Regulation of vascular smooth muscle cell bioenergetic function by protein glutathiolation

AbstractProtein thiolation by glutathione is a reversible and regulated post-translational modification that is increased in response to oxidants and nitric oxide. Because many mitochondrial enzymes contain critical thiol residues, it has been hypothesized that thiolation reactions regulate cell metabolism and survival. However, it has been difficult to differentiate the biological effects due to protein thiolation from other oxidative protein modifications. In this study, we used diamide to titrate protein glutathiolation and examined its impact on glycolysis, mitochondrial function, and cell death in rat aortic smooth muscle cells. Treatment of cells with diamide increased protein glutathiolation in a concentration-dependent manner and had comparably little effect on protein–protein disulfide formation. Diamide increased mitochondrial proton leak and decreased ATP-linked mitochondrial oxygen consumption and cellular bioenergetic reserve capacity. Concentrations of diamide above 200 μM promoted acute bioenergetic failure and caused cell death, whereas lower concentrations of diamide led to a prolonged increase in glycolytic flux and were not associated with loss of cell viability. Depletion of glutathione using buthionine sulfoximine had no effect on basal protein thiolation or cellular bioenergetics but decreased diamide-induced protein glutathiolation and sensitized the cells to bioenergetic dysfunction and death. The effects of diamide on cell metabolism and viability were fully reversible upon addition of dithiothreitol. These data suggest that protein thiolation modulates key metabolic processes in both the mitochondria and cytosol

ANÁLISE MICROSCÓPICA FOLIAR DE MUTAMBA (Guazuma ulmifolia LAM., MALVACEAE)

Guazuma ulmifolia Lam. é uma planta arbórea, nativa das Américas e reposicionada atualmente na família Malvaceae. É conhecida comumente como mutamba ou chico-magro, e utilizada na medicinal popular como cicatrizante e no tratamento de distúrbios gastrointestinais e respiratórios. Estudos fitoquímicos indicaram a presença de taninos e flavonoides nos órgãos vegetativos aéreos. Ensaios farmacológicos comprovaram as atividades gastroprotetora e antibacteriana de extratos da planta. Ao analisar a anatomia foliar de G. ulmifolia, este trabalho buscou estabelecer os caracteres estruturais de fácil reconhecimento para a identificação microscópica dessa potencial droga vegetal. O material foi coletado de espécimes cultivados no Cerrado. Estudaram-se o terço inferior do limbo e o pecíolo de folhas adultas, por meio de observações feitas em microscopia fotônica e eletrônica de varredura. Testes microquímicos foram também conduzidos com reativos usuais. Em ambas as faces epidérmicas, ocorrem tricomas estelares e glandulares. Os primeiros são formados por tricomas tectores unicelulares, reunidos pela base. Os glandulares são capitados, apresentando um pedicelo curto e glândula ovoide. A cutícula é nitidamente estriada e os estômatos são anomocíticos e restritos à superfície abaxial. O mesofilo é dorsiventral e a nervura central tem secção biconvexa e é percorrida por um único feixe vascular em arco aberto, envolto sucessivamente por bainhas esclerenquimática e parenquimática, esta contendo compostos fenólicos. O pecíolo tem contorno circular e um ou mais feixes vasculares colaterais. Ocorrem células com conteúdo mucilaginoso e fenólico na folha, bem como cristais prismáticos de oxalato de cálcio

Review-Development of Huckel Type Anions: From Molecular Modeling to Industrial Commercialization. A Success Story

This paper reviews the battery electrolyte technologies involving Huckel-type salts as a major electrolyte component. The concept was initially proposed by M. Armand in 1995 and then explored by several research groups. In the present review studies on the optimization of the electrolyte composition starting from molecular modeling through enhancing the yield of the salt synthesis to structural characterization and electrochemical performance are described. Furthermore, the use of the optimized electrolytes in a variety of lithium-ion and post-lithium batteries is presented and discussed. Finally, the commercialization of the up to date technology by Arkema is discussed as well as the performance of the present Huckel anion based electrolytes as compared to other marketed electrolyte technologies

LEAF ANATOMY OF YELLOW COTTON TREE: Cochlospermum regium (SCHRANK) PILG., BIXACEAE

