Inhibition of plasma membrane and mitochondrial transmembrane potentials by ethanol

The actions of ethanol and its primary oxidative metabolite, acetaldehyde, on plasma membrane and mitochondrial transmembrane potentials were examined in rat brain using fluorescence techniques. Subchronic treatment of adult rats with ethanol resulted in a significant depolarization of both the plasma and mitochondrial membranes when the mean blood ethanol level of the rats was 59±11 mM (mean±SEM, n=6). Acute dosing of animals (4.5 g/kg, i.p.) failed to show any significant alterations. Various concentrations of ethanol, added in vitro to a crude synaptosomal preparation isolated from the rat cerebrocortex (P₂) from untreated animals, depolarized both the plasma and mitochondrial transmembrane potentials in a dose-related manner. Addition of acetaldehyde in vitro did not reveal any significant effects on plasma or mitochondrial transmembrane potential.     

Nanosecond electric pulses cause mitochondrial membrane permeabilization in Jurkat cells

Nanosecond, high‐voltage electric pulses (nsEP) induce permeabilization of the plasma membrane and the membranes of cell organelles, leading to various responses in cells including cytochrome c release from mitochondria and caspase activation associated with apoptosis. We report here evidence for nsEP‐induced permeabilization of mitochondrial membranes in living cells. Using three different methods with fluorescence indicators—rhodamine 123 (R123), tetramethyl rhodamine ethyl ester (TMRE), and cobalt‐quenched calcein—we have shown that multiple nsEP (five pulses or more, 4 ns duration, 10 MV/m, 1 kHz repetition rate) cause an increase of the inner mitochondrial membrane permeability and an associated loss of mitochondrial membrane potential. These effects could be a consequence of nsEP permeabilization of the inner mitochondrial membrane or the activation of mitochondrial membrane permeability transition pores. Plasma membrane permeabilization (YO‐PRO‐1 influx) was detected in addition to mitochondrial membrane permeabilization. Bioelectromagnetics 33:257–264, 2012. © 2011 Wiley Periodicals, Inc.     

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。     

Effect of temperature on plasma membrane and tonoplast ion channels in Arabidopsis thaliana

The temperature dependence of the activity of ion channels was investigated, by means of the patch-clamp technique in the 'whole-cell' configuration, using protoplasts and vacuoles isolated form Arabidopsis thaliana L. cultured cells. The effect of temperature changes in the range 11–22°C was tested on the hyperpolarization and depolarization-activated K⁺ currents in the plasma membrane and on the hyperpolarization-activated K⁻ currents in the tonoplast (vacuolar membrane). All 3 kinds of currents were unaffected by increasing temperature up to 15°C and were activated between 15 and 20°C.     

Permeability change in transformed mouse fibroblasts caused by ionophores,and its relationship to membrane permeabilization by exogenous ATP

Summary Electrogenic ionophores have been found to induce membrane permeabilization in Swiss mouse 3T3 cells that had undergone spontaneous transformation (3T6 cells). Cells attached to plastic dishes were loaded with [³H] uridine, and then the medium was replaced by buffered salt solution at pH 7.8. The enhancement of membrane permeability was assayed by following the efflux of uridine nucleotides, normally impermeant substances. Titration with electrogenic ionophores, such as carbonylcyanidem-chlorophenylhydrazone (CCCP), SF-6847 and gramicidin D, markedly increased the membrane permeability within a very narrow range of ionophore concentration. Nonelectrogenic ionophores, such as monensin and nigericin, did not affect membrane permeability. Measurements of the distribution of the lipophilic cation tetraphenylphosphonium (TPP⁺) between the cells and their environment implied that the remarkable increase in permeability took place within a narrow range of membrane potential (). The data could be explaine by a threshold value, under which aqueous channels are opened in the plasma membrane. The effects exerted by electrogenic ionophores on the plasma membrane were found to be similar to those induced by exogenous ATP. In both cases rapid efflux of K⁺, influx of Na⁺ and reduction of preceded membrane permeabilization to low molecular weight, charged molecules, such as nucleotides. It is suggested that dissipation of induces conformational alterations in membranal components, and/or topological changes, such as aggregation of protein molecules, to form membranal aqueous channels. Electrogenic ionophores permeabilize both normal (3T3) and transformed (3T6) mouse fibroblasts, whereas ATP effects are specific for transformed cells. Thus, it is postulated that ATP actsvia specific sites on the surface of transformed cells.     

