Introdução: As extrassístoles ventriculares e supraventriculares (EV e ESSV) são frequentes e muitas vezes sintomáticas. O íon magnésio (Mg) desempenha um papel importante na fisiologia do potencial de ação transmembrana celular e do ritmo cardíaco. Objetivo: Avaliar se a administração do Pidolato de Magnésio (PMg) em pacientes com EV e ESSV é superior ao placebo (P) na melhora dos sintomas e densidade das extrassístoles(DES). Métodos: Estudo duplo-cego, randomizado, com 90 pacientes sintomáticos consecutivos, com mais de 240/EV ou ESSV ao Holter de 24 horas e selecionados para receber P ou PMg. Para avaliar a melhora da sintomatologia, foi feito um questionário categórico e específico de sintomas relacionado às extrassístoles. Foi considerada significante uma redução de mais de 70% na DES por hora após o tratamento. A dose do PMg foi de 3,0g/dia por 30 dias, equivalente a 260mg do elemento Mg. Nenhum paciente tinha cardiopatia estrutural ou insuficiência renal. Resultados: Dos 90 pacientes estudados, 49 eram do sexo feminino (54,4%). A faixa etária variou de 16 a 70 anos. No grupo PMg, 77,8% dos pacientes tiveram redução maior que 70%, 6,7% deles entre 50% a 70% e, somente 13,3% dos pacientes com redução menor que 50% na DES . No grupo P, 44,4% dos pacientes tiveram melhora de apenas 30% na frequência de extrassístoles (p<0,001). A melhora dos sintomas foi alcançada em 91,1% dos pacientes do grupo PMg, comparada com somente 15,6% do grupo P(p<0,001). Conclusão: A suplementação de Mg por via oral reduziu a DES, resultando em melhora dos sintomas. Estudos clínicos e moleculares são necessários para avaliar o Mg intracelular e orientar quanto às necessidades diárias deste íon, evidenciar as prováveis deficiências e esclarecer melhor como prevenir e tratar pacientes com extrassístoles sintomáticas e sem cardiopatia estrutural. / Introduction: Premature ventricular and supraventricular complexes (PVC and PsVC) are frequent and often symptomatic. The magnesium (Mg) ion plays a role in the physiology of cell membranes and cardiac rhythm. Objective: We evaluated whether the administration of Mg Pidolate (MgP) in patients with PVC and PsVC is superior to placebo (P) in improving symptoms and arrhythmia frequency. Methods: Randomized double-blind study with 90 consecutive symptomatic patients with more than 240 PVC or PsVC on 24-hour Holter monitoring who were selected to receive placebo or MgP. To evaluate symptom improvement, a categorical and a specific questionnaire for symptoms related to PVC and PsVC was made. Improvement in premature complex density (PCD) per hour was considered significant if percentage reduction was >=70% after treatment. The dose of MgP was 3.0 g/day for 30 days, equivalent to 260mg of Mg element. None of the patients had structural heart disease or renal failure. Results: Of the 90 patients, 49 were female (54,4%). Ages ranged from 16 to 70 years old. In the MgP group, 77.8% of patients had a PCD reduction >70%, 6,7% of them from 50% to 70%, and only 13.3% <50%. In the P group, 44,4% showed slight improvement, <30%, in the premature complexes frequency (p<0.001). Symptom improvement was achieved in 91.1% of patients in the MgP group, compared with only 15.6% in the P group (p<0.001). Conclusions: Oral Mg supplementation decreases PCD, resulting in symptom improvement. Clinical and molecular studies are needed to evaluate intracellular Mg and develop better targets for the daily needs of this ion, show probable deficiencies, and explain how to prevent and better treat patients with symptomatic premature ventricular, and supraventricular complexes and no apparent heart disease.

Os efeitos inibitórios do eugenol (EUG) em canais para Na+ ativados por voltagem (NaV) mostrados anteriormente não são totalmente compatíveis com nossos resultados. Nós estudamos os efeitos do EUG em correntes macroscópicas de Na+ e os comparamos aos da lidocaína, um anestésico local, para referência. O EUG bloqueou, rápida e reversivelmente, correntes de Na+ mistas (TTX-S+TTX-R) assim como as correntes de Na+ TTX-R. As IC50 para a inibição das correntes mistas e TTX-R pelo EUG foram de 2,28 e 2,27 mmol/L, respectivamente. O bloqueio depende da freqüência de despolarizações. Nas correntes mistas, o EUG desloca a curva de ativação para a direita, a de inativação para a esquerda, não altera a cinética de inativação e retarda a recuperação da inativação, rápida e lenta, dos canais. Nas correntes TTX-R, o efeito é semelhante, exceto na curva de ativação, que não é deslocada. Nós concluímos que o EUG bloqueia os NaV por se ligar a estados conformacionais de repouso e inativados, rápido e lento. Os efeitos são semelhantes, mas não idênticos aos da lidocaína. / The previously described inhibitory effects of eugenol (EUG) on voltage-activated Na+ channels (Nav) are not compatible with our results. We have studied the effects of EUG on macroscopic Na+ currents and compared them to the effects of lidocaine, a local anesthetic. EUG blocked both mixed (TTX-S and TTX-R) and TTX-R Na+ currents in a fast and reversible manner. The values of IC50 for the inhibition of mixed and TTX-R currents were 2.28 and 2.27 mmol/L respectively. The blockade depends on frequency of depolarizing pulses. In mixed currents EUG displaced the activation curve to the right, the inactivation curve to the left, does not alter the inactivation kinetics and retards the recovery from inactivation, fast and slow, of the Na+ channels. In TTX-R currents, EUG effects were similar, except on the activation curve, which was not shifted. In conclusion, EUG blocks Nav by binding to the resting and inactivated conformational states of channels, fast and slow. EUG effects resembles lidocaine ones, but are not identical.

