949 resultados para CA2 HOMEOSTASIS
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Specific targeting of the recombinant, Ca2+ -sensitive photoprotein, aequorin to intracellular organelles has provided new insights into the mechanisms of intracellular Ca2+ homeostasis. When applied to small mammalian cells, a major limitation of this technique has been the need to average the signal over a large number of cells. This prevents the identification of inter- or intracellular heterogeneities. Here we describe the imaging in single mammalian cells (CHO.T) of [Ca2+] with recombinant chimeric aequorin targeted to mitochondria. This was achieved by optimizing expression of the protein through intranuclear injection of cDNA and through the use of a charge-coupled device camera fitted with a dual microchannel plate intensifier. This approach allows accurate quantitation of the kinetics and extent of the large changes in mitochondrial matrix [Ca2+] ([Ca2+](m)) that follow receptor stimulation and reveal different behaviors of mitochondrial populations within individual cells. The technique is compared with measurements of [Ca2+](m) using the fluorescent indicator, rhod2. Comparison of [Ca2+](m) with the activity of the Ca2+ -sensitive matrix enzyme, pyruvate dehydrogenase (PDH), reveals that this enzyme is a target of the matrix [Ca2+] changes. Peak [Ca2+](m) values following receptor stimulation are in excess of those necessary for full activation of PDH in situ, but may be necessary for the activation of other mitochondrial dehydrogenases. Finally, the data suggest that the complex regulation of PDH activity by a phosphorylation-dephosphorylation cycle may provide a means by which changes in the frequency of cytosolic (and hence mitochondrial) [Ca2+] oscillations can be decoded by mitochondria.
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Significant progress has been achieved in elucidating the role of the plasma membrane Ca2+-ATPase in cellular Ca2+ homeostasis and physiology since the enzyme was first purified and physiology since the enzyme was first purified and cloned a number of years ago. The simple notion that the PM Ca2+-ATPase controls resting levels of [Ca2+](CYT) has been challenged by the complexity arising from the finding of four major isoforms and splice variants of the Ca2+ pump, and the finding that these are differentially localized in various organs and subcellular regions. Furthermore, the isoforms exhibit differential sensitivities to Ca2+, calmodulin, ATP, and kinase-mediated phosphorylation. The latter pathways of regulation can give rise to activation or inhibition of the Ca2+ pump activity, depending on the kinase and the particular Ca2+ pump isoform. Significant progress is being made in elucidating subtle and more profound roles of the PM Ca2+-ATPase in the control of cellular function. Further understanding of these roles awaits new studies in both transfected cells and intact organelles, a process that will be greatly aided by the development of new and selective Ca2+ pump inhibitors. (C) 1999 Elsevier Science Inc.
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Rsum : Le virus de la maladie de Carr (en anglais: canine distemper virus, CDV) qui est pathogne pour les chiens et autres carnivores, est trs semblable au virus de la rougeole humaine (en anglais MV). Ces deux virus font partie du genre des Morbillivirus qui appartient la famille des Paramyxoviridae. Ils induisent des complications dans le systme nerveux central (SNC). Au stade prcoce et aigu de l'infection du SNC, le CDV induit une dmylinisation (1). Ce stade volue dans certains cas vers une infection chronique avec progression de la dmylinisation. Pendant le stade prcoce, qui suit en gnral de trois semaines les premiers symptmes, le processus de dmylinisation est associ la rplication du virus et n'est pas considr comme inflammatoire (1). Par contre, au stade chronique, la progression des plaques de dmylinisation semble tre plutt lie des processus immunognes caractristiques (2), retrouvs galement dans la sclrose en plaques (SEP) chez les humains. Pour cette raison, le CDV est considr comme un modle pour la SEP humaine et aussi pour l'tude des maladies et complications induites par les Morbillivirus en gnral (3). Dans notre laboratoire, nous avons utilis la souche A75/17-CDV, qui est considre comme le modle des souches neurovirulentes de CDV. Nous avons cherch en premier lieu tablir un systme robuste pour infecter des cultures neuronales avec le CDV. Nous avons choisi les cultures primaires de l'hippocampe du nouveau-n de rat (4), que nous avons ensuite infect avec une version modifie du A75/17, appele rgA75/17-V (5). Dans ces cultures, nous avons prouv que le CDV infecte des neurones et des astrocytes. Malgr une infection qui se diffuse lentement