Zur Charakterisierung der myokardialen SR Ca2+-Transportaktivität nach experimentellem Myokardinfarkt wurde 10-15 Wochen alten Ratten die linke Koronararterie unterbunden und sechs Wochen später die Funktion und Expression der Ca2+-ATPase des sarkoplasmatischen Retikulums untersucht. Ein Teil der Tiere wurde mit dem Carnitin-Palmitoyl-Transferase-1- Inhibitor Etomoxir behandelt, der die mitochondrale Oxidation von langkettigen Fettsäuren hemmt und dadurch verstärkt Glukoseoxidation bewirkt. Diese Stoffwechselumstellung sollte die Schädigung der myokardialen Funktion und die gestörte zelluläre Ca2+-Homöostase nach Myokardinfarkt vermindern. Die mit Etomoxir behandelten Tiere wiesen nach sechs Wochen eine deutlich verminderte Infarktgröße auf. Durch die CPT-1-Hemmung entstand eine starke biventrikuläre Hypertrophie. Darüber hinaus ergaben sich Hinweise auf den Erhalt einer normalen Wandspannung des linken Ventrikels und einer verbesserten Kontraktilität gegenüber der unbehandelten Gruppe. Unter Etomoxir zeigte die für die diastolische Ca2+ Senkung verantwortliche ATP-abhängige Ca2+-Rückbindung in das SR verbesserte Transportraten. Damit korrelierte der immunchemisch gemessene erhöhte SERCA2a-Proteinspiegel. Die Ergebnisse lassen vermuten, daß es infolge einer Behandlung mit Etomoxir nach Myokardinfarkt zu einer verbesserten Zellstoffwechsellage des ischämisch nicht irreversibel geschädigten Myokards kommt. Dafür scheint die verstärkte Glukoseoxidation bei Hemmung der Oxidation langkettiger Fettsäuren im Herzmuskel verantwortlich zu sein. Die Größe des infarzierten Areals wird begrenzt, dadurch lassen sich zum Teil verbesserte hämodynamische Parameter und gesteigerte SR Ca2+-Transportraten und SERCA2a-Proteinspiegel erklären. Auf die Hämodynamik hat sicherlich auch die durch Etomoxir erfolgte myokardiale Hypertrophie einen wesentlichen Einfluß. Für weitere Studien bleibt abzuklären, durch welchen Mechanismus Etomoxir seine verbessernde Wirkung nach Myokardinfarkt entfaltet. Außerdem ist zu prüfen, ob nach akutem Myokardinfarkt beim Menschen mit dem Prinzip der chronischen Verschiebung der myokardialen Substratverwertung von Fettsäure- nach Glukoseoxidation eine verbesserte Überlebensrate und das Hinauszögern bzw. Verhindern einer Dekompensation des Herzens zu erreichen ist. / To characterise the activity of sarcoplasmic reticulum (SR) Ca2+ after myocardial infarction the left coronary artery of 10-15 week old male rats was ligated; six weeks later function and expression of the SR Ca2+-pump ATPase (SERCA2a) transport in the surviving myocardium was investigated. Part of the animals were treated with the carnithin palmitoyltransferase-1 (CPT-1) inhibitor etomoxir (8mg/kg/d for six weeks) to decrease the oxidation of long chain fatty acids. Due to the drug-induced shift from fatty acid to carbohydrate utilization an attenuated myocardial dysfunction and an improved SR Ca2+ handling homeostasis in the surviving myocardium could be expected. The etomoxir treated rats showed a decreased infarct size six weeks after coronary ligation. Due to CPT-1 inhibition a significant biventricular hypertrophy was observed. In addition, the treatment normalized elevated LVEDP and improved contractility. Compared to sham-operated controls treatment with etomoxir caused an enhanced SR Ca2+ uptake activity that correlated with increased immunoreactive SR Ca2+-ATPase levels. The results suggest that chronic inhibition of CPT-1 after myocardial infarction improves the metabolism of reversible damaged tissue. It appears that increased oxidation of glucose and inhibition of long chain fatty acid oxidation is responsible for this effect. Limitation of the infarct size induces improvement of haemodynamic parameters, increases SR Ca2+ uptake and protein levels of SERCA2a. Further studies are required to find out whether etomoxir is able to delay the development of congestive heart failure in humans and whether it could decrease mortality in patients after myocardial infarction.