Plasmalemmale Kalzium- und Kaliumkanäle sind wichtige Regulatoren der kontraktilen Kraft glatter Gefäßmuskelzellen. Bei Untersuchungen an einer Vielzahl nicht humaner glatter Muskelzellen wurden verschiedene Kalzium- und Kaliumkanaltypen identifiziert. Allerdings ist über das Vorkommen, die biophysikalisch-pharmakologischen Eigenschaften, Regulation, Bedeutung und differentielle Expression von spannungsabhängigen Kalzium- und Kaliumkanälen in humanen arteriellen Gefäßmuskelzellen noch wenig bekannt. In dieser Arbeit wurden spannungsabhängige Kalzium- und Kaliumkanalströme in frisch-isolierten Gefäßmuskelzellen humaner Koronararterien analysiert. Die wichtigsten Ergebnisse waren: (1) Gefäßmuskelzellen humaner Koronararterien exprimieren funktionell aktive spannungsabhängige L-Typ-Kalziumkanäle. (2) Die hohe Permeationsrate von L-Typ-Kanälen in Lösungen mit physiologischer Ca2+-Konzentration wird durch die (1- Kanaluntereinheit bestimmt. (3) L-Typ-Kanäle der C-Klasse werden durch G-Proteine und Proteinkinase C moduliert. (4) Die Expression von L-Typ-Kanälen in Gefäßmuskelzellen wird differentiell reguliert. (4) Vier diverse Kaliumkanalströme kommen in Gefäßmuskelzellen humaner Koronararterien vor: IK(dr), IBK(Ca), IK(ATP) und STOCs. (5) Spontane transiente Auswärtsströme (STOCs) werden durch Ca2+-Sparks ausgelöst. (6) Ca2+-Sparks entstehen durch Öffnung von Ca2+-Freisetzungskanälen (Ryanodinrezeptoren) des sarkoplasmatischen Retikulums. (7) KATP-Kanäle fungieren als Zielstruktur für diverse endogene und synthetische Vasodilatatoren. (8) Ca2+-Sparks und STOCs bewerkstelligen die Feineinstellung des myogenen Gefäßtonus. (9) Der Ca2+-Spark/STOC-Signalweg stellt potenziell ein neuartiger Angriffsort für Hormone und Pharmaka dar, über den der Gefäßtonus von geringlumigen Arterien beeinflusst werden kann. Diese Ergebnisse tragen zum Verständnis der Bedeutung von plasmalemmalen Ionenkanälen bei chronischen Gefäßerkrankungen wie Atherosklerose und Hypertonie bei und eröffnen möglicherweise neue therapeutische Möglichkeiten. / Plasmalemmal calcium- and potassium channels represent important regulators of the contractile force of smooth muscle. Studies on various human smooth muscle cells have identified different calcium and potassium channels. However, relatively little is known on the presence, biophysical and pharmacological properties, regulation, function, and differential expression of voltage-dependent calcium and potassium channels in human coronary arteries. In this study, voltage-depedent calcium and potassium channels in human coronary arteries were studied. The main findings are: (1) Smooth muscle cells of human coronary arteries exhibit functional voltage-dependent, L-type calcium channels. (2) The high permeation rate of L-type channels in solutions with physiological calcium concentrations is determined by the (1 channel subunit. (3) C-class L-type channels are modulated by G proteins and protein kinase C. (4) The expression of L-type channels in vascular smooth muscle cells is differentially regulated. (4) Four diverse potassium channel currents are present in smooth muscle cells from human coronary arteries: IK(dr), IBK(Ca), IK(ATP) and STOCs. (5) Spontaneous transient outward currents (STOCs) are induced by Ca2+ sparks. (6) Ca2+ sparks are generated by the opening of calcium release channels (ryanodine receptors) in the sarcoplasmic reticulum. (7) KATP channels function as target for diverse endogeneous and synthetic vasodilators. (8) Ca2+ sparks and STOCs control the myogenic tone of arterial vessels. (9) The Ca2+ spark/STOC signaling pathway represents a novel target for hormones and drugs to regulate the diameter of small, resistance-sized arteries. These results contribute to a better understanding of the role of plasmalemmal ion channels in chronic vascular disease, such as atherosclerosis and hypertension. They may open novel therapeutic possibilites in the treatment of chronic vascular disease.