entre les cellules, cette infection cause une mort massive aussi bien des neurones infects que non infects. En parallle, les astrocytes perdent leur morphologie de type toil pour un type polygonal. Finalment, nous avons trouv une augmentation importante de la concentration en glutamate dans le milieu de culture, qui laisse prsumer une scrtion de glutamate par les cultures infectes (6). Nous avons ensuite tudi le mcanisme des effets cytopathiques induits par le CDV. Nous avons d'abord dmontr que les glycoprotines de surface F et H du CDV s'accumulent massivement dans le rticulum endoplasmique (RE). Cette accumulation dclenche un stress du RE, qui est caractris par une forte expression du facteur de transcription proapoptotique CHOP/GADD 153 et de le la calreticuline (CRT). La CRT est une protine chaperonne localise dans le RE et implique dans l'homostasie du calcium (Ca2+) et dans le repliement des protines. En transfectant des cellules de Vero avec des plasmides codant pour plusieurs mutants de la glycoprotine F de CDV, nous avons dmontr une corrlation entre l'accumulation des protines virales dans le RE et l'augmentation de l'expression de CRT, le stress du RE et la perte de l'homostasie du Ca2+. Nous avons obtenu des rsultats semblables avec des cultures de cellules primaires de cerveau de rat. Ces rsultats suggrent que la CRT joue un rle crucial dans les phnomnes neurodgnratifs pendant l'infection du SNC, notamment par le relazgage du glutamate via le Ca2+. De manire intressante, nous dmontrons galement que l'infection de CDV induit une fragmentation atypique de la CRT. Cette fragmentation induit une re-localisation et une exposition slective de fragments amino-terminaux de la CRT, connus pour tres fortement immunognes la surface des cellules infectes et non infectes. A partir de ce rsultat et des rsultats prcdents, nous proposons le mcanisme suivant: aprs l'infection par le CDV, la rtention dans le RE des protines F et H provoque un stress du RE et une perte de l'homostasie du Ca2+. Ceci induit la libration du glutamate, qui cause une dgnration rapide du SNC (sur plusieurs jours ou semaines) correspondant la phase aigu de la maladie chez le chien. En revanche, les fragments amino-terminaux de la CRT librs la surface des cellules infectes peuvent avoir un rle important dans l'tablissement d'une dmylinisation d'origine immunogne, typique de la phase chronique de l'infection de CDV. Summary : The dog pathogen canine distemper virus (CDV), closely related to the human pathogen measles virus (MV), belongs to the Morbillivirus genus of the Paramyxoviridae family. Both CDV and NIV induce complications in the central nervous system (CNS). In the acute early stage of the infection in CNS, the CDV infection induces demyelination. This stage is sometimes followed by a late persistent stage of infection with a progression of the demyelinating lesions (1). The acute early stage occurs around three weeks after the infection and demyelinating processes are associated with active virus replication and are not associated to inflammation (1). In contrast during late persistent stage, the demyelination plaque progression seems to be mainly due to an immunopathological process (2), which characteristics are shared in many aspects with the human disease multiple sclerosis (MS). For these reasons, CDV is considered as a model for human multiple sclerosis, as well as for the study of Morbillivirus-mediated pathogenesis (3). In our laboratory, we used the A75/17-CDV strain that is considered to be the prototype of neurovirulent CDV strain. We first sought to establish a well characterized and robust model for CDV infection of a neuronal culture. We chose primary cultures from newborn rat hippocampes (4) that we infected with a modified version of A75/17, called rgA75/17-V (5). In these cultures, we showed that CDV infects both neurons and astrocytes. While the infection spreads only slowly to neighbouring cells, it causes a massive death of neurons, which includes also non-infected neurons. In parallel, astrocytes undergo morphological changes from the stellate type to the polygonal type. The pharmacological blocking of the glutamate receptors revealed an implication of glutamatergic signalling in the virus-mediated cytopathic effect. Finally, we found a drastic increase concentration of glutamate in the culture medium, suggesting that glutamate was released from the cultured cells (6). We further studied the mechanism of the CDV-induced cytopathic effects. We first demonstrated that the CDV surface glycoprotein F and H markedly accumulate in the endoplasmic reticulum (ER). This accumulation triggers an ER stress, which is characterized by increased expression of the proapoptotic transcription factor CHOP/GADD 153 and calreticulin (CRT). CRT is an ER resident