Dissertations / Theses on the topic 'Ouabain'

Ouabain and other cardiotonic steroids are known to inhibit Na + ,K + -ATPase (NKA), theion pump responsible for the ionic gradient across the plasma membrane. These steroidsdisplay a selective toxicity towards several tumour cells in comparison to primary humancells, however, the mechanism behind this is not yet understood. Here, we examined theouabain toxicity in renal epithelial cells, proximal tubular cells (PTCs) of different origin. Weinvestigated the relative cytotoxicity in cancer cells (A-498) and papilloma virus-transformedPTCs (HK-2) as well as to primary human PTCs (hPTC) to validate key components in theeffect. In exposure to ouabain, we examined the cell viability and density by MTT and CrystalViolet assays, and cell migration by a scratch assay. The cytotoxic effect was also studied invarious pH, glucose and potassium ion concentrations. In addition, apoptosis was examinedby the TUNEL assay, and if ouabain kills cancer cells through activation of thevolume-regulated anion channel VRAC channel via NKA. We found that there is a decrease in viable cells when cells are exposed to ouabain ≥ 10nM, however, the effect was not seen to be selective towards cancer cells, nor due toapoptosis and the activation of VRAC. The cytotoxic effect was greater in more acidicextracellular pH ~6.8, but independent of glucose concentration in the medium. Interestingly,the effect was also reversed at an increased extracellular concentration of potassium, andouabain did selectively inhibit the cancer cells to migrate. Thus, there could be potential forouabain to act as an anti-cancer agent for renal cancer and to inhibit tumour metastasization.
Ouabain och andra kardiotoniska steroider är kända för att inhibera Na + ,K + -ATPas (NKA),membranpumpen som är ansvarig för den aktiva jontransporten av natrium och kalium ochjongradienten över plasmamembranet. De har påvisat en selektiv toxicitet mot vissatumörceller i jämförelse med primära humana celler, men det är dock inte förstått hurmekanismen bakom denna företeelse fungerar. I denna studien undersökte vi ouabainstoxicitet i njurcancerceller (A-498) och papillomavirustransformerade proximala tubuliceller(hPTC) för att identifiera effektens nyckelkomponenter. Vid exponering av ouabain undersökte vi cellviabiliteten och -densiteten genom MTT- ochkristallviolett-analyser, samt cellmigrering genom scratch-analys. Den cytotoxiska effektenstuderades också under olika pH-förhållanden samt glukos- och kaliumkoncentrationer.Dessutom undersöktes det om apoptos orsakar celldöd genom TUNEL-analys, och omouabain dödar njurcancerceller genom aktivering av den volymreglerade anjonkanalen(VRAC) via NKA. Vi fann minskning av cellernas livskraft vid exponering av ≥ 10 nM ouabain, men effektentycktes dock inte se ut att vara selektiv gentemot cancerceller, inte heller på grund av apoptosoch aktivering av VRAC. Den cytotoxiska effekten var större vid lägre pH, men oberoendeav mediets glukoskoncentrationen. Intressant nog motverkades också effekten vid förhöjdkoncentration av kaliumjoner, och ouabain hämmade selektivt cancercellerna att migrera.Således finns det en viss potential för ouabain att kunna fungera som ett anticancermedel motnjurcancer och att hämma metastasutveckling.

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Initially known as a cardiotonic steroid capable to inhibit the Na+/K+ATPase, ouabain was identified as an endogenous substance present in human plasma, produced by the adrenal, pituitary and hypothalamus and can interfere with various aspects of immune response. In this study, which aimed to study the modulating effect of ouabain on the immune system in vivo and in vitro using mouse models, we demonstrated that treatment for three consecutive days using 0,56 mg/kg ouabain was able to reduce cell migration induced by mitogen Concanavalian A (Con A) to the peritoneum, and this fact reflects a decrease in the number of polymorphonuclear leukocytes, mainly neutrophils. In this same model, ouabain was also able to increase the number of mononuclear leukocytes in the peritoneal cavity. Evaluating the effect of treatment on lymphocytes in peripheral organs, we found that, in lymphocytes from mesenteric lymph nodes, this substance induces a decrease of 20% of T CD3+ lymphocytes, concomitant with an increase in same percentage of B lymphocytes, without, however, modulating the proportion of CD4+ and CD8+ among themselves, as well as the number of regulatory T cells (CD4+/CD25+). In the thymus, the same treatment, does not affect the proportion of lymphocyte subpopulations studied. The analysis qualitative and quantitatively of peripheral blood leukocytes, biometrics and cellularity of spleen, thymus and lymph nodes showed no change in response to ouabain treatment. Comparative studies using treatment for one or two days, with the same dose of 0,56 mg/kg did not trigger modulation, in vivo, in populations of T lymphocytes, B lymphocytes and subpopulations of CD4+ and CD8+ cells in mesenteric lymph nodes. In addition, ouabain was able to inhibit mitochondrial activity of lymphocytes stimulated with Con A, using MTT assay.These findings indicate an immunomodulatory role of ouabain.