A peçonha de anêmonas do mar é uma fonte conhecida de compostos bioativos, incluindo peptídeos, que atuam em canais voltagem-dependentes. Foram descritos 4 tipos de neurotoxinas de anêmonas do mar, que atuam em canais NaV e 4 tipos que atuam em canais KV. Essas toxinas têm permitido discriminar subtipos de canais voltagem-dependentes, estreitamente relacionados, e constituem poderosas ferramentas para estudar o funcionamento e estrutura desses canais. Neste estudo, foram isolados e caracterizados três peptídeos da fração neurotóxica da anêmona do mar Anthopleura cascaia. Esses peptídeos foram nomeados como AcaIII1425, AcaIII2970 e AcaIII3090, onde Aca faz referência a espécie e os números seguem os resultados obtidos nas etapas de purificação. A peçonha foi extraída por meio de estímulos elétricos e purificada por gel-filtração (Sephadex G-50) e fase reversa por HPLC (C-18). As massas moleculares foram obtidas por meio de MALDI-TOF, apresentando 3337,4 Da para a AcaIII1425, 4881,7 Da para a AcaIII2970 e 4880,5 Da para AcaIII3090. Através da técnica de voltage-clamp, esses peptídeos foram testados em diferentes subtipos de canais NaV e KV expressados em ovócito de Xenopus. As toxinas AcaIII2970 e AcaIII3090 retardam, de maneira seletiva, a inativação rápida dos subtipos rNaV1.3, mNaV1.6 e hNaV1.5, enquanto que as outras isoformas testadas permaneceram inalteradas. É importantemente salientar que, a AcaIII2970 e AcaIII3090 também foram examinadas no canal de inseto DmNaV1, revelando uma clara \"filo-seletividade\" na eficácia da atividade das toxinas. A AcaIII2970 e AcaIII3090 inibem fortemente a inativação do canal NaV de inseto, resultando em um aumento na amplitude do pico da corrente e removendo completamente a inativação rápida. Para quantificarmos essa \"filo-seletividade\", foram construídas curvas da dependência da concentração no retardo da inativação induzida pelas toxinas AcaIII2970 e AcaIII3090 nos canais em que apresentaram maior eficácia. Os IC50 foram obtidos após a plotagem dos dados em uma curva sigmoidal. Para a AcaIII2970, os seguintes valores de IC50 foram obtidos: DmNaV1 = 162,19 ± 11,22 nM, mNaV1.6 = 645,92 ± 18,52 nM, rNaV1.3 = 572,56 ± 44,96 nM. Para a AcaIII3090, os seguintes valores de IC50 foram obtidos: DmNaV1 = 99,03 ± 9,25 nM, mNaV1.6 = 158,30 ± 33,86 nM, rNaV1.3 = 371,60 ± 6,48 nM. A AcaIII1425 atua, bloqueando, de modo seletivo os subtipos rKV1.1, rKV1.6 e rKV4.3, enquanto que as outras isoformas testadas permaneceram inalteradas. Devido à maior especificidade da toxina AcaIII1425 pelos subtipos rKV1.1 e rKV1.6, foram realizados ensaios de bloqueio da corrente do canal em função da concentração da toxina (curva dose-resposta). Os valores de IC50 para os subtipos rKV1.1 e rKV1.6 são de 7642,98 ±1601,65 nM e 241,65 ±4,27 nM, respectivamente. Desta forma, a AcaIII1425 é cerca de 32 vezes mais potente em canais do subtipo rKV1.6 do que em relação aos canais do subtipo rKV1.1. A estrutura primária das toxinas foram determinadas por degradação de Edman. A sequência parcial da AcaIII2970 e AcaIII3090 revelou que estas são similares a toxinas de canal de sódio do tipo1 de anêmonas do mar. A sequência completa da AcaIII1425 não apresenta similaridade com toxinas de anêmonas do mar, mas é similar a toxinas de Conus e aranha que possuem um motivo estrutural conhecido como ICK. Dessa forma, propomos que a AcaIII1425 seja um novo grupo de toxinas de anêmonas do mar que bloqueiam KV. Dado o ineditismo da toxina AcaIII1425, foram feitos experimentos in silico para obtermos um maior refinamento do mecanismo de interação entre a toxina e o canal rKV1.6. Estes experimentos indicaram que diferentes regiões dos canais KV são importantes para a seletividade e potência da toxina, corroborando com as propostas que vem sendo descritas / The venom of sea anemones is a known source of bioactive compounds, including peptides that act on voltage-gated ion channels. Four types of neurotoxins from sea anemones, acting on NaV channels, and four types acting on KV channels, have been reported. These toxins have developed the ability to discriminate closely related subtypes of voltage-gated channels, making them powerful tools to studying the function and structure of these channels. In this study, we isolated and characterized three peptides of the neurotoxic fraction from the venom of the sea anemone Anthopleura cascaia. These peptides were named as AcaIII1425, and AcaIII2970 AcaIII3090, where Aca refers to the species and the following numbers refer to results obtained in the purification steps. The venom was milked by electric shock and purified by molecular exclusion (Sephadex G-50) and reverse phase HPLC (C-18). Their molecular