Mitochondrial dysfunction reduces the activity of KIR2.1 K⁺ channel in myoblasts via impaired oxidative phosphorylation

Myoblast fusion depends on mitochondrial integrity and intracellular Ca²⁺ signaling regulated by various ion channels. In this study, we investigated the ionic currents associated with [Ca²⁺]i regulation in normal and mitochondrial DNA-depleted (ρ0) L6 myoblasts. The ρ0 myoblasts showed impaired myotube formation. The inwardly rectifying K⁺ current (I(Kir)) was largely decreased with reduced expression of KIR2.1, whereas the voltage-operated Ca²⁺ channel and Ca²⁺-activated K⁺ channel currents were intact. Sustained inhibition of mitochondrial electron transport by antimycin A treatment (24 h) also decreased the I(Kir). The ρ0 myoblasts showed depolarized resting membrane potential and higher basal [Ca²⁺]ᵢ. Our results demonstrated the specific downregulation of I(Kir) by dysfunctional mitochondria. The resultant depolarization and altered Ca²⁺ signaling might be associated with impaired myoblast fusion in ρ0 myoblasts.

Eixo XPC-P53-H202 e disfunção mitocondrial: qual é o fator central? / XPC-p53-H2O2 axis and mitochondrial disfunction: Which is the key player

A ausência de XPC, uma proteína canonicamente envolvida em reparo de DNA por excisão de nucleotídeos, está associada a vários fenótipos característicos de disfunção mitocondrial como o desequilíbrio entre os complexos da cadeia transportadora de elétrons (CTE), redução no consumo de oxigênio, maior produção de peróxido de hidrogênio, e maior sensibilidade a agentes que causam estresse mitocondrial. Contudo, uma descrição mecanística da relação entre deficiência de XPC e disfunção mitocondrial ainda não está bem estabelecida. Aqui mostramos que a deficiência de XPC está associada ao aumento na expressão do supressor de tumor p53. Essa alteração é acompanhada pelo aumento da expressão de diversas proteínas que participam em importantes funções mitocondriais. A inibição de p53 reverte a superexpressão de algumas dessas proteínas. O tratamento com o inibidor do Complexo III da CTE antimicina A induz aumento da expressão de p53 de forma mais acentuada na linhagem Xpc-/-, enquanto o tratamento com o antioxidante N-acetilcisteína diminue a produção basal de H2O2, expressão de p53 e sensibilidade aumentada ao tratamento com antimicina A. Em conjunto, nossos resultados suportam a hipótese de que o aumento da produção de H2O2 em células Xpc-/- tem um papel causal na regulação da expressão de p53 e na disfunção mitocondrial

Although XPC has been initially implicated in the nucleotide excision DNA repair pathway, its deficiency is associated with mitochondrial dysfunction, including unbalanced electron transport chain (ETC) activity, lower oxygen consumption, increased hydrogen peroxide production, and greater sensitivity to mitochondrial stress. However, a mechanistic understanding of the role of XPC in regulating mitochondrial function is still not well established. Here we show that XPC deficiency is associated with increased expression of the tumor suppressor p53, which is accompanied by increased expression of several proteins that participate in important mitochondrial functions. Inhibition of p53 reverses the overexpression of some of these proteins. In addition, treatment with the ETC inhibitor antimycin A induces p53 expression more robustly in the Xpc-/- cells, while treatment with the antioxidant N-acetylcysteine decreases basal H2O2 production, p53 expression and sensitivity to antimycin A treatment. Together, our results support a model in which increased H2O2 production in Xpc-/- causes upregulation of p53 expression and mitochondrial dysfunction

Mitochondrial Complexes I and II Are More Susceptible to Autophagy Deficiency in Mouse beta-Cells

BACKGROUND: Damaged mitochondria are removed by autophagy. Therefore, impairment of autophagy induces the accumulation of damaged mitochondria and mitochondrial dysfunction in most mammalian cells. Here, we investigated mitochondrial function and the expression of mitochondrial complexes in autophagy-related 7 (Atg7)-deficient beta-cells. METHODS: To evaluate the effect of autophagy deficiency on mitochondrial function in pancreatic beta-cells, we isolated islets from Atg7(F/F):RIP-Cre+ mice and wild-type littermates. Oxygen consumption rate and intracellular adenosine 5'-triphosphate (ATP) content were measured. The expression of mitochondrial complex genes in Atg7-deficient islets and in beta-TC6 cells transfected with siAtg7 was measured by quantitative real-time polymerase chain reaction. RESULTS: Baseline oxygen consumption rate of Atg7-deficient islets was significantly lower than that of control islets (P<0.05). Intracellular ATP content of Atg7-deficient islets during glucose stimulation was also significantly lower than that of control islets (P<0.05). By Oxygraph-2k analysis, mitochondrial respiration in Atg7-deficient islets was significantly decreased overall, although state 3 respiration and responses to antimycin A were unaffected. The mRNA levels of mitochondrial complexes I, II, III, and V in Atg7-deficient islets were significantly lower than in control islets (P<0.05). Down-regulation of Atg7 in beta-TC6 cells also reduced the expression of complexes I and II, with marginal significance (P<0.1). CONCLUSION: Impairment of autophagy in pancreatic beta-cells suppressed the expression of some mitochondrial respiratory complexes, and may contribute to mitochondrial dysfunction. Among the complexes, I and II seem to be most vulnerable to autophagy deficiency.