chaperon involved in the Ca2+ homeostasis and in the response to misfolded proteins. Transfections of Vero cells with plasmids encoding various CDV glycoprotein mutants reveal a correlation between accumulation of viral proteins in the ER, CRT overexpression, ER stress and alteration of ER Ca2+ homeostasis. Importantly, similar results are also obtained in primary cell cultures from rat brain. These results suggest that CRT plays a crucial role in CNS infection, particularly due to CRT involvement in Ca2+ mediated glutamate releases, and subsequent neurodegenerative disorders. Very intriguingly, we also demonstrated that CDV infection induces an atypical CRT fragmentation, with relocalisation and selective exposure of the highly immunogenic CRT N-terminal fragments at the surface of infected and neighbouring non-infected cells. Altogether our results combined with previous findings suggest the following scenario. After CDV infection, F and H retention alter Ca2+ homeostasis, and induce glutamate release, which in turn causes rapid CNS degeneration (within days or a week) corresponding to the acute phase of the disease in dogs. In contrast, the CRT N-terminal fragments released at the surface of infected cells may rather have an important role in the establishment of the autoimmune demyelination in the late stage of CDV infection.
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Evidence has indicated that the sarcoplasmic reticulum (SR) might be involved in the generation of spontaneous electrical activity in atrial pacemaker cells. We report the effect of disabling the SR with ryanodine (0.1 M) on the sinus node recovery time (SNRT) measured in isolated right atria from 4-6-month-old male Wistar rats. Electrogram and isometric force were recorded at 36.5oC. Two methods for sinus node resetting were used: a) pulse: a single stimulus pulse interpolated at coupling intervals of 50, 65 or 80% of the regular spontaneous cycle length (RCL), and b) train: a 2-min train of pulses at intervals of 50, 65 or 80% of RCL. Corrected SNRT (cSNRT) was calculated as the difference between SNRT (first spontaneous cycle length after stimulation interruption) and RCL. Ryanodine only slightly increased RCL (<10%), but decreased developed force by 90%. When the pulse method was used, cSNRT (~40 ms), which represents intranodal/atrial conduction time, was independent of the coupling interval and unaffected by ryanodine. However, cSNRT obtained by the train method was significantly higher for shorter intervals between pulses, indicating the occurrence of overdrive suppression. In this case, ryanodine prolonged cSNRT in a rate-dependent fashion, with a greater effect at shorter intervals. These results indicate that: a) a functional SR, albeit important for force development, does not seem to play a major role in atrial automaticity in the rat; b) disruption of cell Ca2+ homeostasis by inhibition of SR function does not appear to affect conduction; however, it enhances overdrive-induced depression of sinusal automaticity.
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Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human life-threatening monogenic disorders. The disease is characterized by bilateral, progressive renal cystogenesis and cyst and kidney enlargement, often leading to end-stage renal disease, and may include extrarenal manifestations. ADPKD is caused by mutation in one of two genes, PKD1 and PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively. PC2 is a non-selective cation channel permeable to Ca2+, while PC1 is thought to function as a membrane receptor. The cyst cell phenotype includes increased proliferation and apoptosis, dedifferentiation, defective planar polarity, and a secretory pattern associated with extracellular matrix remodeling. The two-hit model for cyst formation has been recently extended by the demonstration that early gene inactivation leads to rapid and diffuse development of renal cysts, while inactivation in adult life is followed by focal and late cyst formation. Renal ischemia/reperfusion, however, can function as a third hit, triggering rapid cyst development in kidneys with Pkd1 inactivation induced in adult life. The PC1-PC2 complex behaves as a sensor in the primary cilium, mediating signal transduction via Ca2+ signaling. The intracellular Ca2+ homeostasis is impaired in ADPKD, being apparently responsible for the cAMP accumulation and abnormal cell proliferative response to cAMP. Activated mammalian target for rapamycin (mTOR) and cell cycle dysregulation are also significant features of PKD. Based on the identification of pathways altered in PKD, a large number of preclinical studies have been performed and are underway, providing a basis for clinical trials in ADPKD and helping the design of future trials.