Transglutaminase overexpression sensitizes neuronal cell lines to apoptosis by increasing mitochondrial membrane potential and cellular oxidative stress

'Tissue' transglutaminase (tTG) selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Considering the central role played by mitochondria in apoptosis, we investigated the relationships existing amongst tTG expression, apoptosis and mitochondrial function. To this aim we studied the mechanisms of apoptosis in a neuronal cell line (SK-N-BE (2)) in which the tTG-expression was driven by a constitutive promoter. Furthermore, a tet-off inducible promoter was also used in 3T3 fibroblastic cells used as control. Both cell lines, when expressing tTG, appeared 'sensitized' to apoptosis. Strikingly, we found major differences in the morphological features of mitochondria among cell lines in the absence of apoptotic stimuli. In addition, these ultrastructural characteristics were associated with specific functional features: (i) constitutively hyperpolarized mitochondria and (ii) increased reactive oxygen intermediates production. Importantly, after mitochondrial-mediated apoptosis by staurosporine, a rapid loss of mitochondrial membrane potential was found in tTG cells only. Taken together, these results seem to suggest that, via hyperpolarization, tTG might act as a 'sensitizer' towards apoptotic stimuli specifically targeted to mitochondria. These results could also be of pathogenetic relevance for those diseases that are characterized by increased tTG and apoptotic rate together with impaired mitochondrial function, e.g. in some neurodegenerative disease.     

Nitric oxide inhibits mitochondrial movement in forebrain neurons associated with disruption of mitochondrial membrane potential

Nitric oxide (NO) has a number of physiological and pathophysiological effects in the nervous system. One target of NO is the mitochondrion, where it inhibits respiration and ATP synthesis, which may contribute to NO-mediated neuronal injury. Our recent studies suggested that impaired mitochondrial function impairs mitochondrial trafficking, which could also contribute to neuronal injury. Here, we studied the effects of NO on mitochondrial movement and morphology in primary cultures of forebrain neurons using a mitochondrially targeted enhanced yellow fluorescent protein. NO produced by two NO donors, papa non-oate and diethylamine/NO complex, caused a rapid cessation of mitochondrial movement but did not alter morphology. Movement recovered after removal of NO. The effects of NO on movement were associated with dissipation of the mitochondrial membrane potential. Increasing cGMP levels using 8-bromoguanosine 3',5'-cyclic monophosphate, did not mimic the effects on mitochondrial movement. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of NO-induced activation of soluble guanylate cyclase, did not block the effects of NO. Thus, neither increasing nor decreasing cGMP levels had an effect on mitochondrial movement. Based on these data, we conclude that NO is a novel modulator of mitochondrial trafficking in neurons, which may act through the inhibition of mitochondrial function.     

Purification and characterization of the voltage-dependent anion channel from the outer mitochondrial membrane of yeast

Summary The outer mitochondrial membranes of all organisms so far examined contain a protein which forms voltage-dependent anion selective channels (VDAC) when incorporated into planar phospholipid membranes. Previous reports have suggested that the yeast (Saccharomyces cerevisiae) outer mitochondrial membrane component responsible for channel formation is a protein of 29,000 daltons which is also the major component of this membrane. In this report, we describe the purification of this 29,000-dalton protein to virtual homogeneity from yeast outer mitochondrial membranes. The purified protein readily incorporates into planar phospholipid membranes to produce ionic channels. Electrophysiological characterization of these channels has demonstrated they have a size, selectivity and voltage dependence similar to VDAC from other organisms. Biochemically, the purified protein has been characterized by determining its amino acid composition and isoelectric point (pI). In addition, we have shown that the purified protein, when reconstituted into liposomes, can bind hexokinase in a glucose-6-phosphate dependent manner, as has been shown for VDAC purified from other sources. Since physiological characterization suggests that the functional parameters of this protein have been conserved, antibodies specific to yeast VDAC have been used to assess antigenic conservation among mitochondrial proteins from a wide number of species. These experiments have shown that yeast VDAC antibodies will recognize single mitochondrial proteins fromDrosophila, Dictyostelium andNeurospora of the appropriate molecular weight to be VDAC from these organisms. No reaction was seen to any mitochondrial protein from rat liver, rainbow trout,Paramecium, or mung bean. In addition, yeast VDAC antibodies will recognize a 50-kDa mol wt protein present in tobacco chloroplasts. These results suggest that there is some antigenic as well as functional conservation among different VDACs.     