Orientador: Prof. Dr. Alexandre Hiroaki Kihara / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, 2013.

Os conhecimentos sobre os mecanismos regulatórios responsáveis pelo crescimento dos fungos filamentosos apresentam lacunas e sua compreensão é necessária para o desenvolvimento de uma terapêutica antifúngica mais adequada, assim como para incrementar a síntese de produtos de interesse comercial. Assim sendo, estudar o envolvimento do Ca2+ na resposta de um fungo modelo como A. nidulans sob fontes de carbono diferentes constitui um meio de gerar conhecimentos sobre as características de crescimento dos fungos filamentosos, de sua resposta a adaptação ambiental e dos mecanismos que controlam essa resposta. Analisou-se na linhagem A26 e na AP27, esta última com ruptura do gene da plcA, o gradiente de Ca2+ citosólico, a morfologia das hifas, a germinação e o ciclo de divisão nuclear quando as linhagens tinham calcineurina ou calmodulina inibidas e quando os canais de Ca2+ estavam bloqueados ou abertos. Os níveis de Ca2+ citosólico na linhagem A26, crescendo em presença de glicose, foram maiores que os detectados em meio suplementado com pectina. O ciclo de germinação e divisão celular no AP27, independentemente da fonte de carbono, mostrou-se mais lento se comparado com a linhagem A26, provavelmente devido ao fato de seus estoques intracelulares de Ca2+, tanto em nível vesicular quanto citosólico, serem menores. A linhagem AP27 apresentou ramificações dicotômicas nas pontas das hifas e nas hifas laterais em ambas as fontes de carbono nas quais foi cultivada, o que não se observou na linhagem A26. Quando calcineurina foi inibida por ciclosporina A, as hifas das duas linhagens, em ambas condições de cultivo, alongaram-se menos e apresentaram-se mais ramificadas, no entanto este efeito foi mais pronunciado em presença de glicose, e entre as duas linhagens pode-se dizer que foi mais intenso na linhagem AP27, demonstrando a importância dos níveis de cálcio na atividade desta enzima e conseqüentemente no desenvolvimento normal das hifas. A abertura dos canais de Ca2+, por ionóforo, produziu hiperramificação em ambas as linhagens, mas principalmente quando cresciam em pectina e ao contrário do efeito observado em presença de verapamil, que bloqueia os canais de Ca2+, não promoveram hifas laterais e nem pontas dicotômicas. No entanto o outro bloqueador dos canais de Ca2+ testado, ácido caurenóico, apresentou efeito morfológico diferente, pois as hifas tornaram-se curvas o que indica perda de polaridade. O inibidor da calmodulina (TFP) retardou a germinação, principalmente no mutante AP27, quando crescendo em presença de glicose. Lembrando que o complexo Ca2+/CaM ativa a calcineurina e que o mutante apresenta menores níveis de cálcio, esse resultado é justificável. A ruptura do gene plcA não impediu o crescimento e desenvolvimento do mutante, provavelmente porque a função desta enzima poder ser provida por outras partes do genoma, mas comprometeu os níveis intracelulares de cálcio e conseqüentemente a sua morfologia. Este estudo mostra a importância da fosfolipase C, para manutenção dos níveis intracelulares de Ca2+, no desenvolvimento normal das hifas de A. nidulans e, pela primeira vez, demonstra que esses níveis são diferentes quando o fungo cresce em presença de uma fonte de carbono, prontamente metabolizável ou não. Esses resultados conferem ao cálcio um papel modulador nessas condições de cultivo. / The knowledge about regulatory mechanisms responsible for filamentous fungi growth presents lacks and its