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Introduction: Familial Hypomagnesaemia with hypercalciuria and nephrocalcinosis, with severe ocular impairment secondary to claudin-19 mutation, is a rare recessive autossomic disorder. Its spectrum includes renal Mg2+ wasting, medullary nephrocalcinosis and progressive chronic renal failure in young people. Objective: To report a case of kidney transplantation father to daughter in a familial occurrence of severe bilateral nephrocalcinosis associated with ocular impairment in a non-consanguineous Brazilian family, in which two daughters had nephrocalcinosis and severe retinopathy. Methods: The index case, a 19 years-old female, had long-lasting past medical history of recurrent urinary tract infections, and the abdominal X-ray revealed bilateral multiple renal calcifications as well as ureteral lithiasis, and she was under haemodialysis. She had the diagnosis of retinitis pigmentosa in the early neonatal period. The other daughter (13 years-old) had also nephrocalcinosis with preserved kidney function, retinopathy with severe visual impairment, and in addition, she exhibited hypomagnesaemia = 0.5 mg/dL and hypercalciuria. The other family members (mother, father and son) had no clinical disease manifestation. Mutation analysis at claudin-19 revealed two heterozygous missense mutations (P28L and G20D) in both affected daughters. The other family members exhibited mutant monoallelic status. In despite of that, the index case underwent intrafamilial living donor kidney transplantation (father). Conclusion: In conclusion, the disease was characterized by an autosomal recessive compound heterozygous status and, after five years of donation the renal graft function remained stable without recurrence of metabolic disturbances or nephrocalcinosis. Besides, donor single kidney Mg2+ and Ca2+ homeostasis associated to monoallelic status did not affect the safety and the usual living donor post-transplant clinical course.
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Lendothlium vasculaire joue un rle prpondrant dans la rgulation du tonus vasculaire en gnrant loxyde nitrique (NO), la prostacycline (PGI2) et les facteurs hyperpolarisants drivs de lendothlium (EDHF) comme puissants vasodilatateurs. Ces mcanismes requirent le calcium (Ca2+) divers niveaux, dmontrant limportance des dynamiques calciques endothliales. Une perturbation de lhomostasie calcique est observe dans une dysfonction endothliale lie lhypertension artrielle. Il est impratif dapprofondir nos connaissances sur les signalisations calciques endothliales impliques dans le contrle du tonus vasculaire. Des tudes rcentes ont montr quune variation locale de la concentration en Ca2+ libre intracellulaire ([Ca2+]i) est suffisante pour gnrer une rponse physiologique importante. Les pulsars calciques sont caractriss par une augmentation de [Ca2+]i spontane et transitoire spcifiquement localise au niveau des projections myoendothliales (PMEs). Ces PMEs sont des sites de communication privilgis entre les cellules endothliales (CEs) et les cellules musculaires lisses vasculaires (CMLVs). Les pulsars calciques sont impliqus dans le mcanisme de lEDHF via lactivation des canaux potassiques Ca2+-dpendant de moyenne conductance (KCa3.1 ou IKCa). Les travaux de cette thse visent amliorer nos connaissances sur les signalisations calciques locales en caractrisant une nouvelle voie de signalisation pouvant tre implique dans la rgulation du tonus vasculaire en condition physiopathologique. Outre les canaux KCa3.1 peu dinformations sont disponibles sur les cibles sensibles aux pulsars calciques. Une premire tude a permis didentifier la protine kinase II dpendante du complexe Ca2+/calmoduline (CaMKII) sous ses isoformes , et dans les CEs dartres natives de souris comme une cible pouvant tre module par les pulsars calciques. Des tudes en immunofluorescence ont permis dobserver la localisation particulire de CaMKII endothliale dans les PMEs, les sites des pulsars calciques. Une stimulation spcifique des pulsars calciques par la phnylphrine (PE) engendre un recrutement de CaMKII dans les PMEs. Sachant que CaMKII active loxyde nitrique synthase endothliale (NOS3), nous avons valu limpact dune stimulation des pulsars calciques sur la production de NO en prsence dun inhibiteur de CaMKII, le KN-93. Nous avons dmontr que la production de NO est en partie dpendante de lactivation de CaMKII par les pulsars calciques. En utilisant un modle dhypertension induite par linfusion chronique de PE, nous avons permis de mettre en vidence une perturbation dans la relation entre les pulsars calciques et CaMKII. Dans