weights are 3337.4 Da to AcaIII1425, 4881.7 Da to AcaIII2970 and 4880.5 Da to AcaIII3090, obtained through a MALDI-TOF. Using the voltage-clamp technique, we have assayed the effects of these peptides on different subtypes of NaV and KV channels expressed in Xenopus oocytes. AcaIII2970 and AcaIII3090 toxins selectively slow down the fast inactivation of rNaV1.3, mNaV1.6 and hNaV1.5 subtypes, while the other mammalian isoforms remained unaffected. Importantly, AcaIII2970 and AcaIII3090 were also examined in insect DmNaV1 channel, revealing a clear phyla-selectivity with regards to the efficacy of the toxin. AcaIII2970 and AcaIII3090 strongly inhibit the inactivation of the insect NaV channel, resulting in an increase in the amplitude of the peak current, and complete removal of the fast and steady-state inactivation. In order to quantify this \"phyla-selectivity\", curves of the concentration dependence of the delayed inactivation induced by AcaIII2970 and AcaIII3090 toxins channels with higher efficacy, were built. After plotting the data on a sigmoidal curve the IC50 values were obtained. For AcaIII2970, the following IC50 values were obtained: DmNaV1 = 162.19 ± 11.22 nM, mNaV1.6 = 645.92 ± 18.52 nM and rNaV1.3 = 572.56 ± 44.96 nM. For AcaIII3090, the following IC50 values were obtained: DmNaV1 = 99.03 ± 9.25 nM, mNaV1.6 = 158.30 ± 33.86 nM and rNaV1.3 = 371.60 ± 6.48 nM. AcaIII1425 acts, selectively, blocking rKV1.1, rKV1.6 and rKV4.3 subtypes, while the others isoforms tested remained unaltered. Due the higher specificity of AcaIII1425 to rKV1.1 and rKV1.6 subtypes, assays were performed to evaluate the blocking channel current versus toxin concentration (dose-response curve). IC50 values for the subtypes rKV1.6 and rKV1.1 are 7642.98 ± 1601.65 nM and 241.65 ± 4.27 nM, respectively. Thus, AcaIII1425 is about 32 times more potent in the rKV1.6 than in the rKV1.1 channel. The primary structure of the toxins was determined by the Edman degradation. The partial sequence of AcaIII2970 and AcaIII3090 revealed that these toxins are similar to the type 1 sodium channel sea anemones neurotoxins. The complete sequence of AcaIII1425 has no similarity with other sea anemone toxins, but is similar to the Conus and spider neurotoxins which have a structural motif known as ICK. Thus, we propose that AcaIII1425 comprises a new group of sea anemones toxins that block KV channels. Given the unprecedented nature of the toxin AcaIII1425, in silico assays were carried out in order to further refining the proposed mechanism underlying the interaction between the toxin and the rKV1.6 channel. The results indicate that, in agreement to what has been proposed elsewhere, different regions of the KV channels are important for the toxin selectivity and potency

No description available.

Yau Ho Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 131-144). / Abstracts in English and Chinese. / DECLARATION --- p.I / ACKNOWLEDGEMENTS --- p.II / ENGLISH ABSTRACT --- p.III / CHINESE ABSTRACT --- p.VI / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Oxidative Stress --- p.1 / Chapter 1.1.1 --- Historical Background of reactive oxygen/nitrogen species --- p.1 / Chapter 1.1.2 --- What is Oxidative Stress? --- p.3 / Chapter 1.1.3 --- Reactive Oxygen Species (ROS) --- p.4 / Chapter 1.1.3.1 --- Superoxide anion (02-) --- p.4 / Chapter 1.1.3.2 --- Hydrogen peroxide (H202) --- p.5 / Chapter 1.1.3.3 --- Hydroxyl radical --- p.6 / Chapter 1.1.3.4 --- Nitric oxide (NO) --- p.7 / Chapter 1.2 --- Cardiovascular System --- p.8 / Chapter 1.2.1 --- Enzymatic and Non-enzymatic Sources of ROS in Cardiovascular System --- p.8 / Chapter 1.2.1.1 --- NADPH oxidase --- p.8 / Chapter 1.2.1.2 --- Hypoxanthine-Xanthine oxidase (HX-XO) --- p.9 / Chapter 1.2.1.3 --- Nitric oxide synthase (NOS) --- p.10 / Chapter 1.2.1.4 --- Mitochondrial electron transport chain (ETC) --- p.11 / Chapter 1.2.1.5 --- Cyclooxygenase --- p.11 / Chapter 1.2.1.6 --- Lipoxygenae --- p.12 / Chapter 1.2.1.7 --- Endoplasmic reticulum --- p.12 / Chapter 1.2.2 --- ROS/RNS Scavenging Systems --- p.13 / Chapter 1.2.2.1 --- Superoxide dismutase (SOD) --- p.13 / Chapter 1.2.2.2 --- Catalase --- p.14 / Chapter 1.2.2.3 --- Glutathione peroxidase --- p.15 / Chapter 1.2.2.4 --- Non-enzymatic antioxidants --- p.15 / Chapter 1.2.3 --- Factors that stimulate ROS production in cardiovascular system --- p.18 / Chapter 1.2.3.1 --- Oxygen tension --- p.18 / Chapter 1.2.3.2 --- "Flow, Shear, and Stretch as an initial stimulus for endothelial oxidant signalling" --- p.18 / Chapter 1.2.3.3 --- Activation of rennin-angiotensin system promote oxidative stress in cardiovascular system --- p.19 / Chapter 1.2.3.4 --- Regulation of