understanding is important to develop adequate antifungal therapy either to contribute the synthesis of interestingly commercial products. By this way, study Ca2+ relationship to a fungal model as A. nidulans about different carbon sources, constitute knowledge about the filamentous fungi growing characteristics, environment adaptation and its control mechanisms. The strains A26 and AP27 was analyzed, this last one with disruption plcA gene, cytosolic Ca2+ gradient hyphal morfology, germination and nuclear division cycle when these strains had calcineurin or calmodulin inhibition and Ca2+ channel were blocked or opened. Cytosolic Ca2+ levels in A26 strain, growing in the presence of glucose was higher than supplemented media with pectin. AP27 strain, independently of carbon source, demonstrated lower germination and cell division than A26 strains, probably due to the fact that intracellular Ca2+ stocks either vesicular as cytosolic levels were lower. AP27 strain presented dichotomous branching at tip-high and lateral hyphae, at both carbon source that was grown, didnt observed at A26 linkage. When calcineurin was inhibited by cyclosporin A, hyphae from both strains, in both growth conditions, had less elongated and showed more branching, however this effect was more pronounced in presence of glucose, and between both strains was more intense at AP27 strain, indicating the importance of Ca2+ levels at this enzymatic activity and therefore the normal development of hyphae. The opening of Ca2+ channel by ionophore, produced hyperbranching in both strains, even when growth in pectin and in contrast of effect observed in the presence of verapamil, that blocks Ca2+ channels, didnt promote lateral or tip high dichotomous branching. However kaurenoic acid, an another Ca2+ channel blocker tested, presented different morphological effect, because hyphae became curved, indicating loss of polarity. Calmodulin inhibitor (TFP) delayed germination mainly at mutant AP27, when growing in the presence of glucose. Remembering that Ca2+/CaM complex activate calcineurin and the mutant exhibit lower Ca2+ levels, justifying this results. The rupture of plcA gene didnt affected growth and development of mutant, probably because the function of this enzyme can be provided by another parts of genoma, damaged the Ca2+ intracellular levels and consequently its morphology. This study shows the importance of fosfolipase C to maintaining the intracellular Ca2+ levels, at the normal hyphae development of A. nidulans and for the first time, demonstrating that this levels are different when fungi are grown in the presence of carbon source, promptly metabolizable or not. This results gives to Ca2+ as modulator at growth conditions.

Electrical potentials exist across the membranes of nearly every cell type in the body. In addition, excitable cells, such as neurons, myocytes and even some endocrine cells elicit electrochemical fluctuations, action potentials (AP), in the cell membrane to initiate cell-to-cell communication or intracellular processes. The basis for the electrical potential is rooted within an array of complex interactions between monovalent ions and their associated membrane channels and transporters that regulate the flux of these charged species across the hydrophobic bilayer. Here, an expansion of our recently published work [1] will serve to explore the modern concepts regarding the origin of the AP as well as to examine the mechanisms by which intracellular calcium ([Ca2+]i) is regulated within the HL-1 mouse cardiac myocyte.