une seconde tude nous avons tabli deux modles (normo- et hypertendus) dinfusion chronique langiotensine II (AngII) afin valuer limpact des ROS et de lhypertension sur la voie de signalisation pulsars/CaMKII/NO. Nos rsultats ont montr une augmentation des pulsars calciques accompagne dun recrutement de CaMKII dans les PMEs. Une stimulation aigue lAngII suggre que les ROS modulent les dynamiques calciques et que lAngII stimule la production de NO. Cette tude propose que ces voies de signalisations impliquent les rcepteurs de type 1 et 2 lAngII (AT1 et AT2). Ltude des pulsars calciques dpend fortement de la structure native des artres qui permet de conserver la formation des PMEs. La dernire tude prsente dans cette thse a permis dtablir une relation entre les PMEs et les pulsars calciques dans trois lits vasculaires distincts (artres msentriques, pulmonaires et coronariennes). Nos rsultats ont montr que les paramtres cintiques des pulsars calciques sont fortement conservs entre les diffrents lits vasculaires. Toutefois, la frquence globale ainsi que le nombre de sites actifs des pulsars calciques diffrent avec une proportion plus leve dans les artres msentriques et coronariennes comparativement aux artres pulmonaires. Ces rsultats corrlent avec le nombre plus lev de PMEs retrouv dans les artres msentriques et coronariennes. Ces travaux suggrent que les pulsars calciques sont fondamentaux pour les artres de rsistance. Les tudes de cette thse ont men lidentification dune nouvelle voie de signalisation impliquant les pulsars calciques et CaMKII endothliale dans la stimulation de la production de NO. Cette nouvelle voie de signalisation pourrait tre implique dans la rgulation du tonus vasculaire en condition physiopathologique. Les pulsars calciques semblent tre fortement conservs entre les diffrentes artres de rsistances et ce malgr la disparit dans les PMEs, suggrant un rle prpondrant dans la fonction vasculaire. Ces travaux ouvrent une avenue pour le dveloppement de potentielles cibles thrapeutiques pouvant contrer la dysfonction endothliale associe lhypertension artrielle.
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Emerging evidence suggests that dietary phytochemicals, in particular flavonoids, may exert beneficial effects in the central nervous system by protecting neurons against stress-induced injury, by suppressing neuroinflammation and by promoting neurocognitive performance, through changes in synaptic plasticity. It is likely that flavonoids exert such effects in neurons, through selective actions on different components within a number of protein kinase and lipid kinase signalling cascades, such as phosphatidylinositol-3 kinase (PI3K)/Akt, protein kinase C and mitogen-activated protein kinase. This review details the potential inhibitory or stimulatory actions of flavonoids within these pathways, and describes how such interactions are likely to affect cellular function through changes in the activation state of target molecules and/or by modulating gene expression. Although, precise sites of action are presently unknown, their abilities to: (1) bind to ATP binding sites on enzymes and receptors; (2) modulate the activity of kinases directly; (3) affect the function of important phosphatases; (4) preserve neuronal Ca2+ homeostasis; and (5) modulate signalling cascades lying downstream of kinases, are explored. Future research directions are outlined in relation to their precise site(s) of action within the signalling pathways and the sequence of events that allow them to regulate neuronal function in the central nervous system.
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Sustained hypoxia alters the expression of numerous proteins and predisposes individuals to Alzheimer's disease (AD). We have previously shown that hypoxia in vitro alters Ca2+ homeostasis in astrocytes and promotes increased production of amyloid beta peptides (Abeta) of AD. Indeed, alteration of Ca2+ homeostasis requires amyloid formation. Here, we show that electrogenic glutamate uptake by astrocytes is suppressed by hypoxia (1% O2, 24h) in a manner that is independent of amyloid beta peptide formation. Thus, hypoxic suppression of glutamate uptake and expression levels of glutamate transporter proteins EAAT1 and EAAT2 were not mimicked by exogenous application of amyloid beta peptide, or by prevention of endogenous amyloid peptide formation (using inhibitors of either beta or gamma secretase). Thus, dysfunction in glutamate homeostasis in hypoxic conditions is independent of Abeta production, but will likely contribute to neuronal damage and death associated with AD following hypoxic events.