vascular ROS production by vasoactive substances --- p.19 / Chapter 1.2.4 --- Regulation of vascular tone in Cardiovascular System by ROS/RNS --- p.20 / Chapter 1.2.4.1 --- Regulation of vascular tone --- p.20 / Chapter 1.2.5 --- Pathophysiological Effects of ROS --- p.23 / Chapter 1.2.5.1 --- Cellular injury by lipid peroxidation --- p.23 / Chapter 1.2.5.2 --- Role of ROS in immune defence --- p.23 / Chapter 1.2.5.3 --- Redox regulation of cell adhesion --- p.24 / Chapter 1.2.6 --- Evidences from Clinical Studies of Oxidative Stress-Related Vascular Diseases --- p.25 / Chapter 1.2.6.1 --- Hyperlipidaemia --- p.25 / Chapter 1.2.6.2 --- Hypertension --- p.25 / Chapter 1.2.6.3 --- Chronic heart failure (CHF) --- p.26 / Chapter 1.2.6.4 --- Chronic renal failure (CRF) --- p.26 / Chapter 1.2.6.5 --- Atherosclerosis --- p.27 / Chapter 1.2.6.6 --- Ischemia/reperfusion (I/R) injury --- p.27 / Chapter 1.2.7 --- Role of Vascular Endothelium in Oxidative Stress --- p.29 / Chapter 1.2.8 --- Role of Ca in oxidative stress in cardiovascular system --- p.29 / Chapter 1.2.8.1 --- Calcium Signaling in Vascular Endothelial Cells --- p.30 / Chapter 1.2.9 --- ROS effect on endothelial Ca2+ --- p.31 / Chapter 1.2.9.1 --- Multiple targets of ROS on intracellular Ca2+ mobilization --- p.32 / Chapter 1.2.9.2 --- Reports of H202-induced Ca2+ release in various cell types --- p.33 / Chapter 1.2.9.3 --- Reported effects of H202 on agonist-induced Ca2+ signal --- p.34 / Chapter 1.2.9.4 --- Differences between macrovessels and microvessels --- p.34 / Chapter 1.3 --- TRP Channel --- p.41 / Chapter 1.3.1 --- Discovery of Drosophila TRP --- p.41 / Chapter 1.3.2 --- Mammalian TRP subfamily --- p.41 / Chapter 1.3.3 --- General topology of TRP channel --- p.42 / Chapter 1.3.4 --- Interactions of oxidative stress with TRP channels --- p.44 / Chapter 1.3.5 --- The role of TRPC3 and TRPC4 in oxidative stress --- p.44 / Chapter 1.3.6 --- TRPM subfamily --- p.44 / Chapter 1.3.6.1 --- Expression of TRPM2 --- p.45 / Chapter 1.3.6.2 --- Dual Role of TRPM´2ؤChannel and Enzyme --- p.45 / Chapter 1.3.6.3 --- Regulatory mechanisms of TRPM2 --- p.46 / Chapter 1.3.6.3.1 --- ADP-ribose (ADPR) directly regulating --- p.46 / Chapter 1.3.6.3.2 --- NAD regulating --- p.46 / Chapter 1.3.6.3.3 --- Oxidative stress regulating independent of ADPR or NAD --- p.47 / Chapter 1.4 --- Cell Death Induced by Oxidative Stress --- p.48 / Chapter 1.4.1 --- Redox status as a factor to determine cell death --- p.48 / Chapter 1.4.2 --- Role of TRPM2 in oxidative stress-induced cell death --- p.48 / Chapter 1.5 --- Aims of the Study --- p.49 / Chapter Chapter 2: --- Materials and Methods --- p.50 / Chapter 2.1 --- Functional Characterization of TRPM2 by Antisense Technique --- p.50 / Chapter 2.1.1 --- Restriction Enzyme Digestion --- p.50 / Chapter 2.1.2 --- Purification of Released Inserts and Cut pcDNA3 Vectors --- p.51 / Chapter 2.1.3 --- "Ligation of TRPM2 Genes into Mammalian Vector, pcDNA3" --- p.52 / Chapter 2.1.4 --- Transformation for the Desired Clones --- p.52 / Chapter 2.1.5 --- Plasmid DNA Preparation for Transfection --- p.53 / Chapter 2.1.6 --- Confirmation of the Clones --- p.53 / Chapter 2.1.6.1 --- Restriction Enzymes Strategy --- p.53 / Chapter 2.1.6.2 --- Polymerase Chain Reaction (PCR) Check --- p.54 / Chapter 2.1.6.3 --- Automated Sequencing --- p.55 / Chapter 2.2 --- Establishing Stable Cell Lines --- p.56 / Chapter 2.2.1 --- Cell Culture --- p.56 / Chapter 2.2.2 --- Geneticin Selection --- p.57 / Chapter 2.3 --- Expression of TRPM2 in Transfected and non-Transfected H5V Cells --- p.57 / Chapter 2.3.1 --- Protein Sample Preparation --- p.57 / Chapter 2.3.2 --- Western Blot Analysis --- p.58 / Chapter 2.3.3 --- Protein Expression Analysis --- p.59 / Chapter 2.4 --- "Immunolocalization of TRPM2 in Human Heart, Cerebral Artery, Renal, Hippocampus and Liver" --- p.59 / Chapter 2.4.1 --- Paraffin Section Preparation --- p.59 / Chapter 2.4.2 --- Immunohistochemistry --- p.60 / Chapter 2.5 --- [Ca2+ ］i Measurement in Confocal Microscopy --- p.62 / Chapter 2.5.1 --- Cytosolic Ca2+ measurement --- p.62 / Chapter 2.5.2 --- Measuring the Ca2+ in the Internal Calcium Stores --- p.63 / Chapter 2.5.3 --- Data Analysis --- p.64 / Chapter 2.6 --- Examining Cell Death Induced by H2O2 by DAPI Staining --- p.65 / Chapter 2.6.1 --- DAPI Staining --- p.65 / Chapter Chapter 3: --- Results --- p.66 / Chapter 3.1 --- Superoxide Anion-Induced [Ca 2+]i rise in H5V Mouse Heart Microvessel Endothelial Cells --- p.66 / Chapter 3.1.1 --- Superoxide Anion-induced [Ca2+ ]i Rise --- p.66 / Chapter 3.1.2 --- Effect of Catalase on the Superoxide Anion-induced [Ca2+]i］］ Rise --- p.66 / Chapter 3.1.3 --- IP3R