Inicialmente, conhecida como um esteróide cardiotônico e por sua propriedade de inibir a Na+/K+ATPase, a ouabaína foi identificada como uma substância endógena presente no plasma humano, produzida pela adrenal, hipófise e hipotálamo e capaz de interferir em vários aspectos da resposta imune. Neste trabalho, que teve como objetivo estudar o efeito modulador da ouabaína no sistema imunológico in vivo e in vitro por meio de modelos murinos, demonstrou-se que o tratamento por três dias consecutivos com ouabaína utilizando a dose de 0,56 mg/kg foi capaz de reduzir a migração celular induzida pelo mitógeno Concanavaliana A (Con A) para o peritôneo, sendo este fato reflexo da redução do número de leucócitos polimorfonucleares, principalmente, neutrófilos. Neste mesmo modelo, a ouabaína também foi capaz de aumentar o número de leucócitos mononucleares no lavado peritoneal. Avaliando-se o efeito desse tratamento no perfil linfocitário de órgãos periféricos, encontrou-se que, em linfócitos de linfonodos mesentéricos, esta substância induz a uma diminuição de 20% de linfócitos T CD3+, concomitante a um aumento de mesmo percentual de linfócitos B, sem, no entanto, modular a proporção de linfócitos TCD4+ e CD8+ entre si, bem como o número de células T regulatórias (CD4+CD25+). No timo, o mesmo tratamento com a ouabaína não interfere na proporção das subpopulações linfocitárias estudadas. As análises qualitativas e quantitativas de leucócitos do sangue periférico, da biometria e celularidade do baço, timo e linfonodos mesentéricos não apresentaram alteração em resposta ao tratamento com a ouabaína. Estudos comparativos utilizando tratamentos de um ou dois dias, com a mesma dose de 0,56 mg/Kg não desencadearam modulação, in vivo, nas populações de linfócitos T, linfócitos B e das subpopulações de linfócitos TCD4+ e CD8+ nos linfonodos mesentéricos. Adicionalmente, a ouabaína foi capaz de inibir a atividade mitocondrial de linfócitos estimulados com Con A, por meio do ensaio de MTT. Estes resultados indicam um papel imunomodulador da ouabaína.

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Ouabain, known as a cardiotonic glycoside capable of inhibiting the Na+/K+ ATPase, was widely used for heart failure treatment. Identified as an endogenous substance, ouabain is capable of interfering with various physiological functions, including immune system modulation. Besides that, little is known about the involvement of this substance in nociceptive and inflammatory processes. The present study investigated the role of ouabain in acute peripheral inflammation induced by intraplantar administrartion of different phlogistic agents (carrageenan, compound 48/80, histamine, bradykinin, and PGE2) and in nociceptive processes (abdominal writhing induced by acetic acid and hot plate). Ouabain produced a significant reduction in the mouse paw edema induced by carrageenan and compound 48/80. This antiinflammatory effect of ouabain is associated to the inhibition of PGE2, bradykinin, and mast-cell degranulation, but not to histamine. Ouabain also presented a central and peripheral anti-nociceptive activity. This analgesic potential might be related to the inhibition of inflammatory mediators and to activation of opioid receptors, since it was reversed by naloxone, an opioid antagonist. Additionally, the analgesic effect of ouabain was not related to sedative effect or to motor function. Taken together, the present work demonstrated for the first time, in vivo, the antiinflammatory and analgesic potential of ouabain
A Ouabaína é um glicosídeo cardiotônico, inibidor da Na+/K+-ATPase, utilizada na clínica para o tratamento de insuficiência cardíaca. Atualmente, sabe-se que essa substância é endógena, e capaz de interferir em várias funções fisiológicas, inclusive em diversos aspectos do sistema imunológico. Apesar disso, pouco se sabe sobre seu envolvimento em processos inflamatórios e nociceptivos. Neste trabalho, foi avaliada a atividade da Ouabaína na inflamação aguda desencadeada pela administração de diversos agentes flogísticos (carragenina, composto 48/80, histamina, PGE2 e bradicinina) e em modelos nociceptivos (contorções abdominais induzidas por ácido acético e placa quente). A Ouabaína produziu uma redução no edema de pata produzido por carragenina e pelo composto 48/80. Esse potencial anti-inflamatório está relacionado ao bloqueio da degranulação de mastócitos, bem como pela inibição da via da PGE2 e da bradicinina, porém é independente da via da histamina. A Ouabaína também apresentou uma atividade anti-nociceptiva central e periférica. Esse efeito está vinculado à inibição da via dos mediadores inflamatórios e a mecanismos opióides, visto que foi revertido pela administração da naloxona, um inibidor dos receptores opióides. Adicionalmente, foi descrito que a inibição da dor pela Ouabaína não possui envolvimento com sedação ou diminuição da capacidade motora. O conjunto desses dados demonstra pela primeira vez, in vivo, a atividade anti-inflamatória e anti-nociceptiva da Ouabaína.