线粒体膜电位与皮质酮对原代培养海马细胞的毒性作用

采用MTT法和激光共聚焦显微术观察皮质酮对原代培养海马神经细胞的存活率及其线粒体膜电位的影响。结果表明,在低糖、无血清培养条件下,皮质酮可剂量依赖地降低海马神经元及神经胶质细胞的存活率,在同等剂量下以神经元损伤更为显著。给予高浓度葡萄糖（25mmol/L）可明显拮抗皮质酮对海马神经元的毒性作用。进一步研究表明,皮质酮（10^-6-10^-5mol/L）可引起海马神经元线粒体膜电位明显下降,此作用亦可被高浓度葡萄糖所对抗。结果提示,在相同处理因素条件下,皮质酮以损伤神经元为主。皮质酮可降低海马神经元的存活率及线粒体膜电位,给予高浓度葡萄糖具有明显的改善作用。线粒体膜电位的下降可能是皮质酮引起神经元损伤的机制之一。     

Ethyl-nitrosourea transformed astrocytes exhibit mitochondrial membrane hyperpolarization and constrained apoptosis

Recent studies have shown the mitochondria and mitochondrial DNA are altered in gliomas, studied either as primary tissues or in culture. Few studies have been performed which evaluate the mitochondria during the development of glial malignancy. We used an ethyl-nitrosourea (ENU) in vitro model to assess the changes in mitochondrial parameters with progression to astrocyte transformation. When compared to the untreated control cells mitochondrial mass of the ENU treated cells significantly decreased ontologically early with concurrent increase in mitochondrial membrane potential, resulting in hyperpolarization of the mitochondrial membrane. At successive divisions, the degree of spontaneous apoptosis during astrocyte transformation was significantly diminished in the ENU treated cells. With 24 h pre- and co-treatment of ENU cells with citrate, an allosteric inhibitor of phosphofructokinase, the astrocytes still became immortal, but did not manifest any of the mitochondrial changes nor acquire the transformed properties of the ENU treated cells without the inhibition. Indeed, the degree of apoptosis noted in these dually treated cells was increased, associated with a loss of anchorage independence and low density growth. Transformed subclones exposed to citrate after the development of malignant properties also exhibited increased apoptosis, and did not form colonies in low density plating conditions. These results suggest that the development of transformed properties in an ENU model is associated with marked hyperpolarization of mitochondrial membrane potential and diminished spontaneous apoptosis. Exposure to citrate attenuated these mitochondrial changes and in vitro growth properties, with increases in apoptosis. The development of transformed astrocytes involve constraints on apoptosis related to alterations in mitochondrial membrane potential and mass.     

In vitro effect of pulsed 900 MHz GSM radiation on mitochondrial membrane potential and motility of human spermatozoa

Ejaculated, density purified, human spermatozoa were exposed to pulsed 900 MHz GSM mobile phone radiation at two specific absorption rate levels (SAR 2.0 and 5.7 W/kg) and compared with controls over time. Change in sperm mitochondrial membrane potential was analysed using flow cytometry. Sperm motility was determined by computer assisted sperm analysis (CASA). There was no effect of pulsed 900 MHz GSM radiation on mitochondrial membrane potential. This was also the case for all kinematic parameters assessed at a SAR of 2.0 W/kg. However, over time, the two kinematic parameters straight line velocity (VSL) and beat-cross frequency (BCF) were significantly impaired (P < 0.05) after the exposure at SAR 5.7 W/kg and no exposure by time interaction was present. This result should not be ascribed to thermal effects, due to the cooling methods employed in the RF chamber and temperature control within the incubator.     