inhibitor Inhibits Superoxide anion-induced [Ca 2+]i Rise --- p.67 / Chapter 3.1.4 --- Effect of Phospholipase A2 Inhibitor on Superoxide anion- induced [Ca2+]i Rise --- p.67 / Chapter 3.1.5 --- Effect of Hydroxyl Radical Scavenger on Superoxide Anion- induced [Ca2+]i Rise --- p.68 / Chapter 3.2 --- Hydrogen Peroxide-induced Ca2+ Entry in Mouse Heart Microvessel Endothelial Cells --- p.74 / Chapter 3.2.1 --- Hydrogen Peroxide Induces [Ca2 +]i rise in H5V Mouse Heart Microvessel Endothelial Cells --- p.74 / Chapter 3.2.2 --- Hydrogen Peroxide Induces [Ca 2+]i rise in two phases (Rapid and Slow response) --- p.74 / Chapter 3.2.3 --- Hydrogen Peroxide Induces [Ca 2+]i rise in a Extracellular Ca + Concentration Dependent Manner --- p.77 / Chapter 3.3 --- Hydrogen Peroxide Reduces Agonist-induced [Ca2+]i rise --- p.79 / Chapter 3.3.1 --- Hydrogen Peroxide Reduces ATP-induced [Ca2+ ]i rise in a H2O2 Concentration Dependent Manner --- p.79 / Chapter 3.3.2 --- Hydrogen Peroxide Reduces ATP-induced [Ca 2+]i rise in a H2O2 Incubation Time Dependent Manner --- p.79 / Chapter 3.3.3 --- Hydrogen Peroxide Reduces the ATP-induced Intracellular Ca2+ Release --- p.80 / Chapter 3.3.4 --- XeC Inhibited H202-induced [Ca2+]i rise --- p.80 / Chapter 3.3.5 --- Hydrogen Peroxide Partially Depletes Internal Ca2+ Stores --- p.81 / Chapter 3.4 --- Dissecting Signal Transduction Pathways in H202-induced [Ca2+]i rise --- p.82 / Chapter 3.4.1 --- Effect of Phospholipase C Inhibitor on H202-induced [Ca2 +]i rise --- p.82 / Chapter 3.4.2 --- Effect of Phospholipase A2 Inhibitor on H202-induced [Ca 2+]i rise --- p.83 / Chapter 3.4.3 --- Effect of hydroxyl radical scavenger on H2O2-induced [Ca 2+]i rise --- p.83 / Chapter 3.5 --- Functional Role of TRPM2 Channel in H202-induced [Ca2+]i Rise in H5V Cells --- p.92 / Chapter 3.5.1 --- Expression of TRPM2 and the Effect of TRPM2 Antisense Construct on TRPM2 Protein Expression --- p.92 / Chapter 3.5.2 --- Effect of Antisense TRPM2 on H202-induced Ca2+ Entry --- p.94 / Chapter 3.6 --- H202-induced Cell Death --- p.101 / Chapter 3.7 --- Expression Pattern of TRPM2 Channel in Vascular System --- p.104 / Chapter 3.7.1 --- Immunolocalization of TRPM2 in Human Cerebral Arteries --- p.104 / Chapter 3.7.2 --- Immunolocalization of TRPM2 in Human Cardiac Muscles --- p.105 / Chapter 3.7.3 --- Immunolocalization of TRPM2 in Human Kidney --- p.105 / Chapter Chapter 4: --- Discussion --- p.113 / Chapter 4.1 --- Oxidative modification of Ca2+ homeostasis --- p.113 / Chapter 4.2 --- Pathophysiological effects of ROS on endothelium --- p.113 / Chapter 4.3 --- Effects of ROS on microvascular endothelial Ca2+ reported by other investigators --- p.115 / Chapter 4.4 --- Studies of the effect of HX-XO on cytosolic [Ca2+]i --- p.116 / Chapter 4.4.1 --- Role of 0´2Ø- and H202 in HX-XO-induced [Ca2+]i elevation --- p.116 / Chapter 4.4.2 --- IP3R involvement in HX-XO-evoked Ca + movements in H5V cells --- p.118 / Chapter 4.4.3 --- PLA2 involvement in HX-XO experiment --- p.119 / Chapter 4.5 --- Studies of the effect of direct H202 application on cytosolic [Ca2+]i --- p.120 / Chapter 4.5.1 --- Hydrogen Peroxide Induced [Ca2 +]i rise in a Extracellular Ca2 + Concentration Dependent Manner --- p.120 / Chapter 4.5.2 --- Hydrogen Peroxide Induced [Ca 2+]i rise in two phases (Rapid and Slow response) --- p.121 / Chapter 4.6 --- Effect of H202 on ATP-induced Ca2+ response --- p.121 / Chapter 4.6.1 --- H202 inhibited ATP-induced Ca2+ release in a concentration and time dependent manner --- p.121 / Chapter 4.6.2 --- IP3R involvement and store depletion in H202 experiment --- p.123 / Chapter 4.7 --- Dissecting Signal Transduction Pathways in H202-induced [Ca2+]i rise --- p.124 / Chapter 4.7.1 --- PLC involvement in H2O2 experiment --- p.124 / Chapter 4.7.2 --- PLA2 involvement in H2O2 experiment --- p.125 / Chapter 4.7.3 --- Hydroxyl radical did not involve in H2O2 experiment --- p.125 / Chapter 4.8 --- Functional Studies of TRPM2 --- p.127 / Chapter 4.8.1 --- Expression of TRPM2 in H5V on protein level --- p.127 / Chapter 4.8.2 --- TRPM2 involvement in the Ca2+ signalling in response to H2O2 in H5V cells --- p.127 / Chapter 4.9 --- H202 concentration in my projec´tؤphysiological or pathological? --- p.128 / Chapter 4.10. --- H20´2ؤTRPM´2ؤCell death --- p.129 / Chapter 4.11 --- Expression of TRPM2 in human blood vessels and other tissues --- p.130 / References --- p.131