Preconditioning is the phenomenon whereby tolerance to lethal insults is induced by exposing the tissue to a prior sublethal stimulus. This exists in several forms, such as ischaemic preconditioning, adenosine preconditioning and excitotoxic preconditioning. Adenosine preconditioning is known to be mediated by activation of A1 receptors and ATP-sensitive potassium channels whilst excitotoxic preconditioning mainly involves stimulation of NMDA receptors, nitric oxide and most likely ATP-sensitive potassium channel activation. ATP-sensitive potassium channel openers such as pinacidil and diazoxide are also known to exert preconditioning against various types of insults. There have been several models of ischaemia used to study preconditioning in vivo and in vitro leading to some confusion over the effects of preconditioning agents. High concentrations of glutamate or NMDA have been used as models of excitotoxicity in many experimental paradigms. Some molecular changes are associated with preconditioning phenomena, the most prominent being an increased expression of heat shock protein 72 (HSP72). The aims of the current study were to: 1) investigate the effects of exogenous glutamate and other depolarizing agents in the slice preparation and their validity for use as toxic agents 2) examine any potential preconditioning neuroprotection induced by adenosine against various depolarizing agents and elucidate the underlying mechanisms where relevant 3) examine the excitotoxic preconditioning phenomenon and possible underlying mechanisms 4) look at the effectiveness of other known preconditioning agents e.g. ATP-sensitive potassium channel openers against depolarizing agents and identify the underlying mechanisms of protection 5) identify any molecular changes that may occur during acute models of chemical ischemia or acute preconditioning. The rat hippocampal slice preparation was used to investigate the effects of depolarizing agents and preconditioning paradigms upon the extracellularly evoked field epsps, orthodromic and antidromic population spikes. Western blotting was used to detect any changes in the levels of HSP72 in the slices that may have occurred as a result of the depolarizing agents or the preconditioning treatments. It was first established that 5mM and 10mM glutamate induced depressions in the amplitudes of orthodromic population spikes which recovered to a stable plateau. The degree of recovery of the spikes depended partially upon the initial size of the response. As adenosine is known to be released in response to glutamate receptor stimulation, the effects of 5mM glutamate upon the orthodromic spikes were studied in the presence of the A1 receptor antagonist, DPCPX. It was observed that DPCPX did not attenuate the depression of the response during glutamate perfusion but there was a significant elevation in the post-glutamate recovery of the response. This effect was not observed when the protocol was applied to antidromic population spikes and field epsps, both of which showed a depression in response during 5mM glutamate perfusion but recovered fully when glutamate was removed. The field epsps showed a trend whereby smaller epsps recovered to a far greater degree than population spikes. Although this effect was not significant, the NMDA receptor blocker, MK-801, was co-perfused with glutamate during epsp recordings to examine this further. The degree to which MK-801 alone affected the response correlated with the post-glutamate recovery. To study this effect, isolated NMDA-receptor mediated epsps were recorded and the effects of 5mM glutamate upon them were studied. There was a similar tendency for small NMDA-receptor mediated epsps to recover to a higher level following glutamate treatment compared with larger potentials. In the presence of DPCPX, the larger potentials showed a significant elevation in recovery following treatment with glutamate. It was also shown that the post-5mM glutamate recovery of the orthodromic population spikes was elevated by the presence of the A2a receptor antagonist, SCH 58261. Further experiments using the ATP-sensitive potassium channel blocker, glibenclamide, indicated that this effect may be due to increasing the opening of these channels. Adenosine preconditioning was attempted using 10mM glutamate as an insult. It was shown that adenosine could not precondition against this effect in antidromic or orthodromic population spikes. The effects of the sodium-potassium ATPase inhibitor, ouabain, upon the evoked responses were studied as an alternative insult. It was shown that ouabain induced depressions in field epsps, orthodromic and antidromic population spikes. The antidromic population spikes showed significantly smaller depressions than the orthodromic responses. Further experiments using the glutamate receptor antagonist, kynurenic acid, showed that glutamate receptors mediated the effects of ouabain upon the orthodromic population spikes but not the antidromic spikes. Adenosine preconditioning was attempted against ouabain. It was shown that adenosine preconditioned against the effects of ouabain upon orthodromic and antidromic population spikes but not field epsps. Further experiments were conducted using antidromic population spikes. It was shown using various antagonists, that adenosine protection