Cochlospermum regium é uma espécie nativa encontrada no Cerrado, conhecida comumente como algodãozinho devido ao aspecto lanuginoso das sementes. É empregada na medicina popular como analgésico, anti-inflamatório e anti-infeccioso. Estudos farmacológicos comprovaram as atividades antinociceptiva, antimicrobiana e antipirética, justificando a sua utilização na terapêutica. Este trabalho objetivou investigar os caracteres anatômicos foliares, úteis para a caracterização e o controle de qualidade farmacognóstico dessa espécie medicinal, bem como para o conhecimento da flora brasileira. O material botânico foi preparado de acordo com técnicas de microscopia fotônica e eletrônica de varredura, incluindo-se testes microquímicos. A folha é anfiestomática, possuindo estômatos anomocíticos na face abaxial e raros estômatos do tipo paracítico na superfície oposta. Tricomas tectores pluricelulares, com células apicais longas, e glandulares peltados estão presentes. O mesofilo é dorsiventral, a nervura central é biconvexa e percorrida por diversos feixes vasculares colaterais dispostos em V, e ocorrem idioblastos com compostos lipofílicos e fenólicos, bem como drusas de oxalato de cálcio. Esses aspectos são relevantes para a caracterização da espécie, porém pesquisa complementar sobre o gênero é necessária para atribuição de valor diagnóstico aos mesmos

A mitochondria-targeted mass spectrometry probe to detect glyoxals: implications for diabetes

The glycation of protein and nucleic acids that occurs as a consequence of hyperglycaemia disrupts cell function and contributes to many pathologies, including those associated with diabetes and aging. Intracellular glycation occurs following the generation of the reactive 1,2-dicarbonyls methylglyoxal and glyoxal and disruption to mitochondrial function is associated with hyperglycemia. However, the contribution of these reactive dicarbonyls to mitochondrial damage in pathology is unclear due to uncertainties about their levels within mitochondria in cells and in vivo. To address this we have developed a mitochondria-targeted reagent (MitoG) designed to assess the levels of mitochondrial dicarbonyls within cells. MitoG comprises a lipophilic triphenylphosphonium cationic function, which directs the molecules to mitochondria within cells and an o-phenylenediamine moiety that reacts with dicarbonyls to give distinctive and stable products. The extent of accumulation of these diagnostic heterocyclic products can be readily and sensitively quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), enabling changes to be determined. Using the MitoG-based analysis we assessed the formation of methylglyoxal and glyoxal in response to hyperglycaemia in cells in culture and in the Akita mouse model of diabetes in vivo. These findings indicated that the levels of methylglyoxal and glyoxal within mitochondria increase during hyperglycaemia in both cells and in vivo, suggesting that they can contribute to the pathological mitochondrial dysfunction that occurs in diabetes and aging

Bioenergetic profile of human coronary artery smooth muscle cells and effect of metabolic intervention

Bioenergetics of artery smooth muscle cells is critical in cardiovascular health and disease. An acute rise in metabolic demand causes vasodilation in systemic circulation while a chronic shift in bioenergetic profile may lead to vascular diseases. A decrease in intracellular ATP level may trigger physiological responses while dedifferentiation of contractile smooth muscle cells to a proliferative and migratory phenotype is often observed during pathological processes. Although it is now possible to dissect multiple building blocks of bioenergetic components quantitatively, detailed cellular bioenergetics of artery smooth muscle cells is still largely unknown. Thus, we profiled cellular bioenergetics of human coronary artery smooth muscle cells and effects of metabolic intervention. Mitochondria and glycolysis stress tests utilizing Seahorse technology revealed that mitochondrial oxidative phosphorylation accounted for 54.5% of ATP production at rest with the remaining 45.5% due to glycolysis. Stress tests also showed that oxidative phosphorylation and glycolysis can increase to a maximum of 3.5 fold and 1.25 fold, respectively, indicating that the former has a high reserve capacity. Analysis of bioenergetic profile indicated that aging cells have lower resting oxidative phosphorylation and reduced reserve capacity. Intracellular ATP level of a single cell was estimated to be over 1.1 mM. Application of metabolic modulators caused significant changes in mitochondria membrane potential, intracellular ATP level and ATP:ADP ratio. The detailed breakdown of cellular bioenergetics showed that proliferating human coronary artery smooth muscle cells rely more or less equally on oxidative phosphorylation and glycolysis at rest. These cells have high respiratory reserve capacity and low glycolysis reserve capacity. Metabolic intervention influences both intracellular ATP concentration and ATP:ADP ratio, where subtler changes may be detected by the latter

