牵张作用对成骨样细胞MG63的线粒体跨膜电位的影响

目的:探讨在低渗透压形成的静牵张应力环境下线粒体跨膜电位与细胞增殖分化及凋亡的关系。方法:用成骨样细胞MG63细胞株进行体外培养、扩增,在对数生长期采用不同的低渗透压对细胞进行刺激,检测不同作用务件下线粒体跨膜电位(ΔΨm)、细胞增殖比例(S期百分比)以及凋亡指数。结果:240mOsm组ΔΨm呈上升趋势,4h达到峰值,6h逐渐下降,但仍高于对照组;163 mOsm组ΔΨm在6 h时明显降低。277和240 mOsm组S期百分比在6 h和8 h达到峰值(26.54±0.71,28.10±0.39:26.96±0.33,28.55±0.26)。三个实验组的凋亡峰均提前,且大于对照组,尤以163 mOsm组为最(54.87±0.78)。结论:成骨样细胞MG63ΔΨm的变化与时间和力学刺激强度有一定的依赖性,预示线粒体跨膜电位的变化与细胞增殖活性之间存在一定的关系。     

SIRT1 deficiency attenuates MPP+-induced apoptosis in dopaminergic cells

One of the functions mediated by sirtuin 1 (SIRT1), the NAD⁺-dependent protein deacetylase, has been suggested to be neuroprotective since resveratrol, a SIRT1 activator, inhibits 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced cytotoxicity. In this study, we show that SIRT1 siRNA transfection blocks MPP⁺-induced apoptosis in SH-SY5Y cells. The ratio of potential pro-apoptotic BNIP2 to antiapoptotic BCL-xL was attenuated in SIRT1-deficient cells following MPP⁺ treatment. In addition, BNIP2 shRNA-transfected cells showed reduced cleavage of PARP-1, while BNIP2 overexpression intensified the cleavage in MPP⁺-treated SH-SY5Y cells, suggesting that BNIP2 participates in the MPP⁺-induced apoptosis. Overall, these data imply that SIRT1 may mediate MPP⁺-induced cytotoxicity, possibly through the regulation of BNIP2.     

MPTP/MPP+ 诱导神经元凋亡的机制研究

1982年人们发现1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)能诱发PD,它的有效成分是1-甲基-4-苯基吡啶离子(MPP+)。目前,MPTP/MPP+广泛的被用作诱导PD实验模型的有效药物,可诱导神经元细胞发生凋亡性死亡。MPTP/MPP+诱导细胞凋亡的机制牵涉Bcl-2、p53、caspase家族、JNK通路、ERK通路和PARP等多种机制,它们共同参与了MPTP/MPP+诱导的细胞凋亡的调控和执行阶段。本文主要综述MPTP/MPP+诱导的神经元细胞凋亡机制。     

Early Sr2+-induced effects on membrane potential,proton pumping- and ATP hydrolysis-activities of plasma membrane vesicles from maize root cells

When released in plant environment, strontium (Sr²⁺) can be absorbed predominantly by the plant roots. As the plasma membrane of root cells is amongst the first barriers encountered by Sr²⁺ during its soil/plant transfer and the main entry point of Sr²⁺ into the roots, the main objective of this work aimed to enlighten on some of the Sr²⁺-induced effects at this level in Zea mays L. cv. “Liberal”.Thus this study focused on the Sr²⁺-induced changes on membrane potential of cortical root cells and on proton fluxes in maize roots, in order to determine whether the activity of some of the ion transport systems present in the plasma membrane of maize root cell could be among the first targets of Sr²⁺. We focused in particular on the plasma membrane H⁺-ATPase, known to be one of the major transport systems found in the plasmalemma where it generates a proton motive force (contributing to membrane potential maintaining, and providing energy for ion transport through membrane).The data presented here showed that Sr²⁺ triggered an early and transient membrane depolarisation whose magnitude and duration were dependent on the Sr²⁺-concentration. The time course pattern of a second longer lasting depolarisation could be examined in perspective with the Sr²⁺-induced decrease of the spontaneous proton extrusion observed in root tissues, suggesting a relationship between Sr²⁺-effects on membrane potential and H⁺ excretion. Furthermore, the inhibitory effect exerted by Sr²⁺ on the fusicoccin (FC)-enhanced proton extrusion strongly suggested an inhibition of the plasma membrane H⁺-ATPase. This hypothesis was supported by the inhibition induced by Sr²⁺ on proton pumping- and ATP hydrolysis-activities measured in plasma membrane vesicles (PMV) prepared from maize roots.Taken together the data reported here evidence that, with however a lower efficiency, Sr²⁺ behaved in a quite similar way to Ca²⁺ when inhibiting the H⁺-ATPase activity, and suggest that Sr²⁺ could partially mimic Ca²⁺ onto regulation of the H⁺-ATPase activity.     