The Neuroprotective Effects of Carnosine in Early Stage of Focal Ischemia Rodent Model

OBJECTIVE: This study was conducted to elucidate neuroprotective effect of carnosine in early stage of stroke. METHODS: Early stage of rodent stroke model and neuroblastoma chemical hypoxia model was established by middle cerebral artery occlusion and antimycin A. Neuroprotective effect of carnosine was investigated with 100, 250, and 500 mg of carnosine treatment. And antioxidant expression was analyzed by enzyme linked immunosorbent assay (ELISA) and western blot in brain and blood. RESULTS: Intraperitoneal injection of 500 mg carnosine induced significant decrease of infarct volume and expansion of penumbra (p<0.05). The expression of superoxide dismutase (SOD) showed significant increase than in saline group in blood and brain (p<0.05). In the analysis of chemical hypoxia, carnosine induced increase of neuronal cell viability and decrease of reactive oxygen species (ROS) production. CONCLUSION: Carnosine has neuroprotective property which was related to antioxidant capacity in early stage of stroke. And, the oxidative stress should be considered one of major factor in early ischemic stroke.

Effects of Mitochondrial Reactive Oxygen Species on Neuronal Excitability in Rat Spinal Substantia Gelatinosa Neurons

Recent studies indicate that reactive oxygen species (ROS) are critically involved in persistent pain primarily through spinal mechanisms, and that mitochondria are the main source of ROS in the spinal dorsal horn. To investigate whether mitochondrial ROS can induce changes in membrane excitability on spinal substantia gelatonosa (SG) neurons, we examined the effects of mitochondrial electron transport complex (ETC) substrates and inhibitors on the membrane potential of SG neurons in spinal slices. Application of ETC inhibitors, rotenone or antimycin A, resulted in a slowly developing and slight membrane depolarization in SG neurons. Also, application of both malate, a complex I substrate, and succinate, a complex II substrate, caused reversible membrane depolarization and enhanced firing activity. Changes in membrane potential after malate exposure were more prominent than succinate exposure. When slices were pretreated with ROS scavengers such as phenyl-N-tert-buthylnitrone (PBN), catalase and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), malate-induced depolarization was significantly decreased. Intracellular calcium above 100 microM increased malateinduced depolarization, witch was suppressed by cyclosporin A, a mitochondrial permeability transition (MPT) inhibitor. These results suggest that enhanced production of spinal mitochondrial ROS can induce nociception through central sensitization.

Progress in small-molecule inhibitors of Bcl-2 family proteins / 药学学报

Apoptosis is an essential factor in keeping homeostasis of the organism. Apoptosis is regulated by a series of cytokines. Bcl-2 family proteins are key regulators of apoptosis. The Bcl-2 family includes both anti- and pro-apoptotic proteins with opposing biological functions. Their interaction regulates the transmission of the apoptosis signal. High expression of anti-apoptotic members such as Bcl-2 and Bcl-xL are commonly found in human cancers. In recent years, following the disclosing of the crystal structures of Bcl-2 family proteins, researchers have paid attention to the development of the small molecule inhibitors of Bcl-2 family proteins. This article reviews the progress in this field from the view of drug design.

Inhibitory effect of agarohexaose on antimycin A induced generation of reactive oxygen species / 药学学报

<p><b>AIM</b>To evaluate the hepatocyte protective effect of agarohexaose against indirect oxidative stress injury induced by antimycin A.</p><p><b>METHODS</b>Antimycin A was used to induce oxidative injury of human hepatocyte L-02. The oxidative degree in cells was detected by dichlorofluorescin diacetate (DCFH-DA) and the fluorescence generation was recorded by flow cytometer and fluorescent microscope. The apoptosis of L-02 cells induced by oxidation was identified by TUNEL test, and the morphologic features of cells were also observed.</p><p><b>RESULTS</b>Agarohexaose at concentration of 1 mg x mL(-1) inhibited the oxidation of DCFH into DCF significantly. The fluorescence intensity and oxidized cell number decreased after the incubation with agarohexaose. The photomicrographs of antimycin A and agarohexaose treated cells revealed that agarohexaose could reduce the apoptotic morphologic features. The TUNEL results also indicated that the number of apoptotic cells decreased significantly after the treatment of agarohexaose.</p><p><b>CONCLUSION</b>Agarohexaose could inhibit the sudden increase reactive oxygen species (ROS) in cells significantly, and it also protected cells against oxidative stress injury in vitro.</p>

