelular. Nesse contexto, nosso grupo tem reportado o envolvimento da bradicinina (BK) em processos do desenvolvimento neural. Recentemente, observou-se que a BK desempenha um papel importante na determinação do destino neural, favorecendo a neurogênese em detrimento da gliogênese em diversos modelos de diferenciação, além de potencializar a migração celular observada no modelo de neuroesferas de rato (Trujillo et al, 2012). Essas descobertas motivaram, como objetivo geral dessa tese, a investigação dos mecanismos subjacentes à BK que determinam seus efeitos. Dessa forma, o principal modelo de diferenciação utilizado foi as células precursoras neurais (CPNs) isoladas do telencéfalo de embriões de camundongos. Estas células proliferam na presença dos fatores de crescimento (GFs) EGF + FGF2, mantendo-se multipotentes e formando as neuroesferas, ao passo que migram e diferenciam em neurônios e glias pela remoção desses GFs, com boa proximidade aos eventos do desenvolvimento do cortex in vivo. Como resultados do presente trabalho, observou-se, inicialmente, que a BK também influencia efetivamente na diferenciação neural no modelo de CPNs murinas. Ao término da diferenciação, observou-se que esta cinina favoreceu a migração e promoveu o enriquecimento neuronal, evidenciado pelo aumento da expressão das proteínas β3-Tubulina e MAP2. Constatou-se também, que se observa uma baixa taxa de proliferação ao término da diferenciação na presença de BK (Trujillo et al, 2012), em consequência da grande proporção de neurônios em cultura estimulada por esta cinina. Esta relação causal foi evidenciada pelo ensaio de incorporação de EdU e concomitante imuno-detecção dos marcadores β3-Tubulina, GFAP e Nestina. Fatores que promovem a neurogênese podem promovê-la suprimindo a proliferação celular em CPNs indiferenciadas, mais especificamente, alongando a fase G1 do ciclo celular que resulta na divisão de diferenciação. Assim, investigou-se também se a BK influencia nesse processo. Análises por citometria de fluxo demonstraram que esta cinina suprimiu a proliferação estimulada pelos GFs, levando ao acúmulo de células na fase G1 do ciclo celular. Esse acúmulo não provém do bloqueio do ciclo, uma vez que se observam grandes proporções de células nas fases subsequentes à G1, indicando que essa fase foi apenas prolongada pela BK e, assim, corroboraria no favorecimento da neurogênese. Outra face dos mecanismos adjacentes à BK para seus efeitos na diferenciação neural se refere às vias de sinalização disparadas por esta cinina. Observou-se que a BK induz a produção de AMPc por intermédio de proteínas G sensíveis à toxina pertussis (TP) (provavelmente através da subunidade βγ de proteínas Gi) e promove a mobilização de cálcio dos estoques intracelulares, evidenciando o envolvimento da família de proteínas Gq. Esses resultados sugerem que o receptor B2 de cinina acopla-se tanto às proteínas Gi quanto às proteínas Gq em CPNs. A exposição dessas células à BK também ativou as vias da PI3K/Akt e da MAPK p38, mas não influenciou na ativação de STAT3 e JNK. Destaca-se o potencial da rota da MAPK ERK como uma das principais cascatas responsáveis por decodificar sinais de mensageiros externos em respostas celulares. O tratamento com BK em CPNs ativou a ERK por tempo prolongado e estimulou sua translocação para o núcleo. O efeito de BK na glio- e neurogênese de CPNs foi dependente da atividade de ERK, porque o bloqueio farmacológico dessa enzima impediu esse efeito de BK. Por outro lado, o favorecimento da migração induzido por esta cinina foi dependente da atividade da p38, enquanto, o seu efeito antiproliferativo foi condicionado à atividade das suas duas MAPKs, ERK e p38. Além disso, a via da PI3K/Akt ativada por BK não influenciou nos três eventos avaliados. Finalmente, utilizou-se nessa tese uma abordagem reducionista da diferenciação, porém amplamente utilizada por estudos mecanísticos de neurogênese, as células PC12. Assim, observou-se que a BK também ativa a ERK por tempo prolongado e com translocação nuclear, sendo que tal forma de ativação dessa quinase é proposta na literatura como necessária e suficiente para induzir a neurogênese dessas células. Demonstrou-se ainda que o bloqueio apenas da ativação sustentada de ERK, pela inibição das atividades das PKCs clássicas, impede o favorecimento da neurogênese por BK em células PC12. Juntos, esses resultados contribuem para elucidação dos mecanismos de ação da BK na regulação da diferenciação neural, colaborando para melhor entender esse processo e prevendo possíveis aplicações em terapias de reparo neuronal em pacientes com doenças, por exemplo, de Parkinson, Alzheimer, Esclerose Múltipla e lesões isquêmicas. / During CNS development cells perform the task of proliferating, migrating, differentiating, dying or maturing in highly accurate patterns. Such accuracy is reached as a result of the perfect interaction among the cells that constantly communicate with each other through cell-cell contact or through chemical messengers present in the