Na+/Ca2+ exchanger (NCX) is a membrane protein which can mediate either Ca2+ entry (reverse mode) or exit (forward mode) in cells. As one of the major Ca2+ transport systems, NCX is postulated to play a critical role in the vascular smooth muscle cell. The aims of the present study are to firstly demonstrate the functional existence of NCX in vascular smooth muscle (including aorta and arteriole); to clarify the modulation of NCX; to explore the selectivity of NCX inhibitor KB-R7943; and lastly to investigate the role of NCX in the myogenic response. KB-R7943 has been widely used as a NCX inhibitor. The study investigated its pharmacological actions in rat aorta on a variety of Ca2+ dependent systems. Rat aortic rings were used. The constriction to low extracellular [Na+] is a functional response mediated by NCX operating in reverse mode. The data demonstrate that 10 µM KB-R7943 inhibited L-type Ca2+ channel, the capacitative Ca2+ entry and  adrenergic receptor pathway. Nevertheless, KB-R7943 can be used as a selective inhibitor of NCX at the lower concentration of 1 µM in rat aortic rings. The study investigated whether the endothelium could modulate NCX in rat aortic rings. Lowering extracellular [Na+] to 1.18 mM induced constriction in endothelium denuded rat aortic rings, but only a small constriction in endothelium intact rat aortic rings. In endothelium intact rat aortic rings, the guanylate cyclise inhibitor ODQ (1 µM) and the nitric oxide synthase inhibitor L-NAME (50 µM) greatly amplified the vasoconstriction to lowering extracellular [Na+], but had no effect when the endothelium was removed. The adenylate cyclise inhibitor SQ 22536 (100 µM) and the cyclooxygenase inhibitor indomethacin (10 M) showed no significant effect on the low-Na+ induced vasoconstriction in either endothelium denuded or intact aortic rings. The results suggest that endothelium modulated the NCX operation via the nitric oxide/guanylate cyclase, not the adenylate cyclase system; further prostanoids including prostacyclin was not involved. The interaction between nitric oxide and NCX was furt her explored using the nitric oxide donor sodium nitroprusside. Endothelium denuded rat aortic rings were preconstricted to the same extent with either low Na+ (1.18 mM), or the thromboxane A2 agonist U46619 (0.1 µM) or high K+ (80 mM). The vasorelaxation of SNP (30 nM) in low Na+ constriction was significantly larger compared to other agents. This indicates that NO has a special antagonism of low Na+ constriction and a hypothesis is proposed involving Na+/K+ ATPase. The investigation of NCX is mainly conducted in large vessels; much less evidence is available for small resistance vessels. The study investigated the role of NCX on myogenic response in pressurized cremaster muscle arterioles. Reducing extracellular [Na+] resulted in graded vasoconstriction which was inhibited by NCX inhibitor SEA0400 (1 µM). Myogenic vasoconstriction and the concomitant rise in internal [Ca2+] were induced by a transmural pressure increase from 70 to 120 mmHg which was prevented by NCX inhibitor: SEA0400 (1 µM). In conclusion, the present study suggests that NCX contributes to the myogenic response in cremaster arteriole.

Le puissant anti-paludéen, l'artémisinine a été décrit comme inhibiteur de l'activité ATPasique de PfATP6 après son expression dans des ovocytes de Xénope. PfATP6 est l'unique ATPase Ca2+ du réticulum endo/sarcoplasmique de P. falciparum, le parasite responsable du paludisme. Quand un acide aminé de SERCA1a de lapin (E255) est muté en son équivalent dans PfATP6 (L), l'activité de ce mutant exprimé en ovocyte de Xénope est inhibée en présence d'artémisinine. Après expression de ce mutant et de PfATP6 dans S. cerevisiae puis leur purification, nous avons constaté qu'aucune de ces deux protéines n'était sensible à l'artémisinine. En parallèle, nous montrons que PfATP6 purifiée est sensible aux principaux inhibiteurs de SERCA mais elle est moins sensible à la thapsigargine que ne l'est SERCA1a. Les résultats présentés ici suggèrent que le méchanisme d'action de l'artémisinine est complexe et ne peut pas être du à une interaction directe entre l'artémisinine et PfATP6. D'autre part, la purification de PfATP6 laisse entrevoir l'opportunité de mieux caractériser cette protéine voire même de développer de nouveaux anti-paludéens en recherchant des inhibiteurs de cette protéine.