against ouabain was mediated by A1 receptors, ATP-sensitive potassium channels, NMDA receptors and nitric oxide. To extend these results further, preconditioning using the ATP-sensitive potassium channel opener, pinacidil, was attempted against 10mM glutamate and ouabain. It was shown that pinacidil was able to precondition the antidromic population spike against either insult. Using the NMDA receptor antagonist, DL-AP5, showed that the preconditioning effect of pinacidil against ouabain was mediated by NMDA receptors. Another preconditioning paradigm was attempted to see if glutamate could precondition against ouabain. It was shown that pre- treatment with glutamate resulted in enhancing the depressant effect of ouabain upon field epsps and antidromic population spikes. To further examine the effects of ouabain upon antidromic population spikes, ouabain was co-perfused in the presence of the intracellular calcium chelator, BAPTA-AM. This resulted in enhancing the depressant effect of ouabain upon the response. A similar result was observed when the calcium concentration in the perfusion medium was lowered to 0.5mM from 2.5mM whereas increasing the concentration to 5mM attenuated the depressant effect. Ouabain was also co-perfused in the presence of charybdotoxin, a blocker of large-conductance calcium activated potassium channels. It was observed that charybdotoxin enhanced the effect of ouabain upon the antidromic spikes. No changes were detected in HSP72 expression in the slices in response to ouabain treatment, 10mM glutamate treatment, pinacidil preconditioning treatment or glutamate preconditioning. The present results show that glutamate and ouabain can induce depressions in the evoked responses from the rat hippocampal slice and that the effects of 5mM glutamate can be attenuated by adenosine receptor antagonists. In addition, adenosine can precondition against ouabain but not glutamate and this effect involves A1 receptors, NMDA receptors, nitric oxide and ATP-sensitive potassium channels. It has also been observed that pinacidil can precondition against ouabain or glutamate and NMDA receptors may be involved in this effect. The inability of glutamate to precondition against ouabain in evoked responses was also demonstrated. The study highlights the effectiveness of preconditioning agents against different depolarizing agents and the interactions between adenosine and glutamate receptors which play a role in preconditioning.

Cardiotonic steroids (CTSs), such as digoxin, are used to treat heart failure as they inhibit the sodium potassium pump (NKP). However, sporadic studies report NKP stimulation by a CTS called ouabain. This thesis investigates such reports and explores mechanisms for stimulation. Rabbit cardiomyocytes were isolated and voltage clamped, and the NKP current (Ip) identified. Exposure of myocytes to 5 nM or 10 nM ouabain for ~1-minute significantly increased Ip to 0.69±0.09 pA/pF (N=6) and 0.64±0.05 pA/pF respectively vs. 0.46±0.03 pA/pF in 25 controls. Rostafuroxin, a putative ouabain antagonist, abolished this stimulation (Ip=0.40±0.04 pA/pF, N=7). Higher ouabain concentrations, or a longer ouabain exposure duration of 5 m, also abolished stimulation. 1 h incubation with 10 nM ouabain caused NKP inhibition. NKP activity is regulated by glutathionylation, a reversible oxidative modification; Cysless FXYD3 inclusion in pipette solutions, preventing de-glutathionylation, abolished ouabain-induced stimulation. Stimulation was strongly dependent on the presence of the rhamnose sugar moiety of ouabain. 10-500 nM ouabagenin (ouabain without a sugar moiety) did not increase Ip, nor did 5–30 nM digoxin which possesses a different steroid and sugar moiety. Dihydroouabain, differing from ouabain by possessing a saturated lactone, increased Ip at 1 nM to 0.65±0.02 pA/pF, N=6 (p<0.01). Effects of long-term CTS exposure were investigated using cell viability to indirectly measure NKP activity. 24 h low-dose ouabain exposure significantly increased viability in MCF-7 and MDA-MB-468 human breast cancer cells, albeit to a greater degree in MCF-7 cells which are comparatively FXYD-3 rich. This may implicate FXYD in the mechanism of stimulation. As ouabain was previously used therapeutically in humans, this study supports exploration of ouabain as a treatment for new onset atrial fibrillation, to lower high levels of intracellular sodium.

The microvasculature plays a significant role in the regulation of blood pressure and regional blood supply. Cardiotonic steroids like the adrenal cortical hormone (ouabain) have been proposed to play a role in some forms of hypertension. The purpose of this study was to determine the effect of different agonists on arteriolar diameter in the presence and absence of ouabain. In Vitro studies on isolated intact rat mesenteric arterioles were performed by administering different concentrations of the vasoconstrictor norepinephrine (NE) and the vasorelaxant acetylcholine (Ach) in the presence and absence of ouabain. NE induced constriction was not significantly enhanced in the presence of ouabain. Ach completely reversed NE-induced constriction without ouabain, which was significantly impaired in ouabain presence (p+Channel blockade almost completely abolished Ach-induced relaxation in presence and absence of ouabain.