Dimer ribbons of ATP synthase shape the inner mitochondrial membrane

ATP synthase converts the electrochemical potential at the inner mitochondrial membrane into chemical energy, producing the ATP that powers the cell. Using electron cryo-tomography we show that the ATP synthase of mammalian mitochondria is arranged in long approximately 1-microm rows of dimeric supercomplexes, located at the apex of cristae membranes. The dimer ribbons enforce a strong local curvature on the membrane with a 17-nm outer radius. Calculations of the electrostatic field strength indicate a significant increase in charge density, and thus in the local pH gradient of approximately 0.5 units in regions of high membrane curvature. We conclude that the mitochondrial cristae act as proton traps, and that the proton sink of the ATP synthase at the apex of the compartment favours effective ATP synthesis under proton-limited conditions. We propose that the mitochondrial ATP synthase organises itself into dimer ribbons to optimise its own performance.     

Effects of MPTP, MPP+ and paraquat on mitochondrial potential and oxidative stress

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridinium (MPP+) and 1,1-dimethyl-4,4-bipyridinium (paraquat) upon the electrical potential across the plasma and mitochondrial membranes within synaptosomes has been investigated. MPTP selectively depressed plasma membrane potential while MPP+ specifically reduced mitochondrial potential. The structurally similar compound paraquat had no effect on either membrane potential. Enhancement of the lipid peroxidative activity with an Fe-ADP complex depressed both potentials. Paraquat effected increased peroxidative activity in brain homogenates that was less pronounced than that due to Fe-ADP. MPTP reduced basal but stimulated Fe-ADP enhanced peroxidation. The mechanisms underlying the toxicity of MPP+ are likely to differ from those of paraquat, primarily involving impaired mitochondrial function rather than increased oxidative stress.     

Acetylsalicylic acid and acetaminophen protect against MPP+-induced mitochondrial damage and superoxide anion generation

The effects of 1-methyl-4-phenylpyridinium (MPP+) has been extensively researched due to its selective toxicity to dopaminergic neurons. Mitochondrial dysfunction which is common in the etiology of Parkinson's disease (PD), has been widely implicated in MPP+-induced toxicity. MPP+-induced mitochondrial dysfunction is believed to result in the generation of free radicals. This study was therefore performed to assess the effect of MPP+ on mitochondrial function and the ability of MPP+ to generate superoxide free radicals. Furthermore, we assessed the ability of the non-narcotic analgesics, acetaminophen and acetylsalicylic acid to prevent any diliterious effects of the potent neurotoxin, MPP+, on mitochondrial function and superoxide anion generation, in vivo. Acetylsalicylic acid and acetaminophen prevented the MPP+-induced inhibition of the electron transport chain and complex I activity. In addition, acetylsalicylic acid and acetaminophen significantly attenuated the MPP+-induced superoxide anion generation. Furthermore the results provide novel data explaining the ability of these agents to prevent MPP+-induced mitochondrial dysfunction and subsequent reactive oxygen species generation. While these findings suggest the usefulness of non-narcotic analgesics in neuroprotective therapy in neurodegenerative diseases, acetylsalicylic acid appears to be a potential candidate in prophylactic as well as in adjuvant therapy in Parkinson's disease.     

Mechanism of ATP synthesis by mitochondrial ATP synthase from beef heart

Previous studies of the rate constants for the elementary steps of ATP hydrolysis by the soluble and membrane-bound forms of beef heart mitochondrial F₁ supported the proposal that ATP is formed in high-affinity catalytic sites of the enzyme with little or no change in free energy and that the major requirement for energy in oxidative phosphorylation is for the release of product ATP.The affinity of the membrane-bound enzyme for ATP during NADH oxidation was calculated from the ratio of the rate constants for the forward binding step (k ₊₁) and the reverse dissociation step (k _–1).k _–1 was accelerated several orders of magnitude by NADH oxidation. In the presence of NADH and ADP an additional enhancement ofk _–1 was observed. These energy-dependent dissociations of ATP were sensitive to the uncoupler FCCP.k ₊₁ was affected little by NADH oxidation. The dissociation constant (K _d ATP) increased many orders of magnitude during the transition from nonenergized to energized states.     

MPTP, MPP+ and mitochondrial function

1-Methyl-4-phenylpyridinium (MPP+), the putative toxic metabolite of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), inhibited NAD(H)-linked mitochondrial oxidation at the level of Complex I of the electron transport system. MPTP and MPP+ inhibited aerobic glycolysis in mouse striatal slices, as measured by increased lactate production; MPTP-induced effects were prevented by inhibition of monoamine oxidase B activity. Several neurotoxic analogs of MPTP also form pyridinium metabolites via MAO; these MPP+ analogs were all inhibitors of NAD(H)-linked oxidation by isolated mitochondria. 2'-Methyl-MPTP, a more potent neurotoxin in mice than MPTP, was also more potent than MPTP in inducing lactate accumulation in mouse brain striatal slices. Overall, the studies support the hypothesis that compromise of mitochondrial oxidative capacity is an important factor in the mechanisms underlying the toxicity of MPTP and similar compounds.     