L'ATPase-Ca2+ du réticulum sarcoplasmique de muscle squelettique rapide (SERCA1a) est une protéine membranaire qui permet le transport actif de deux ions calcium depuis le cytosol jusque dans la lumière du réticulum. Depuis 2000, l'obtention de nombreuses structures à résolution atomique de cette enzyme a été un atout majeur pour comprendre son cycle catalytique (Toyoshima, Nakasako et al. 2000). Toutefois, avant 2007, les formes de cette enzyme cristallisées en absence de calcium avaient été obtenues en présence d'un inhibiteur fixé au domaine membranaire. Nous avons d'abord montré, notamment par des techniques de spectroscopie de fluorescence et de protéolyse ménagée, que l'interaction de la protéine avec ces inhibiteurs, qui rend l'ATPase incapable d'adopter certaines des conformations présentes au cours de son cycle catalytique, avait certainement induit des biais significatifs dans la structure de certaines des formes cristallisées avant 2007 (Montigny, Picard et al. 2007). Nos résultats ont stimulé la recherche de structures nouvelles, en absence ou en présence d'inhibiteurs, dont l'analyse a pleinement confirmé nos conclusions. L'utilisation de ces inhibiteurs pendant la cristallisation de la protéine solubilisée était jusque là jugée utile pour protéger celle-ci de son éventuelle inactivation irréversible en présence de détergent (Lund, Orlowski et al. 1989). Utilisant le glycérol pour ralentir cette inactivation, nous avons mis en évidence des conséquences inattendues de l'interaction de l'ATPase avec deux des détergents communément utilisés pour sa cristallisation, son isolement ou sa simple étude (Montigny, Arnou et al. 2008). En présence de glycérol, nous avons également pu étudier certains traits de fonctionnement de trois mutants de l'ATPase, purifiés après expression hétérologue dans la levure S. cerevisiae : un mutant d'un des sites de fixation du calcium (i.e. capable de fixer un seul des deux calciums) (Montigny, Arnou et al. 2008) et deux autres mutants bloquant l'enzyme dans des états particuliers du cycle catalytique (Marchand, Winther et al. 2008). Parallèlement à ces études, nous avons élucidé le mystère des propriétés de fluorescence anormales d'une forme particulière de l'enzyme SERCA1a marquée au FITC dans son site nucléotidique. Nous avons mis en évidence une phosphorylation imprévue du FITC lié, conduisant à une très faible fluorescence, particulièrement stable dans le temps. Les conditions de cette phosphorylation impliquent une réorganisation de l'orientation relative des domaines cytosoliques de l'ATPase lors de l'étape de relargage des ions calcium vers la lumière du réticulum (McIntosh, Montigny et al. 2008). Dans toutes nos études, il nous a fallu évidemment tenir compte de la possible chélation des cations divalents interagissant avec l'enzyme (calcium, magnésium, ...) par les anions présents. Par pH métrie et à l'aide de sondes colorées, nous avons vérifié que le magnésium n'était PAS chélaté par le tampon Mops classiquement utilisé, contrairement à ce qui était rapporté dans certaines tables de constantes d'association (Montigny and Champeil 2007). A cette occasion, mettant notre savoir-faire au service de collègues du laboratoire, nous avons également précisé les différents complexes formés par association entre le cadmium (Cd2+) et le glutathion réduit (GSH-) (Leverrier, Montigny et al. 2007), afin de mieux comprendre lesquels de ces complexes étaient le plus susceptibles de se former au cours de la détoxication cellulaire du Cd2+. Abréviations : ATPase, AdénosineTriPhosphatase ; SERCA1a, Sarco-Endoplasmic Reticulum Ca2+-ATPase, isoforme 1a ; FITC, fluorescéine isothiocyanate ; Mops, acide 4-morpholinopropanesulfonique.