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A regimen of low-protein diet induces a reduction of pancreatic islet function that is associated with development of metabolic disorders including diabetes and obesity afterward. In the present study, the influence of leucine supplementation on metabolic parameters, insulin secretion to glucose and to amino acids, as well as the levels of proteins that participate in the phosphatidylinositol 3-phosphate kinase (PI3K) pathway was investigated in malnourished rats. Four groups were fed with different diets for 12 weeks: a normal protein diet (17%) without (NP) or with leucine supplementation (NPL) or a low (6%)-protein diet without (LP) or with leucine supplementation (LPL). Leucine was given in the drinking water during the last 4 weeks. As indicated by the intraperitoneal glucose tolerance test, LPL rats exhibited increased glucose tolerance as compared with NPL group. Both NPL and LPL rats had higher circulating insulin levels than controls. The LPL rats also showed increased insulin secretion by pancreatic islets in response to glucose or arginine compared with those observed in islets from LP animals. Glucose oxidation was significantly reduced in NPL, LP, and LPL isolated islets as compared with NP; but no alteration was observed for leucine and glutamate oxidation among the 4 groups. Western blotting analysis demonstrated increased PI3K and mammalian target protein of rapamycin protein contents in LPL compared with LP islets. A significant increase in insulin-induced insulin receptor substrate I associated PI3K activation was also observed in LPL compared with LP islets. These findings indicate that leucine supplementation can augment islet function in malnourished rats and that activation of the PI3K/maminalian target protein of rapamycin pathway may play a role in this process. (C) 2010 Elsevier Inc. All rights reserved.
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High protein content in the diet during childhood and adolescence has been associated to the onset insulin-dependent diabetes mellitus. We investigated the effect of interleukin-1 beta (IL-I beta) on insulin secretion, glucose metabolism, and nitrite formation by islets isolated from rats fed with normal protein (NP, 17%) or low protein (LP, 6%) after weaning. Pretreatment of islets with IL-1 beta for 1 h or 34 h inhibited the insulin secretion induced by glucose in both groups, but it was less marked in LP than in NP group. Islets from LP rats exhibited a decreased IL-1 beta -induced nitric oxide (NO) production, lower inhibition of D-[(UC)-C-14]-glucose oxidation to (CO2)-C-14, and less pronounced effect of IL-1 beta on alpha -ketoisocaproic acid-induced insulin secretion than NP islets. However, when the islets were stimulated by high concentrations of K+ the inhibitory effect of IL-1 beta on insulin secretion was not different between groups. In conclusion, protein restriction protects beta -cells of the deleterious effect of IL-1 beta, apparently, by decreasing NO production. The lower NO generation in islets from protein deprived rats may be due to increased free fatty acids oxidation and consequent alteration in Ca2+ homeostasis. (C) 2001 Elsevier B.V. All rights reserved.
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FUNDAMENTO: Vrios autores mostraram que a deteriorao da funo cardaca associa-se com o grau e a durao da obesidade. Os padres de expresso gnica aps longos perodos de obesidade precisam ser estabelecidos. OBJETIVO: Este estudo testou a hiptese de que a exposio prolongada obesidade leva reduo nos nveis de RNAm de protenas envolvidas na homeostase do Ca2+ miocrdico. Alm disso, este estudo avaliou se uma diminuio no hormnio tireoidiano causava reduo na expresso de RNAm. MTODOS: Ratos Wistar machos de 30 dias de idade foram distribudos em dois grupos: controle (C) e obeso (Ob). O grupo C recebeu uma dieta padro e o grupo Ob recebeu dietas hiperlipdicas por 15, 30 e 45 semanas. A obesidade foi definida pelo ndice de adiposidade. A expresso gnica foi avaliada por PCR em tempo real quantitativa. RESULTADOS: O ndice de adiposidade foi maior no grupo Ob do que no C em todas as etapas. Enquanto a obesidade nas semanas 15 e 45 determinou uma reduo no RNAm de Ca2+-ATPase do retculo sarcoplasmtico (SERCA2a), trocador Na+/Ca2+ (NCX) e calsequestrina (CSQ), observou-se aumento da expresso do RNAm de canal de Ca2+ do tipo L, receptor de rianodina, SERCA2a, fosfolamban (PLB), NCX e CSQ aps a semana 30, em comparao ao grupo C. No houve associao significativa entre os nveis de T3 e a expresso de RNAm. CONCLUSES: Nossos dados indicam que a obesidade por curtos ou longos perodos de tempo pode promover alterao na expresso gnica de protenas reguladoras da homeostase do Ca2+ sem influncia do hormnio tireoidiano