Thesis (M.S.)--University of Toledo, 2009.
"In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Science." Title from title page of PDF document. Bibliography: p. 39-48.

Na,K-ATPase é uma proteína de membrana que tem como função manter o equilíbrio osmótico nas células pela hidrólise de ATP. A ouabaína (OUA) se liga a Na,K-ATPase e é capaz de ativar cascatas de sinalização. As subunidades a da Na,K-ATPase possuem 4 isoformas que são distribuídas de forma diferenciada nos tecidos. As células da glia são importantes na resposta contra lesões no cérebro e também controlam a inflamação. Dados na literatura mostram que a OUA tem efeito protetor em alguns tipos de dano. O objetivo do estudo é avaliar a função da isoforma a2 na cultura de células da glia em resposta à OUA e ao LPS. Nós investigamos a ação da OUA em diversas concentrações e LPS (1g/mL) na viabilidade celular (LDH) e proliferação celular (MTT). O LPS foi utilizado como modelo de inflamação e uma das perguntas era se o tratamento prévio com OUA, seria capaz de reverter a ativação do fator de transcrição NF-kB que está envolvido com inflamação. O pré-tratamento com OUA diminuiu a ativação do NF-kB induzida pelo LPS. Após, nós silenciamos a isoforma a2 das células da glia com RNAi. Os nossos dados mostram que o pré-tratamento com OUA reverte o efeito na ativação do NF-kB causado pelo LPS. Provavelmente, a isoforma a2 está relacionada com alguma via de sinalização que interage com a via do LPS.
Na,K-ATPase is a conserved membrane protein which maintains the osmotic balance in the cell by the hydrolysis of ATP. Ouabain (OUA) binds to Na,K-ATPase and it can activate signaling pathways. The a subunits of Na,K-ATPase have 4 isoforms which are distributed in a different pattern in the tissues. Glial cells have an important role in the response against injury and they also control inflammation. Some data have reported that OUA can protect against some types of injury. The aim of this study is to evaluate the role of a2 isoform in glial cells in response to OUA and LPS stimulus. We investigated the action of OUA and LPS in cell viability (LDH) and cell proliferation (MTT). LPS was used as a model of inflammation and one of our questions was if the treatment with OUA before LPS was capable of reduce the activation of the transcription factor NF-kB which is involved in inflammation. The pre-treatment with OUA decreased the NF-kB activation induced by LPS. We also silenced the a2 isoform in culture glial cells with iRNA. Taken together our data showed that OUA pretreatment reversed the NF-kB activation induced by LPS in primary cultures of glial cells from mice. Probably,the a2 isoform is related with some signaling pathway that interacts with the LPS pathway.

The mechanisms mediating the hypertension of preeclampsia (PE) are unclear. Endogenous digitalis-like factors (EDLFs) are specific sodium pump (SP) inhibitors implicated in essential and experimental hypertension, but they have not been fully explored in the setting of PE. This study uses a digoxin antibody Fab fragment to address the question of whether such factors are present and increased in PE, to investigate a possible treatment of PE, and to isolate and characterize all EDLFs present in PE. Sera and placenta from women with PE did show a significant increase in SP inhibition in comparison to women with normal pregnancy and Digibind® was found to bind EDLFs and essentially block or reverse SP inhibition. Sera were collected in a Phase II, double-blind, placebo controlled clinical study in which women with severe preeclampsia were dosed with Digibind®, as a therapeutic, and the SP activity measured. Sera and placenta from women with PE was also investigated for their inhibitory effects on the SP. Known candidates for EDLFs were investigated for their SP inhibitory effects, as well as how digitalis antibody immune Fab fragments, Digibind® and DigiFab™, bound them and affected the SP activity. Digibind® is also a sufficient affinity material used to isolate and purify PE EDLFs. Additionally, the placentas of preeclamptic women have high levels of similar EDLFs. These studies provide evidence for the existence of EDLFs that circulate in women with PE, and Digibind® is an effective and novel tool to bind, isolate and purify EDLFs in PE.