Dans cette thèse est présentée une nouvelle stratégie de purification de SERCA1a après son expression hétérologue chez S. cerevisiae.. La protéine est fusionnée à un domaine accepteur de biotine, biotinylé in vivo par la levure. La procédure de purification, basée sur la forte interaction entre avidine et biotine, permet d'obtenir une protéine active pure à 40-50%. Une étape supplémentaire de filtration sur gel en HPLC a permis d'augmenter la pureté d'environ 70%, tout en conservant une très bonne activité spécifique. Les cristaux obtenus de SERCA1a ainsi purifiée diffractent les rayons X à 3,1Å. <br />Cette nouvelle méthode de purification a été appliquée avec succès au mutant SERCA1a-E309Q. De petits cristaux de ce mutant ont pu être isolés. Cette méthode a également permis de purifier SERCA3a, bien que le faible taux d'expression de la protéine de fusion chez S. cerevisiae limite la quantité purifiée. En parallèle, des essais d'immunolocalisation cellulaire de SERCA3a dans différentes lignées cellulaires et dans des coupes de peau ont été réalisés

The four main steps of bacterial viruses (bacteriophages) lytic infection are (i) specific recognition and genome entry into the host bacterium, (ii) replication of the viral genome, (iii) assembly of viral particles, and (iv) their release, leading in most cases to cell lysis. Although the course of individual steps of the viral infection cycle has been relatively well established, the details of how viral DNA transits from the virion to the host cytoplasm and of how the cellular environment catalyzes and possibly organizes the entire process remain poorly understood.Tailed bacteriophages are by far the most abundant viruses that infect Eubacteria. The first event in their infection is recognition of a receptor on the surface of host bacterium by the phage adsorption machinery. The barriers that the infectious particle overcomes subsequently are the cell wall and the cytoplasmic membrane of bacteria. This implies a localized degradation of the wall and the flow of its double stranded DNA (dsDNA) through a hydrophilic pore in the membrane. The lineards DNA molecule is most frequently circularized in the cytoplasm followed by its replication. In this study we used bacteriophage SPP1 that infects the Gram-positive bacterium Bacillus subtilis as a model system to dissect the different steps leading to transfer of the phage genome from the viral capsid to the host cell cytoplasm.normally to B. subtilis but do not trigger depolarization of the CM. Attachment of intact SPP1 particles is thus required for phage-induced depolarization.The beginning of B. subtilis infection by bacteriophage SPP1 was followed inspace and time. The position of SPP1 binding at the cell surface was imaged by fluorescence microscopy using virus particles labeled with "quantum dots". We found that SPP1 reversible adsorption occurs preferentially at the cell poles. This initial binding facilitates irreversible adsorption to the SPP1 phage receptor protein YueB,which is encoded by a putative type VII secretion system gene cluster.Immunostaining and YueB - GFP fusion showed that the phage receptor protein YueB is found over the entire cell surface. It concentrates at the bacterial poles too,and displays a punctate distribution over the sidewalls. The dynamics of SPP1 DNA entry and replication was visualised in real time by assaying specific binding of a fluorescent protein to tandem sequences present in the SPP1 genome. During infection, most of the infecting phages DNA entered and replicated near the bacterial poles in a defined focus. Therefore, SPP1 assembles a replication factory at a specific location in the host cell cytoplasm. DNA delivery to the cytoplasm depends on millimolar concentrations of Ca2+ allowing uncoupling it from the precedent steps of SPP1 adsorption to the cell envelope and CM depolarization that require only micromolar amounts of this divalent cation. A model describing the early events of bacteriophage SPP1 infection is presented.

Biogenic amines and their receptors regulate and modulate many physiological and behavioural processes in animals. In vertebrates, octopamine is only found in trace amounts and its function as a true neurotransmitter is unclear. In protostomes, however, octopamine can act as neurotransmitter, neuromodulator and neurohormone. In the honeybee, octopamine acts as a neuromodulator and is involved in learning and memory formation. The identification of potential octopamine receptors is decisive for an understanding of the cellular pathways involved in mediating the effects of octopamine. Here we report the cloning and functional characterization of the first octopamine receptor from the honeybee, Apis mellifera . The gene was isolated from a brain-specific cDNA library. It encodes a protein most closely related to octopamine receptors from Drosophila melanogaster and Lymnea stagnalis . Signalling properties of the cloned receptor were studied in transiently transfected human embryonic kidney (HEK) 293 cells. Nanomolar to micromolar concentrations of octopamine induced oscillatory increases in the intracellular Ca2+ concentration. In contrast to octopamine, tyramine only elicited Ca2+ responses at micromolar concentrations. The gene is abundantly expressed in many somata of the honeybee brain, suggesting that this octopamine receptor is involved in the processing of sensory inputs, antennal motor outputs and higher-order brain functions.