Cerium oxide nanoparticles prevent apoptosis in primary cortical culture by stabilizing mitochondrial membrane potential

Abstract

Cerium oxide nanoparticles (CNPs) of spherical shape have unique antioxidant capacity primarily due to alternating + 3 and + 4 oxidation states and crystal defects. Several studies revealed the protective efficacies of CNPs in cells and tissues against the oxidative damage. However, its effect on mitochondrial functioning, downstream effectors of radical burst and apoptosis remains unknown. In this study, we investigated whether CNPs treatment could protect the primary cortical cells from loss of mitochondrial membrane potential (Δψ_m) and Δψ_m-dependent cell death. CNPs with spherical morphology and size range 7–10 nm were synthesized and utilized at a concentration of 25 nM on primary neuronal culture challenged with 50 μM of hydrogen peroxide (H₂O₂). We showed that optimal dose of CNPs minimized ROS content of the cells and also curbed related surge in cellular calcium flux. Importantly, CNPs treatment prevented apoptotic loss of cell viability. Reduction in the apoptosis could be successfully attributed to the maintenance of Δψ_m and restoration of major redox equivalents NADH/NAD⁺ ratio and cellular ATP. These findings, therefore, suggest possible route of CNPs protective efficacies in primary cortical culture.     

Identification of anion and cation pathways in the inner mitochondrial membrane by patch clamping of mouse liver mitoplasts

Summary Alkalinization of the matrix side of the mitochondrial inner membrane by pH shifts from 6.8 to 8.3 caused a reversible increase in current of 3.2±0.2 pA (mean±se,n=21) at±40 mV measured using patch-clamp techniques. The current increase was reversed in a graded fashion by the addition of Mg²⁺ in 0.15m KCl corresponds to approximately 15 pS. Reversal potentials derived from whole patch currents indicated that the inner mitochondrial membrane was primarily cation selective at pH 6.8 with aP _k/P _Cl=32 (n=6). Treatment with alkaline pH (8.3) increased the current and anion permeability (P _K/P _Cl=16,n=6). The membrane becomes completely cation selective when low concentrations (12 m) of the drug propranolol are added. The amphiphilic drugs amiodarone (4 m), propranolol (70 m) and quinine (0.6mm) blocked almost all of the current. The pH-dependent current was also inhibited by tributyltin. These results are consistent with the presence of two pathways in the inner mitochondrial membrane. One is cation selective and generally open and the other is anion selective and induced by alkaline pH. The alkaline pH-activated channel likely corresponds to the inner membrane anion channel postulated by others from suspension studies.     

Modulation of mitochondrial membrane potential and reactive oxygen species production by copper in astrocytes

In monolayers of cultured rat astrocytes a number of agents that induce oxidative stress act synergistically with exposure to copper leading to rapid depolarization of the mitochondrial membrane potential (Psi m) and increased reactive oxygen species (ROS) production. Copper sensitized astrocytes to the action of menadione, an intracellular generator of superoxide anion radical, exogenous hydrogen peroxide (H2O2) and rotenone, an inhibitor of mitochondrial electron transport chain complex I. However, significant differences were observed in the ability to modulate the copper-enhanced oxidative stress depending on which stressor was used. The inhibitor of mitochondrial permeability transition cyclosporin A attenuated the effect of copper and rotenone, but had no protective action in the case of H2O2/copper and menadione/copper combinations. The H2O2 scavenger pyruvate was effective at protecting mitochondria against damage associated with the combined exposure to H2O2/copper and menadione/copper but not to the rotenone/copper combination. The antioxidant Trolox was ineffective at protecting against any of these actions and indeed had a damaging effect when combined with copper. The membrane-permeable copper chelator neocuproine combined with sensitizing concentrations of menadione caused a decrease in Psi m, mimicking the action of copper. Penicillamine, a membrane-impermeable copper chelator, was effective at reducing copper sensitization. Endogenous copper, mobilized during periods of oxidative stress, may play a role in the pathophysiology of brain injury. Our results suggest that this might be particularly dangerous in dysfunctional conditions in which the mitochondrial electron transport chain is compromised.     