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Abamectin (ABA), which belongs to the family of avermectins, is used as a parasiticide; however, ABA poisoning can impair liver function. In a previous study using isolated rat liver mitochondria, we observed that ABA inhibited the activity of adenine nucleotide translocator and FoF1-ATPase. The aim of this study was to characterize the mechanism of ABA toxicity in isolated rat hepatocytes and to evaluate whether this effect is dependent on its metabolism. The toxicity of ABA was assessed by monitoring oxygen consumption and mitochondrial membrane potential, intracellular ATP concentration, cell viability, intracellular Ca2+ homeostasis, release of cytochrome c, caspase 3 activity and necrotic cell death. ABA reduces cellular respiration in cells energized with glutamate and malate or succinate. The hepatocytes that were previously incubated with proadifen, a cytochrome P450 inhibitor, are more sensitive to the compound as observed by a rapid decrease in the mitochondrial membrane potential accompanied by reductions in ATP concentration and cell viability and a disruption of intracellular Ca2+ homeostasis followed by necrosis. Our results indicate that ABA biotransformation reduces its toxicity, and its toxic action is related to the inhibition of mitochondrial activity, which leads to decreased synthesis of ATP followed by cell death. 2012 Elsevier Ltd.
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Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES)
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The mitochondrion is an essential cytoplasmic organelle that provides most of the energy necessary for eukaryotic cell physiology. Mitochondrial structure and functions are maintained by proteins of both mitochondrial and nuclear origin. These organelles are organized in an extended network that dynamically fuses and divides. Mitochondrial morphology results from the equilibrium between fusion and fission processes, controlled by a family of mitochondria-shaping proteins. It is becoming clear that defects in mitochondrial dynamics can impair mitochondrial respiration, morphology and motility, leading to apoptotic cell death in vitro and more or less severe neurodegenerative disorders in vivo in humans. Mutations in OPA1, a nuclear encoded mitochondrial protein, cause autosomal Dominant Optic Atrophy (DOA), a heterogeneous blinding disease characterized by retinal ganglion cell degeneration leading to optic neuropathy (Delettre et al., 2000; Alexander et al., 2000). OPA1 is a mitochondrial dynamin-related guanosine triphosphatase (GTPase) protein involved in mitochondrial network dynamics, cytochrome c storage and apoptosis. This protein is anchored or associated on the inner mitochondrial membrane facing the intermembrane space. Eight OPA1 isoforms resulting from alternative splicing combinations of exon 4, 4b and 5b have been described (Delettre et al., 2001). These variants greatly vary among diverse organs and the presence of specific isoforms has been associated with various mitochondrial functions. The different spliced exons encode domains included in the amino-terminal region and contribute to determine OPA1 functions (Olichon et al., 2006). It has been shown that exon 4, that is conserved throughout evolution, confers functions to OPA1 involved in maintenance of the mitochondrial membrane potential and in the fusion of the network. Conversely, exon 4b and exon 5b, which are vertebrate specific, are involved in regulation of cytochrome c release from mitochondria, and activation of apoptosis, a process restricted to vertebrates (Olichon et al., 2007). While Mgm1p has been identified thanks to its role in mtDNA maintenance, it is only recently that OPA1 has been linked to mtDNA stability. Missense mutations in OPA1 cause accumulation of multiple deletions in skeletal muscle. The syndrome associated to these mutations (DOA-1 plus) is complex, consisting of a combination of dominant optic atrophy, progressive external ophtalmoplegia, peripheral neuropathy, ataxia and deafness (Amati- Bonneau et al., 2008; Hudson et al., 2008). OPA1 is the fifth gene associated with mtDNA breakage syndrome together with ANT1, PolG1-2 and TYMP (Spinazzola et al., 2009). In this thesis we show for the first time that specific OPA1 isoforms associated to exon 4b are important for mtDNA stability, by anchoring the nucleoids to the inner mitochondrial membrane. Our results clearly demonstrate that OPA1 isoforms including exon 4b are intimately associated to the maintenance of the mitochondrial genome, as their silencing leads to mtDNA depletion. The mechanism leading to mtDNA loss is associated with