A Na+K+-ATPase (NKA) é uma proteína de membrana que participa de mecanismos de transporte nos túbulos renais para a reabsorção de sódio e outros substratos. Sabe-se que a administração de ouabaína (OUA), um inibidor da NKA, induz hipertensão arterial em ratos. No entanto, o papel dos rins nesse modelo de hipertensão não está bem elucidado. Desta forma, o objetivo do presente estudo foi avaliar as possíveis alterações na função renal induzidas pelo tratamento crônico com OUA por 5 ou 20 semanas. Sendo assim, foi observado que o tratamento com OUA promoveu hipertensão de mesma magnitude nos dois grupos avaliados. Além disso, a administração de OUA induziu o aumento da ingestão de água, do fluxo urinário e da expressão protéica da isoforma 1 da NKA. Porém, não foi capaz de alterar de maneira significativa o ritmo de filtração glomerular, assim como a fração de excreção de Na+ e K+. Pode-se concluir que, o tratamento crônico com OUA induz hipertensão, porém parece que os rins não contribuem de forma importante para o processo hipertensivo neste modelo de hipertensão.
Na+K+-ATPase (NKA) is an integral membrane protein that participates in transport mechanisms along renal tubules for sodium reabsorption and other substrates. Its known that ouabain (OUA) administration, a NKA inhibitor, induces hypertension in Wistar rats. However, the role of kidneys in this model of hypertension is not elucidated. So, the aim of this study was to evaluate the possibles alterations in renal function induced by chronic treatment with OUA by 5 or 20 weeks. Chronic treatment with OUA induced hypertension in a similar magnitude in both experimental groups. Moreover, OUA administration was able to increase water intake, urinary flow, and protein expression of 1 isoform of NKA. However, OUA treatment did not alter significantly the glomerular filtration rate, likewise the fractional excretion of Na+ and K+. In summary, chronic OUA treatment induces mild hypertension independent of the period of administration, but the kidneys dont play an important role in the hypertensive process in this model of hypertension.

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Epilepsy is a syndrome characterized by spontaneous recurrent seizures, result of paroxys-mal discharges , excessive or synchronous a neural population . Despite the good prognosis, the high number of patients with epilepsy who have seizures refractory to medication, reflects the lack of a bet-ter understanding of excitotoxic disorders characteristic of this disease. Thus, it becomes important to understand the mechanisms for induction and maintenance of seizures as well, the search for new antiepileptic compounds that may prevent the development of this pathology. As nociception and epi-lepsy have mechanisms in common and several anticonvulsant drugs are used in treatment of pain , we investigated the effect of triterpene 3β , 6β , 16β Trihidroxilup -20 ( 29) -ene (TTHL), a compound with antinociceptive properties in convulsions induced by pentylenetetrazol (PTZ). The TTHL admin-istration ( 30 mg / kg , po) increased the latency to the first myoclonic seizures and tonic- clonic sei-zure and decreased the duration of generalized seizures induced by PTZ . In addition , the administra-tion of TTHL reduced lipid peroxidation and protein carbonylation , as well as protected from inhibition of glutamate uptake and activity of the Na+, K+-ATPase (α1and α2/α3 subunits ) caused by PTZ . Alt-hough the TTHL showed no antioxidant activity per se and not alter the binding of [ 3H ] flunitrazepam to the site of the GABAA receptor bezodiazepínico , this protected compound of convulsions and inhi-bition of Na+, K+-ATPase activity induced by ouabain . These results suggest that the anticonvulsant action is due TTHL s maintenance of Na+, K+-ATPase . In fact , the experiments performed in the cer-ebral cortex in vitro showed that PTZ ( 10 mM ) reduced the activity of Na+, K+-ATPase and that prein-cubation with TTHL ( 10 mM ) protected from this inhibition.Thus , these data indicate that the protec-tion exerted by TTHL this seizure model is not related to antioxidant activity or GABAergic activity . However, these results demonstrate that effective protection of Na+, K+-ATPase activity induced by this compound protects against oxidative and excitotoxic damage induced by PTZ .
A epilepsia é uma síndrome caracterizada por crises espontâneas e recorrentes, resultado de descargas paroxísticas, excessivas e sincrônicas de uma população neural. Apesar do bom prognós-tico, o elevado número de pacientes com epilepsia, que apresentam convulsões refratárias aos medi-camentos, reflete a falta de um melhor entendimento dos distúrbios excitotóxicos característico desta doença. Desta forma, torna-se importante o entendimento dos mecanismos de indução e manuten-ção das convulsões, bem como, a busca por novos compostos anticonvulsivantes que possam evitar o desenvolvimento desta patologia. Como a nocicepção e a epilepsia possuem mecanismos em co-mum, e vários anticonvulsivantes são usados no tratamento da dor, foi investigado o efeito do Triter-peno 3β,6β,16β Trihidroxilup-20(29)-eno (TTHL), um composto com propriedades antinociceptivas, nas convulsões induzidas pelo pentilenotetrazol (PTZ). A administração do TTHL (30 mg/kg; p.o) au-mentou a latência para a primeira convulsão mioclônica e tônico-clônica e reduziu a duração das convulsões generalizadas induzidas pelo PTZ. Além disso, a administração do TTHL reduziu a pero-xidação lipídica e a carbonilação de proteínas, assim como, protegeu da inibição da captação de glu-tamato e da atividade da Na+,K+-ATPase (subunidades α1 e α2/α3) causadas pelo PTZ. Embora, o TTHL não mostrou uma atividade antioxidante per se e não alterou a ligação do [3H]flunitrazepam ao sítio para bezodiazepínico do receptor GABAA, este composto protegeu das convulsões e da inibição da atividade da Na+, K+- ATPase induzidos pela ouabaina. Estes resultados sugerem que a ação an-ticonvulsivante do TTHL é devido s manutenção da atividade da Na+,K+-ATPase. De fato, os experi-mentos realizados no córtex cerebral in vitro mostraram que o PTZ (10 mM) reduziu a atividade da Na+,K+-ATPase e que a incubação prévia com TTHL (10 μM) protegeu desta inibição. Dessa forma, estes dados indicam que a proteção exercida pela TTHL neste modelo de convulsão não está relaci-onado com atividade antioxidante ou a atividade GABAérgica. No entanto, estes resultados demons-traram que a proteção eficaz da atividade da Na+,K+-ATPase , induzida por este composto, protege contra os danos excitotóxic e oxidativos induzidos pelo PTZ.