extracellular medium. In this context, our group has reported the involvement of bradykinin (BK) in neural differentiation of stem cell models (Trujillo et al, 2012). Recently, it has been observed that BK plays an important role in determining neural destination, favoring neurogenesis over gliogenesis in several models of differentiation, besides potentializing cell migration observed in the model of rat neurospheres. These discoveries have motivated, as the general objective of this thesis, the investigation of the mechanisms underlying BK-promoted effects on neural differentiation using neural precursor cells (NPCs) isolated from the telencephalon of mice embryos. These cells proliferate in the presence of growth factors (GFs) EGF + FGF2, remaining multipotent and forming neurospheres, while they migrate and differentiate in neurons and glias following removal of these GFs, resembling in simplified conditions events of the development of the cortex in vivo. As results of the present thesis, it was initially observed that BK also effectively influences neural differentiation fate of the mouse NPC model. This kinin favored migration and promoted neuronal enrichment, evidenced by increased expression of β3-Tubulin and MAP2 marker proteins. Moreover, proliferation rates were largely decreased following differentiation in the presence of BK (Trujillo et al, 2012), due to the large proportion of neurons in the culture stimulated by this kinin. This causal relation was evidenced by the EdU incorporation assay and the concomitant immunodetection rates of β3- Tubulin, GFAP and Nestin markers. Factors which promote neurogenesis can promote it by suppressing cell proliferation in undifferentiated NPCs, more specifically, prolonging the G1 phase of the cell cycle that result in the division of differentiation. Thus, it was further investigated whether BK influences this process. Flow cytometry analyses showed that this kinin suppressed the proliferation stimulated by GFs, resulting in the accumulation of cells in the G1 phase of the cell cycle. This accumulation is not caused by a cycle block, since wide proportions of cells are observed in phases subsequent to the G1, indicating that this phase was only prolonged by BK, thus corroborating for favoring neurogenesis. Another aspect of the mechanisms adjacent to BK for its effects on neural differentiation refers to the signaling pathways triggered by this kinin. Here, we show that the kinin B2 receptor couples to both Gi and Gq proteins in NPCs. BK induced the production of intracellular cAMP by activation of G proteins sensitive to pertussis toxin (PT) (probably through βγ subunit of Gi proteins) and promoted the mobilization of calcium from intracellular stocks, demonstrating the involvement of YM-254890-sensitive Gq proteins. Exposure of these cells to BK also activated PI3K/Akt and MAPK p38 pathways, but did not affect the activation of STAT3 and JNK. It is important to note the potential MAPK-ERK route as one of the main cascades responsible for decoding signals from external messengers into cellular responses. NPC treatment with BK activated ERK for prolonged time and stimulated its translocation into the nucleus. The effect of BK on glio- and neurogenesis of NPCs depended plainly on ERK activity, because the pharmacological blockade of this enzyme prevented the BK-exerted effects. On the other hand, the favoring of migration induced by this kinin was dependent on p38 activity, while its antiproliferative effect was conditioned to the activity of both the MAPKs ERK and p38. In addition, the PI3K/Akt pathway activated by BK did not affect any of the three evaluated events. Finally, we used in this thesis a reductionist approach of differentiation based on the use of PC12 cells, which has been widely used for mechanistic studies of neurogenesis. Thus, it was observed that BK also activated ERK for prolonged time and with nuclear translocation, considering that such form of kinase activation is proposed in the literature as necessary and sufficient to induce neurogenesis in these cells. This study also demonstrated that blockade only of the sustained ERK activation, through the inhibition of the activity of classic PKCs, prevents the favoring of neurogenesis by BK in PC12 cells. Together, these results compose novel mechanisms of action of BK on events of neural development in vitro, contributing to the better understanding of this process and foreseeing possible applications in the future for neuronal repair strategies