O sistema colinérgico possui papel importante na modulação dos processos de aprendizagem e memória. Nesse trabalho, avaliamos a evocação da memória de ratos de 3, 6, 12, 18 e 22 meses em esquiva inibitória e, também, analisamos a densidade dos receptores nicotínicos a7 e da subunidade b2 no hipocampo, além de enzimas colinérgicas, a colina acetiltransferase e a acetilcolinesterase. Os grupos com 18 e 22 meses apresentaram uma redução na evocação da memória de longa duração (MLD) e um aumento na densidade de receptores a7 nas piramidais de CA3 e nas células de PoDG, este apenas em 22 meses. Em contrapartida, os demais marcadores colinérgicos estavam inalterados nas diferentes idades. Assim, a redução na evocação da MLD dos animais de 18 e 22 meses acompanhada do aumento na densidade de receptores a7 pode ser caracterizada como: 1) estratégia adaptativa do envelhecimento (plasticidade colinérgica em neurônios glutamatérgicos) ou 2) modulação nos interneurônios GABAérgicos desencadeando, assim, um bloqueio no processamento da informação e o declínio da memória. / The cholinergic system plays an important role in modulating learning and memory. In this work, we evaluated the memory recall of 3, 6, 12, 18 and 22 months old rats in passive avoidance apparatus and we also evaluated, in the hippocampus, the density of a7 nicotinic receptors, b2 subunit receptor, choline acetiltransferase (ChAT) and acetilcholinesterase (AChE). The 18 and 22 months old showed a deficit in long-term memory (LTM) and an increase in a7 density in CA 3 pyramidals cells (py CA3) and polymorphic cells of Dentate Gyrus (PoDG).On the other hand, the other cholinergic markers were unchanged with aging. Thus, the deficit in LTM observed in 18 and 22 months combined with the increased a7 receptors in PoDG and py CA 3 in the hippocampus could be explained by: 1) as an adaptative strategy of aging (cholinergic plasticity in glutamatergic neurons) or 2) as a modulation in GABA interneurons triggering inhibition in the memory evocation.

Control of intracellular ion homeostasis is pivotal to plant salt tolerance. Plants have developed a number of mechanisms to keep ions at appropriate concentrations. Both transporters and channels on the plasma membrane play important roles in this function. Plant cyclic nucleotide-gated channels (CNGCs) in the plasma membrane are non-selective monovalent and divalent cation channels. So far, most studies on plant CNGCs have been conducted on heterologous systems. In planta, reverse genetic studies revealed the role of different CNGCs in cation uptake, transport and homeostasis. However, there is little information available about the functional characteristics of plant CNGCs. Among the 20 members of this protein family in Arabidopsis, only AtCNGC2 has been functionally identified as an ion channel; therefore, more functional characterization needs to be done on other members of this protein family. Several CNGCs were suggested to be involved in K+, Ca2+ and Na+ uptake and transport, but available information is scarce. This study investigated the relationship between CNGC10 and ion transport in Arabidopsis, with a particular emphasis on the involvement of CNGC10 in salt tolerance. Arabidopsis thaliana wild type (WT) and two AtCNGC10 antisense lines (A2 and A3) were used to characterise the impact of different level of salt stress on (i) root growth, ion concentration in tissues, ion fluxes across the root surface and intracellular ion concentration and pH at the seedling stage, and (ii) photosynthesis and ion concentration in tissues at the flowering stage. Plants of both antisense lines had higher K+ and lower Ca2+ and Mg2+ concentrations in shoots than WT plants when grown in non-salt control 1/4 Hoagland solution. Altered K+, Ca2+ and Mg2+ internal concentrations in AtCNGC10 antisense lines compared with WT plants under non-salt conditions indicated disturbed long distance ion transport, especially xylem loading/retrieval and/or phloem loading. The results of ion fluxes across the root surface also suggested that AtCNGC10 might be involved in transport of K+, Ca2+ and Mg2+ in tissue. Under sudden salt exposure, higher Na+ efflux and smaller K+ efflux in both antisense lines suggested that AtCNGC10 channels are involved in Na+ and K+ transport. The shoots of AtCNGC10 antisense lines A2 and A3 contained higher Na+ concentrations and significantly higher Na+/K+ ratios compared to WT, resulting in impaired photosynthesis and increased salt sensitivity in A2 and A3 than in WT plants. In contrast, seedlings of both antisense lines exposed to salt stress had lower shoot Na+/K+ ratios and longer roots than WT seedlings, indicating that A2 and A3 were more salt-tolerant than WT in the seedling stage, likely because growth is less dependent on photosynthesis in the seedling than in the flowering stage. These results suggested CNGC gene might play a different role during different developmental stages and in various plant organs.