Comunicação entre mitocôndria e núcleo controla a transcrição do gene GAL1 de Saccharomyces cerevisae / Communication between mitochondrial and nuclear controls Saccharomyces cerevisiae GAL1 gene transcription

O gene nuclear GAL1 de Saccharomyces cerevisiae codifica uma galactoquinase induzida por galactose e reprimida por glicose. Três evidências indicam que a transcrição de GAL1 é dependente da atividade mitocondrial. Linhagens petite, com deleção no DNA da organela (pî) ou rompimento em gene nuclear, que codifica a farnesil transferase mitocondrial, são incapazes de induzir GAL1. Os inibidores de respiração antimicina-A e azoteto de sódio (NaN3), que atuam, respectivamente, nos complexos III e IV da cadeia de transporte de elétrons, impedem a indução de GAL1. Em células crescidas em glicose ou glicerol, o oligômero formado pela proteína URF13, na presença de metomil, produz um poro na membrana mitocondrial...

Ischemia-responsive Protein(irp94) Gene Expression in a Neuronal Cell Culture Model of Ischemia

BACKGROUND: The ischemia responsive protein 94 kDa(irp94) gene belongs to the heat shock protein 110 family and was isolated in 1999 from rat brain by transiently induced forebrain ischemia. The PC12 cell is the pheochromocytoma cell line of rat, which is differentiated to a sympathetic neuron-like cell by the stimulation of a nerve growth factor. This study is to determine whether irp94 is expressed when an ischemia-like condition is induced by ATP depletion in cultured PC12 cells in vitro. METHODS: PC12 cells were maintained as monolayer cultures in RPMI-1640 medium(Sigma) supplemented with 10% horse serum, 5% fetal bovine serum, 5 mg/ml transferrin, and 1 mg/ml insulin in a humidified 5% CO2 incubator at 37degrees C. The ATP depleting agent antimycin A was added at concentrations of 1, 2.5, and 5 microM to simulate ischemia, and 10 microgram/ml of tunicamycin, which is expected to express heat shock protein maximally, was used as a positive control. The cells were harvested after a 60-minute incubation, and the total RNA was extracted. The reverse transcription polymerase chain reaction(RT-PCR) was performed to use 501 bp irp94 cDNA as a molecular probe, and the expression of irp94 mRNA was analyzed by northern blotting. RESULTS: The irp94 mRNA expression was enhanced, compared to the negative control group, as the concentration of antimycin A was increased. CONCLUSION: This study suggests that irp94 mRNA expression is enhanced as the severity of ischemia is increased. Thus, it is possible to investigate the mechanism of ischemic neuronal injury indirectly by using this in-vitro model of neuronal ischemia.

Effects of dexamethasone and DHEA on the responses of rat cerebral cortical astrocytes to lipopolysaccharide and antimycin A

As part of a study on the effects of dexamethasone and dehydroepiandrosterone (DHEA) on the biological roles of astrocytes in brain injury, this study evaluated the effects of dexamethasone and DHEA on the responses of primary cultured rat cortical astrocytes to lipopolysaccharide (LPS) and antimycin A. Dexamethasone decreased spontaneous release of LDH from astrocytes, and the dexamethasone effect was inhibited by DHEA. However, the inhibitory effect of DHEA on the dexamethasone-induced decrease of LDH release was not shown in astrocytes treated with LPS, and antimycin A-induced LDH release was not affected by dexamethasone or DHEA. Unlike dexamethasone, DHEA increased MTT value of astrocytes and also attenuated the antimycin A-induced decrease of MTT value. Glutamine synthetase activity of astrocytes was not affected by DHEA or LPS but increased by dexamethasone, and the dexamethasone-dependent increase was attenuated by DHEA. However, antimycin A markedly decreased glutamine synthetase activity, and the antimycin A effect was not affected by dexamethasone or DHEA. Basal release of (3H)arachidonic acid from astrocytes was moderately increased by LPS and markedly by antimycin A. Dexamethasone inhibited the basal and LPS-dependent releases of (3H)arachidonic acid, but neither dexamethasone nor DHEA affected antimycin A-induced (3H)arachidonic acid release. Basal IL-6 release from astrocytes was not affected by dexamethasone or DHEA but markedly increased by LPS and antimycin A. LPS-induced IL-6 release was attenuated by dexamethasone but was little affected by DHEA, and antimycin A-induced IL-6 release was attenuated by DHEA as well as dexamethasone. At the concentration of dexamethasone and DHEA which does not affect basal NO release from astrocytes, they moderately inhibited LPS-induced NO release but little affected antimycin A-induced decrease of NO release. Taken together, these results suggest that dexamethasone and DHEA, in somewhat different manners, modulate the astrocyte reactivity in brain injuries inhibitorily.