Contexto: Alguns agentes farmacológicos em estudos de modelos animais demonstraram proteção nas lesões de isquemia e reperfusão do miocárdio. Entre eles, a bradicinina desempenha papel cardioprotetor durante a reperfusão do miocárdio. Os efeitos desta substância no músculo esquelético isquêmico não são conhecidos. Objetivo: Demonstrar os efeitos da bradicinina administrada após 2 horas de isquemia total seguidas de 4 horas de reperfusão em músculo esquelético de ratos com base nos efeitos sobre enzimas musculares (aspartato aminotransferase - AST, lactato desidrogenase - LDH, e creatinino fosfoquinase - CPK), membrana celular, recrutamento de leucócitos e apoptose. Materiais e Métodos: Foram estudados 18 ratos da linhagem Wistar distribuídos em 3 grupos com 06 animais e submetidos a 2 horas de isquemia total pelo método do torniquete do membro pélvico e 4 horas de reperfusão. Durante o período de reperfusão o grupo Controle recebeu solução salina, o grupo Bradicinina recebeu solução de bradicinina na dose de 1mg/Kg e o grupo HOE recebeu solução de icatibant (HOE 140), um antagonista do receptor B2, na dose de 125g/kg. Coletou-se 1,5ml de sangue da veia cava inferior antes do início da isquemia, ao final do período de isquemia e após a reperfusão para dosagem das enzimas AST, LDH e CPK. Colheuse também, após a reperfusão, biópsia do músculo gastrocnêmio (membro pélvico esquerdo) para dosagem tissular de malondialdeído (MDA), mieloperoxidase (MPO) e para avaliação imunohistoquímica da apoptose (TUNEL). Resultados: Houve aumento significativo da AST após a reperfusão apenas no grupo Bradicinina. O valor da LDH no grupo Bradicinina foi maior em relação aos demais antes da isquemia, após a isquemia e após a reperfusão. Observou-se que nos três grupos ocorreu diminuição significativa de LDH após a reperfusão. Ocorreu aumento significativo dos valores da CPK após a reperfusão nos três grupos. Não houve diferença dos valores de CPK entre os grupos antes da isquemia. Os valores de CPK no grupo Bradicinina foram maiores quando comparados aos grupos Controle e HOE após a isquemia e após a reperfusão. O MDA elevou-se significativamente após a reperfusão no grupo Bradicinina em relação aos grupos Controle e HOE. Após a reperfusão, a MPO, elevou-se significativamente no grupo Bradicinina em relação ao grupo Controle. O número de nucleos apoptóticos foi semelhante entre os grupos. Conclusão: A bradicinina (1mg/Kg) aplicada continuamente, intra-arterial, durante 4 horas de reperfusão agrava a lesão de isquemia e reperfusão em musculatura esquelética de ratos submetidos a 2 horas de isquemia total, com base na elevação de enzima muscular (CPK), no aumento de indicador de lesão de membrana celular (MDA) e no maior recrutamento neutrofílico (MPO). / Background: Some studies of pharmacological agents in animal models have demonstrated protection in ischemia and reperfusion of the myocardium. Among them, bradykinin plays a cardioprotective effect during myocardial reperfusion. The effects of Bradykinin on ischemic skeletal muscle are unknown. Objective: To demonstrate the effects of bradykinin administered after 2 hours of total ischemia followed by 4 hours of reperfusion on rat skeletal muscle based on the effects on the muscle enzymes (aspartate aminotransferase - AST, lactate dehydrogenase - LDH and creatinine phosphokinase - CPK), on the cell membrane, on the recruitment of leukocytes and on apoptosis. Materials and Methods: Eighteen Wistar rats were studied and distributed to three groups of 06 animals each and submitted to 2 hours of total ischemia by the tourniquet method of hind limb and to 4 hours of reperfusion. During the reperfusion period the Control group received saline solution, the Bradykinin group received bradykinin solution at the dose of 1mg/Kg and the HOE group received icatibant (HOE 140) solution, a B2 receptor antagonist, at the dose of 125 g/Kg. A blood volume of 1,5 ml was collected from the inferior vena cava before the onset of ischemia, at the end of the period of ischemia and after reperfusion for the measurement of AST, LDH and CPK. A gastrocnemius muscle biopsy (left pelvic limb) was collected at the end of the reperfusion period for the determination of tissue malondialdehyde (MDA), myeloperoxidase (MPO) and immunohistochemical evaluation of apoptosis (TUNEL). Results: There was a significant increase in AST after reperfusion only in Bradykinin group. LDH was higher in the Bradykinin group compared to the others before ischemia, after ischemia and after reperfusion. All three groups showed a significant decrease in LDH after reperfusion. There was a significant increase in CPK values after reperfusion in all three groups. There was no difference in CPK values between groups before ischemia. CPK values were higher in the Bradykinin group when compared to the Control and HOE groups after ischemia and after reperfusion. MDA increased significantly after reperfusion in the Bradykinin group compared to the Control and HOE groups. After reperfusion, MPO increased significantly in the Bradykinin group compared to the control group. The number of apoptotic nuclei was similar in all groups. Conclusion: Bradykinin (1mg/Kg) continuously applied intra-arterially for 4 hours of reperfusion aggravates ischemia and reperfusion in skeletal muscle of rats submitted to 2 hours of total ischemia, based on the elevation of muscle enzyme (CPK), on the increase in the indicator of cell membrane damage (MDA) and on the greater neutrophil recruitment (MPO).