Na+ sensitive hypertension in Dahl salt sensitive rats (Dahl S) or spontaneously hypertensive rats (SHR) is linked to intrinsic changes in the brain that favour increased Na+ entry into the cerebrospinal fluid (CSF) followed by increases in sympathetic hyperactivity and hypertension (Huang et al 2004). Similar responses are observed in salt resistant and Wistar rats that receive an intracerebroventricular (icv) infusion of Na+ rich artificial cerebrospinal fluid (aCSF) (Huang et al 2001, 2006). Downstream to increased CSF[Na+], a pathway has been described involving mineralocorticoid receptors (MRs), benzamil sensitive Na+ channels, “ouabain”, and angiotensin II type 1 receptors (AT1-R) (Huang et al 1998, Zhao et al 2001, Wang and Leenen 2003, Huang et al 2008). Blood pressure (BP) responses to increased CSF[Na+] may involve activation of AT1-R in the subfornical organ (SFO) as the BP response to injection of NaCl into a lateral ventricle can be blocked by AT1-R blockade in the SFO (Rohmeiss et al 1995a). The role of aldosterone and AT1-R in the SFO was investigated in mediating the BP and heart rate (HR) response to increases in CSF[Na+] and local [Na+]. Results show that infusion of 0.45M and 0.6M Na+ rich aCSF into the SFO increases BP but not HR. The BP is unchanged by infusion of a mannitol solution osmotically equivalent to 0.6M Na+ rich aCSF indicating that the SFO is Na+ sensitive. The BP response to a lower concentration of Na+ (0.45M) is enhanced by prior infusion of aldosterone while BP response to 0.6M is not further enhanced suggesting that the SFO may have maximal responsiveness to acute increases in [Na+] at 0.6M. The BP responses to Na+ rich aCSF in the SFO and the enhancement of those responses by aldosterone can be blocked by infusion of the AT1-R blocker Candesartan in the SFO. This response appears therefore to be mediated in the SFO through AT1-R activation, likely through Ang II release in the SFO. ICV infusion of Na+ rich aCSF increases BP but not HR and this response is partially blocked by infusion of the AT1-R blocker Candesartan in the SFO. This indicates that nearly half the BP responses to icv infusion of Na+ rich aCSF is mediated through AT1-R activation in the SFO. Lastly, contrary to icv, PVN and MnPO studies (Huang and Leenen 1996, Budzikowski and Leenen 2001, Gabor and Leenen 2009) ouabain in the SFO does not increase BP or HR. In conclusion, these results show that the SFO is Na+ sensitive and mediates half the BP responses to changes in CSF[Na+] through a mechanism that involves AT1-R activation. The SFO is further sensitized to Na+ by aldosterone presumably through its genomic effects. Lastly, ouabain in the SFO does not increase BP or HR suggesting that endogenous ouabain in the SFO is not involved in modulating BP or HR responses.