Effects of dexamethasone and DHEA on the changes of glutamate and polyamine uptake in rat astrocytes by lipopolysaccharide and antimycin A

Interactions among dexamethasone, dehydroepiandrosterone (DHEA), lipopolysaccharide (LPS), and antimycin A on the glutamate uptake and the polyamine uptake were investigated in primary cultures of rat cerebral cortical astrocytes to examine the effects of dexamethasone and DHEA on the regulatory role of astrocytes in conditions of increased extracellular concentrations of glutamate or polyamines. 1. (3H)Glutamate uptake: LPS and antimycin A decreased Vmax, but both drugs had little effect on Km. Dexamethasone also decreased basal Vmax without any significant effect on Km. And dexamethasone further decreased the antimycin A-induced decrease of Vmax. DHEA did not affect the kinetics of basal glutamate uptake and the change by LPS or antimycin A. 2. (14C)Putrescine uptake: LPS increased Vmax, and antimycin A decreased Vmax. They showed little effect on Km. Dexamethasone decreased Vmax of basal uptake and further decreased the antimycin A-induced decrease of Vmax, and also decreased Vmax to less than control in LPS-treated astrocytes. DHEA did not affect Km and the change of Vmax by LPS or antimycin A. 3. (14C)Spermine uptake: Antimycin A decreased Vmax, and LPS might increase Vmax. Km was little affected by the drugs. Dexamethasone decreased basal Vmax and might further decrease the antimycin A-induced decrease of Vmax. And dexamethasone also decreased Vmax to less than control in LPS-treated astrocytes. DHEA might increase basal Vmax and Vmax of LPS-treated astrocytes. 4. Vmax of glutamate uptake by astrocytes was increased by putrescine (1000 muM & 2000 muM) and spermidine (200 muM, 500 muM & 2000 muM). Spermine, 200 muM (and 100 muM), also increased Vmax, but a higher dose of 2000 muM decreased Vmax. Km of glutamate uptake was not significantly changed by these polyamines, except that higher doses of spermine showed tendency to decrease Km of glutamate uptake. In astrocytes, dexamethasone inhibited the glutamate uptake and the polyamine uptake in normal or hypoxic conditions, and the polyamine uptake might be stimulated by LPS and DHEA. Polyamines could aid astrocytes to uptake glutamate.

Calcium homeostasis in Trypanosoma cruzi

By using the fluorescent Ca2+ indicator fura 2, submicromolar levels of intracellular Ca2+ have been detected in Trypanosoma cruzi different stages. The intracellular transport mechanisms involved in maintaining Ca2+ homeostasis in T. cruzi have been characterized by measuring Ca2+ transport in digitonin-permeabilized cells. Two intracellular calcium transport systems have been detected. Ca2+ uptake by the mitochondria occurs by an electrophoretic mechanism, is inhibited by antimycin A, FCCP, and ruthenium red, and stimulated by respiratory substrates, phosphate and acetate. This pool has a high capacity and low affinity for Ca2+ and is able to buffer external Ca2+ at concentrations in the range of 0.6-0.7 microM. Ca2+ uptake by the endoplasmic reticulum is inhibited by high concentrations of vanadate and anticalmodulin agents, and stimulated by ATP. This pool has a low capacity and a high affinity for Ca2+ and is able to buffer external Ca2+ at concentrations in the range of 0.05-1.0 microM. In addition, calmodulin has been purified from T. cruzi epimastigotes and shown to stimulate the homologous plasma membrane Ca(2+)-ATPase and cyclic-AMP phosphodiesterase. The gene encoding this protein has been cloned and sequenced and shown to have a great homology to mammalian calmodulin. The role of the plasma membrane of T. cruzi in the regulation of [Ca2+]i has been studied using fura 2-loaded epimastigotes or plasma membrane vesicles prepared from epimastigotes. Plasma membrane vesicles transport Ca2+ in the presence of Mg2+ and have a high affinity, vanadate-sensitive (Ca(2+)-Mg2+)-ATPase with an apparent Km for free Ca2+ of 0.3 microM.(ABSTRACT TRUNCATED AT 250 WORDS)