Understanding protein-ligand interactions is highly important in drug development. In the present work the objective is to comprehend the link between structure and function using molecular modelling. Specifically, this thesis has been focused on implementation of receptor flexibility in molecular docking and studying structure-activity relationships of potassium ion channels and their blockers. In ligand docking simulations protein motion and heterogeneity of structural waters are approximated using an ensemble of protein structures. Four methods of combining multiple target structures within a single grid-based lookup table of interaction energies are tested. Two weighted average methods permit consistent and accurate ligand docking using a single grid representation of the target protein structures. Quaternary ammonium ions (QAIs) are well known K+ channel blockers. Conformations around C–N bonds at the quaternary centre in tetraalkylammonium ions in water solution are investigated using quantum mechanical methods. Relative solvation free energies of QAIs are further estimated from molecular dynamics simulations. The torsion barrier for a two-step interconversion between the conformations D2d and S4 is calculated to be 9.5 kcal mol–1. Furthermore D2d is found to be more stable than the S4 conformation which is in agreement with experimental studies. External QAI binding to the K+ channel KcsA is also studied. Computer simulations and relative binding free energies of the KcsA complexes with QAIs are calculated. This is done with the molecular dynamics free energy perturbation approach together with automated ligand docking. In agreement with experiment, the Et4N+ blocker in D2d symmetry has better binding than the other QAIs. Binding of blockers to the human cardiac hERG potassium channel is studied using a combination of homology modelling, automated docking and molecular dynamics simulations. The calculations reproduce the relative binding affinities of a set of drug derivatives very well and indicate that both polar interactions near the intracellular opening of the selectivity filter as well as hydrophobic complementarity in the region around F656 are important for blocker binding. Hence, the derived model of hERG should be useful for further interpretations of structure-activity relationships.

<p>Disruption of coordinated impulse propagation in the heart as a result of fibrosis or myocardial infarction can create an asynchronous substrate with poor conduction and impaired contractility. This can ultimately lead to cardiac failure and make the heart more vulnerable to life-threatening arrhythmias and sudden cardiac death. The transplantation of exogenous cells into the diseased myocardium, "cardiac cell therapy," has been proposed as a treatment option to improve compromised cardiac function. Clinical trials of stem cell-based cardiac therapy have shown promising results, but also raised concerns about our inability to predict or control the fate of implanted cells and the electrical consequences of their interactions with host cardiomyocytes. Alternatively, genetically engineered somatic cells could be implanted to selectively and safely modify the cardiac electrical substrate, but their unexcitable nature makes them incapable of electrically repairing large conduction defects. The objective of this thesis was thus to develop a methodology to generate actively conducting excitable cells from an unexcitable somatic cell source and to demonstrate their utility for studies of basic electrophysiology and cardiac cell therapy.</p><p>First, based on the principles of cardiac action potential propagation, we applied genetic engineering techniques to convert human unexcitable cells (HEK-293) into an autonomous source of excitable and conducting cells by the stable forced expression of only three genes encoding an inward rectifier potassium (Kir2.1), a fast sodium (Na_v1.5), and a gap junction (Cx43) channel. Systematic pharmacological and electrical pacing studies in these cells revealed the individual contributions of each expressed channel to action potential shape and propagation speed. Conduction slowing and instability of induced arrhythmic activity was shown to be governed by specific mechanisms of I_Na inhibition by TTX, lidocaine, or flecainide. Furthermore, expression of the Na_v1.5 A1924T mutant sodium channel or Ca_v3.3 T-type calcium channel was utilized to study the specific roles of these channels in action potential conduction and demonstrate that genetic modifications of the engineered excitable cells in this platform allow quantitative correlations between single-cell patch clamp data and tissue-level function.</p><p>We further performed proof-of-concept experiments to show that networks of biosynthetic excitable cells can successfully repair large conduction defects within primary excitable tissue cultures. Specifically, genetically engineered excitable cells supported active action potential propagation between neonatal rat ventricular myocytes (NRVMs) separated by at least 2.5 cm in 2-dimensional and 1.3 cm in 3-dimensional cocultures. Using elastic films with micropatterned zig-zag NRVM networks that mimicked the tortuous conduction patterns observed in cardiac fibrosis, we showed that electrical resynchronization of cardiomyocyte activation by application of engineered excitable cells improved transverse conduction by 370% and increased cardiac twitch force amplitude by 64%. This demonstrated that despite being noncontractile, engineered excitable cells could potentially improve both the electrical and mechanical function of diseased myocardial tissue. </p><p>Lastly, we investigated how activation and repolarization gradients at the interface between cardiomyocytes and other excitable cells influence the vulnerability to conduction block. Microscopic optical mapping of action potential propagation was used to quantify dispersion of repolarization (DOR) in micropatterned heterocellular strands in which either well-coupled or poorly-coupled engineered excitable cells with a short action potential duration (APD), seamlessly interfaced with NRVMs that had a significantly longer APD. The resulting electrical gradients originating from the underlying heterogeneity in intercellular coupling and APD dispersion were further manipulated by the application of barium chloride (BaCl2) to selectively prolong APD in the engineered cells. We measured how the parameters of DOR affected the vulnerable time window (VW) of conduction block and found a strong linear correlation between the size of the repolarization gradient and VW. Reduction of DOR by BaCl2 significantly reduced VW and showed that VW correlated directly with dispersion height but not width. Conversely, at larger DOR, VW was inversely correlated with the dispersion width but independent of the dispersion height. In addition, despite their similar APDs, poorly-coupled excitable cells were found to significantly increase the maximum repolarization gradient and VW compared to well-coupled excitable cells, but only at larger DOR.</p><p>In summary, this thesis presents the novel concept of genetically engineering membrane excitability and impulse conduction in previously unexcitable somatic cells. This biosynthetic excitable cell platform is expected to enable studies of ion channel function in a reproducible tissue-level setting, promote the integration of theoretical and experimental studies of action potential propagation, and stimulate the development of novel gene and cell-based therapies for myocardial infarction and cardiac arrhythmias.</p> / Dissertation