Epigenetics continues to be redefined and new discoveries are likely to revolutionise the field still further. This thesis explores different aspects of how epigenetic regulation of gene expression contributes to human disease. Paper I explores the function of the IKKα kinase in regulating gene expression through the nuclear retinoic acid receptor (RAR). We define a set of genes requiring IKKα for their expression and found recruitment of IKKα to the RAR dependent on structural motifs in its protein sequence. This interplay between the NFκB pathway and nuclear receptor regulated transcription is important to consider when designing therapeutic strategies. Papers II and III focus on the plasma cell malignancy multiple myeloma (MM) and define a gene regulatory circuit defining an underexpressed gene profile in MM dependent on the Polycomb proteins. We provide proof-of-principle that the use of small chemical inhibitors may be operational in reactivating genes silenced by H3K27me3 and that this leads to decreased tumour load and increased survival in the 5T33 in vivo model of MM. We explored the genome-wide distribution of H3K27me3 and H3K4me3, and defined their association with gene expression in freshly-isolated malignant plasma cells from MM patients. Importantly, H3K27me3-marked genes in MM associated with more aggressive stages of the disease and less favourable survival. We present evidence that gene targeting by H3K27me3 is likely to not only involve a small population of tumour cells, but rather represent a common MM profile and further provide a rationale for evaluating epigenetic therapeutics in MM. Paper IV shows that pro-inflammatory gene expression in monocytes of psychiatric patients can be induced in vitro by sodium pump inhibitors, as the steroid hormone ouabain. We suggest that the ouabain-induced gene expression is regulated by an intricate network involving microRNAs, Polycomb and the H3K27me3 demethylase JMJD3. Our data indicates that epigenetic regulators play a role in transmitting cues between intrinsic and/extrinsic stimuli and gene expression in psychiatric illness. This thesis provides novel insights on how seemingly unrelated pathways may converge on transcriptional regulation and evidence that epigenetic modifiers contribute to the pathogenesis of human complex diseases such as multiple myeloma and mood disorders.

A enzima Na+, K+-ATPase é uma proteína de membrana altamente conservada em eucariotos, capaz de gerar um gradiente eletroquímico, fundamental para o balanço osmótico das células, o potencial de repouso das membranas e a propriedade excitatória das células musculares e nervosas. Além de seu papel regulatório na homeostasia iônica, desempenha um papel na transdução de sinal e na ativação de transcrição gênica, modulando na presença de ouabaína o crescimento celular, migração e morte celular programada. A Ouabaína (OUA) é um esteróide cardiotônico, produzido no córtex da adrenal e no hipotálamo. Em linhas gerais, a sinalização da Na+, K+-ATPase promovida pela OUA parece ativar vias associadas à modulação de fatores de transcrição como a via da Src, MAPK, Ca2+ e NF-kB. Evidências indicam que o NF-kB exerça algum tipo de modulação na via canônica do WNT, no entanto os mecanismos moleculares ainda são desconhecidos. A via de sinalização WNT desempenha função importante na embriogênese e na homeostase de tecidos adultos. Assim, o objetivo do presente projeto é verificar se a administração intrahipocampal de OUA é capaz de modular a atividade das vias canônicas do NF-kB e da WNT. Estas vias foram estudadas em um decurso temporal imediato (1 -2 horas) e tardio (10, 24 e 48 horas) utilizando técnicas como Western Blotting, RT-PCR e EMSA. Os resultados encontrados mostram que a OUA (10 nM) foi capaz de ativar a via de sinalização NF-kB, após 1 hora, 10, 24 e 48 horas. A OUA também foi capaz de ativar a via canônica do WNT, sendo que após 10 horas ocorreu aumento da proteína pGSK-3b, enquanto que em 24 horas, observamos aumento da translocação nuclear da b-CATENINA. Além disso, pode-se verificar aumento de BDNF ao longo de todo o decurso temporal.
The enzyme Na+,K+-ATPase is an integral membrane protein, highly conserved in eukaryotes, that establishes the electrochemical gradient across the plasma membrane, which is essential to maintain the osmotic balance of cells, the resting membrane potential and the excitatory property of nerve and muscle cells. Besides its role in ion homeostasis, several recent studies suggest that this pump may also act as a signal transducer and transcription activator involved in cell growth, differentiation and programmed cell death. Ouabain (OUA), the ligand of Na+,K+-ATPase, is a steroid derivative that is produced by the adrenal cortex and hypothalamus. After OUA binding, the Na+,K+-ATPase signaling seems to activate pathways such as Src, MAPK, NF-kB and Ca2+. Some evidences indicate a possible crosstalk between the NF-kB signaling pathway and the canonical WNT pathway, however the molecular mechanisms are still unknown. The canonical WNT play important roles during embryogenesis and in adult tissue homeostasis. The aim of this project is to verify if the intrahipocampal administration of OUA is able to modulate the activity of the canonical pathways of NF-kB and WNT. Both pathways were studied after 1 and 2 hours, and after 10, 24 and 48 hours by methods such Western blot, RT-PCR and Electrophoretic mobility shift assays. The results show that the OUA (10 nM) was able to activate the signaling pathway NF-kB after 1, 10, 24 and 48 hours. The OUA was also able to activate the canonical WNT pathway, since after 10 hours there was an increased in pGSK-3b protein, whereas in 24 hours, we observed increased nuclear translocation of b-CATENIN. Moreover, we found increased levels of BDNF throughout the time course.