replication inhibition in cells where exon 4b containing isoforms were down-regulated. Furthermore silencing of exon 4b associated isoforms is responsible for alteration in mtDNA-nucleoids distribution in the mitochondrial network. In this study it was evidenced that OPA1 exon 4b isoform is cleaved to provide a 10kd peptide embedded in the inner membrane by a second transmembrane domain, that seems to be crucial for mitochondrial genome maintenance and does correspond to the second transmembrane domain of the yeasts orthologue encoded by MGM1 or Msp1, which is also mandatory for this process (Diot et al., 2009; Herlan et al., 2003). Furthermore in this thesis we show that the NT-OPA1-exon 4b peptide co-immuno-precipitates with mtDNA and specifically interacts with two major components of the mitochondrial nucleoids: the polymerase gamma and Tfam. Thus, from these experiments the conclusion is that NT-OPA1- exon 4b peptide contributes to the nucleoid anchoring in the inner mitochondrial membrane, a process that is required for the initiation of mtDNA replication and for the distribution of nucleoids along the network. These data provide new crucial insights in understanding the mechanism involved in maintenance of mtDNA integrity, because they clearly demonstrate that, besides genes implicated in mtDNA replications (i.e. polymerase gamma, Tfam, twinkle and genes involved in the nucleotide pool metabolism), OPA1 and mitochondrial membrane dynamics play also an important role. Noticeably, the effect on mtDNA is different depending on the specific OPA1 isoforms down-regulated, suggesting the involvement of two different combined mechanisms. Over two hundred OPA1 mutations, spread throughout the coding region of the gene, have been described to date, including substitutions, deletions or insertions. Some mutations are predicted to generate a truncated protein inducing haploinsufficiency, whereas the missense nucleotide substitutions result in aminoacidic changes which affect conserved positions of the OPA1 protein. So far, the functional consequences of OPA1 mutations in cells from DOA patients are poorly understood. Phosphorus MR spectroscopy in patients with the c.2708delTTAG deletion revealed a defect in oxidative phosphorylation in muscles (Lodi et al., 2004). An energetic impairment has been also show in fibroblasts with the severe OPA1 R445H mutation (Amati-Bonneau et al., 2005). It has been previously reported by our group that OPA1 mutations leading to haploinsufficiency are associated in fibroblasts to an oxidative phosphorylation dysfunction, mainly involving the respiratory complex I (Zanna et al., 2008). In this study we have evaluated the energetic efficiency of a panel of skin fibroblasts derived from DOA patients, five fibroblast cell lines with OPA1 mutations causing haploinsufficiency (DOA-H) and two cell lines bearing mis-sense aminoacidic substitutions (DOA-AA), and compared with control fibroblasts. Although both types of DOA fibroblasts maintained a similar ATP content when incubated in a glucose-free medium, i.e. when forced to utilize the oxidative phosphorylation only to produce ATP, the mitochondrial ATP synthesis through complex I, measured in digitonin-permeabilized cells, was significantly reduced in cells with OPA1 haploinsufficiency only, whereas it was similar to controls in cells with the missense substitutions. Furthermore, evaluation of the mitochondrial membrane potential (DYm) in the two fibroblast lines DOA-AA and in two DOA-H fibroblasts, namely those bearing the c.2819-2A>C mutation and the c.2708delTTAG microdeletion, revealed an anomalous depolarizing response to oligomycin in DOA-H cell lines only. This finding clearly supports the hypothesis that these mutations cause a significant alteration in the respiratory chain function, which can be unmasked only when the operation of the ATP synthase is prevented. Noticeably, oligomycin-induced depolarization in these cells was almost completely prevented by preincubation with cyclosporin A, a well known inhibitor of the permeability transition pore (PTP). This results is very important because it suggests for the first time that the voltage threshold for PTP opening is altered in DOA-H fibroblasts. Although this issue has not yet been addressed in the present study, several are the mechanisms that have been proposed to lead to PTP deregulation, including in particular increased reactive oxygen species production and alteration of Ca2+ homeostasis, whose role in DOA fibroblasts PTP opening is currently under investigation. Identification of the mechanisms leading to altered threshold for PTP regulation will help our understanding of the pathophysiology of DOA, but also provide a strategy for therapeutic intervention.