Investigation of Mitochondrial Dysfunction by Sequential Microplate-Based Respiration Measurements from Intact and Permeabilized Neurons

Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria

Endothelial Cell and Platelet Bioenergetics: Effect of Glucose and Nutrient Composition

It has been suggested that cells that are independent of insulin for glucose uptake, when exposed to high glucose or other nutrient concentrations, manifest enhanced mitochondrial substrate oxidation with consequent enhanced potential and generation of reactive oxygen species (ROS); a paradigm that could predispose to vascular complications of diabetes. Here we exposed bovine aortic endothelial (BAE) cells and human platelets to variable glucose and fatty acid concentrations. We then examined oxygen consumption and acidification rates using recently available technology in the form of an extracellular oxygen and proton flux analyzer. Acute or overnight exposure of confluent BAE cells to glucose concentrations from 5.5 to 25 mM did not enhance or change the rate of oxygen consumption (OCR) under basal conditions, during ATP synthesis, or under uncoupled conditions. Glucose also did not alter OCR in sub-confluent cells, in cells exposed to low serum, or in cells treated with added pyruvate. Likewise, overnight exposure to fatty acids of varying saturation had no such effects. Overnight exposure of BAE cells to low glucose concentration decreased maximal uncoupled respiration, but not basal or ATP related oxygen consumption. Labeled glucose oxidation to CO2 increased, but only marginally after high glucose exposure while oleate oxidation to CO2 decreased. Overnight exposure to linolenic acid, but not oleic or linoleic acid increased extracellular acidification consistent with enhanced glycolytic metabolism. We were unable to detect an increase in production of reactive oxygen species (ROS) from BAE cells exposed to high medium glucose. Like BAE cells, exposure of human platelets to glucose did not increase oxygen consumption. As opposed to BAE cells, platelet mitochondria demonstrate less respiratory reserve capacity (beyond that needed for basal metabolism). Our data do not support the concept that exposure to high glucose or fatty acids accelerates mitochondrial oxidative metabolism in endothelial cells or platelets

Bezielle Selectively Targets Mitochondria of Cancer Cells to Inhibit Glycolysis and OXPHOS

Bezielle (BZL101) is a candidate oral drug that has shown promising efficacy and excellent safety in the early phase clinical trials for advanced breast cancer. Bezielle is an aqueous extract from the herb Scutellaria barbata. We have reported previously that Bezielle was selectively cytotoxic to cancer cells while sparing non-transformed cells. In tumor, but not in non-transformed cells, Bezielle induced generation of ROS and severe DNA damage followed by hyperactivation of PARP, depletion of the cellular ATP and NAD, and inhibition of glycolysis. We show here that tumor cells' mitochondria are the primary source of reactive oxygen species induced by Bezielle. Treatment with Bezielle induces progressively higher levels of mitochondrial superoxide as well as peroxide-type ROS. Inhibition of mitochondrial respiration prevents generation of both types of ROS and protects cells from Bezielle-induced death. In addition to glycolysis, Bezielle inhibits oxidative phosphorylation in tumor cells and depletes mitochondrial reserve capacity depriving cells of the ability to produce ATP. Tumor cells lacking functional mitochondria maintain glycolytic activity in presence of Bezielle thus supporting the hypothesis that mitochondria are the primary target of Bezielle. The metabolic effects of Bezielle towards normal cells are not significant, in agreement with the low levels of oxidative damage that Bezielle inflicts on them. Bezielle is therefore a drug that selectively targets cancer cell mitochondria, and is distinguished from other such drugs by its ability to induce not only inhibition of OXPHOS but also of glycolysis. This study provides a better understanding of the mechanism of Bezielle's cytotoxicity, and the basis of its selectivity towards cancer cells