The spatio-temporal organization of mitochondrial F1FO ATP synthase in cristae depends on its activity mode

F₁F_O ATP synthase, also known as complex V, is a key enzyme of mitochondrial energy metabolism that can synthesize and hydrolyze ATP. It is not known whether the ATP synthase and ATPase function are correlated with a different spatio-temporal organisation of the enzyme. In order to analyze this, we tracked and localized single ATP synthase molecules in situ using live cell microscopy. Under normal conditions, complex V was mainly restricted to cristae indicated by orthogonal trajectories along the cristae membranes. In addition confined trajectories that are quasi immobile exist. By inhibiting glycolysis with 2-DG, the activity and mobility of complex V was altered. The distinct cristae-related orthogonal trajectories of complex V were obliterated. Moreover, a mobile subpopulation of complex V was found in the inner boundary membrane. The observed changes in the ratio of dimeric/monomeric complex V, respectively less mobile/more mobile complex V and its activity changes were reversible. In IF1-KO cells, in which ATP hydrolysis is not inhibited by IF1, complex V was more mobile, while inhibition of ATP hydrolysis by BMS-199264 reduced the mobility of complex V. Taken together, these data support the existence of different subpopulations of complex V, ATP synthase and ATP hydrolase, the latter with higher mobility and probably not prevailing at the cristae edges. Obviously, complex V reacts quickly and reversibly to metabolic conditions, not only by functional, but also by spatial and structural reorganization.     

Reduced mitochondrial membrane potential and altered responsiveness of a mitochondrial membrane megachannel in p53-induced senescence.

There is accumulating evidence that mitochondrial membrane potential (DeltaPsi(M)) is reduced in aged cells. In addition, a decrease of DeltaPsi(M) has been shown to be an early event in many forms of apoptosis. Here we use a mitochondrial potentiometric dye with in situ laser scanning confocal microscopic (LSCM) imaging to demonstrate that DeltaPsi(M) is dramatically decreased in both the p53-overexpressing, senescent EJ tumor cells and in pre-apoptotic PC12 cells compared to controls. Treatment with cyclosporin A (CSA), which facilitates closure of the mitochondrial permeability transition pore (PTP), was able to reverse the decrease in DeltaPsi(M) in pre-apoptotic PC12 cells but not in the senescent EJ-p53 cells. The capacity to prevent dissipation of DeltaPsi(M) in response to agents that facilitate PTP closure may differentiate cells entering apoptosis from those participating in senescence. Therefore, regulation of the closure of the mitochondrial PTP in the presence of decreased DeltaPsi(M) may be a decisional checkpoint in distinguishing between growth arrest pathways.     

Synthesis and hydrolysis of ATP by the mitochondrial ATP synthase

A brief summary of the factors that control synthesis and hydrolysis of ATP by the mitochondrial H+-ATP synthase is made. Particular emphasis is placed on the role of the natural ATPase inhibitor protein. It is clear from the existing data obtained with a number of agents that there is no correlation between variations of the rate of ATP hydrolysis and ATP synthesis as driven by respiration. The mechanism by which each condition differentially affects the two activities is not entirely known. For the case of the natural ATPase inhibitor protein, it appears that the protein controls the kinetics of the enzyme. This control seems essential for achieving maximal accumulation of ATP during electron transport in systems that contain relatively high concentrations of ATP.     

Characterization of the mitochondrial ATP synthase from yeast Saccharomyces cerevisae

The mitochondrial ATP synthase from yeast S. cerevisiae has been genetically modified, purified in a functional form, and characterized with regard to lipid requirement, compatibility with a variety of detergents, and the steric limit with rotation of the central stalk has been assessed. The ATP synthase has been modified on the N-terminus of the β-subunit to include a His(6) tag for Ni-chelate affinity purification. The enzyme is purified by a two-step procedure from submitochondrial particles and the resulting enzyme demonstrates lipid dependent oligomycin sensitive ATPase activity of 50 units/mg. The yeast ATP synthase shows a strong lipid selectivity, with cardiolipin (CL) being the most effective activating lipid and there are 30 moles CL bound per mole enzyme at saturation. Green Fluorescent Protein (GFP) has also been fused to the C-terminus of the ε-subunit to create a steric block for rotation of the central stalk. The ε-GFP fusion peptide is imported into the mitochondrion, assembled with the ATP synthase, and inhibits ATP synthetic and hydrolytic activity of the enzyme. F(1)F(o) ATP synthase with ε-GFP was purified to homogeneity and serves as an excellent enzyme for two- and three-dimensional crystallization studies.