Effects of phenothiazines on inhibition of plasma membrane ATPase and hyperpolarization of cell membranes in the yeast Saccharomyces cerevisiae

The transmembranal potential, in Saccharomyces cerevisiae, has been calculated from the distribution of the lipophilic cation tetraphenylphosphonium (TPP⁺) between the intracellular and extracellular water. Trifluoperazine at concentrations of 10 to 50 μM, caused a substantial increase in the membrane potential (negative inside). This increase was observed only in the presence of a metabolic substrate and was eliminated by the addition of the protonophores 2,4-dinitrophenol and sodium azide, removal of glucose, replacement of glucose by the nonmetabolizable analog 3-O-methyl glucose, or by the addition of 100mM KCl. An increase in ⁴⁵CaCl₂ accumulation from solutions of low concentrations (1 μM) was observed under all conditions where membrane potential was increased. Proton ejection activity was monitored by measurements of the rates of the decrease in the pH of unbuffered cell suspensions in the presence of glucose. Trifluoperazine inhibited the changes in medium pH; this inhibition was not the result of an increase in the permeability of cell membranes to protons since in the absence of glucose, trifluoperazine did not cause a change in the rate of pH change generated by proton influx. The activity of plasma membrane ATPase was measured in crude membrane preparations in the presence of sodium azide to inhibit mitochondrial ATPase. Trifluoperazine strongly inhibited the activity of the plasma membrane ATPase. The effect of phenothiazines on transport and on membrane potential reported in this study and in the previous one (Eilam, Y. (1983) Biochim. Biophys. Acta 733, 242–248) were observed only in the presence of a metabolic substrate. The possibility that energy is required for the uptake of phenothiazines into the cells was eliminated by results showing energy-independent uptake of [³H]chlorpromazine. The results strongly suggest that phenothiazines activate energy-dependent K⁺-extrusion pumps, which lead to increased membrane potential. Increased influx of calcium seems to be energized by membrane potential, and therefore stimulated under all conditions where membrane potential is increased. The analog which does not bind to calmodulin, trifluoperazine sulfoxide, had no effect on the cells, but the involvement of calmodulin in the processes altered by trifluoperazine cannot as yet, be determined.     

Steady-state and transient membrane potentials in human red cells determined by protonophore-mediated pH changes

Summary Protonophores have been used frequently to determine changes in membrane potential in suspensions of red cells, since such changes are reflected by changes in extracellular pH, due to proton and consequently protonophore reequilibration.In a previous paper (Bennekou, P. 1988.J. Membrane Biol. 102:225–234) a kinetic model for the translocation of a protonophore, CCCP, across the human red cell membrane was established. This model accounts for the protonophore reequilibration following abrupt changes in membrane potential.In this paper, the limitations of the method with regard to the estimation of transient membrane potentials are examined, using the transport model to simulate changes in extracellular pH in response to noninstantaneous changes in membrane potential. The temperature and time resolution calculated from the model are reported.Furthermore, it is shown that the transport model established for CCCP is valid for another protonophore, TCS, thus indicating the general validity of the transport scheme for the entire class of protonophores.     

VDAC electronics: 2. A new,anaerobic mechanism of generation of the membrane potentials in mitochondria

Mitochondrial hexokinase (HK) and creatine kinase (CK) known to form complexes with a voltage dependent anion channel (VDAC) have been reported to increase cell death resistance under hypoxia/anoxia. In this work we propose a new, non-Mitchell mechanism of generation of the inner and outer membrane potentials at anaerobic conditions. The driving force is provided by the Gibbs free energy of the HK and CK reactions associated with the VDAC–HK and the ANT (adenine nucleotide translocator)–CK–VDAC complexes, respectively, both functioning as voltage generators. In the absence of oxygen, the cytosolic creatine phosphate can be directly used by the ANT–CK–VDAC contact sites to produce ATP from ADP in the mitochondrial matrix. After that, ATP released through the fraction of unbound ANTs in exchange for ADP is used in the mitochondrial intermembrane space by the outer membrane VDAC–HK electrogenic complexes to convert cytosolic glucose into glucose-6-phosphate. A simple computational model based on the application of Ohm's law to an equivalent electrical circuit showed a possibility of generation of the inner membrane potential up to − 160 mV, under certain conditions, and of relatively high outer membrane potential without wasting of ATP that normally leads to cell death. The calculated membrane potentials depended on the restriction of ATP/ADP diffusion in narrow cristae and through the cristae junctions. We suggest that high inner membrane potential and calcium extrusion from the mitochondrial intermembrane space by generated positive outer membrane potential prevent mitochondrial permeability transition, thus allowing the maintenance of mitochondrial integrity and cell survival in the absence of oxygen.     

以人皮肤成纤维细胞作为饲养层细胞培养人胚胎干细胞

目的：比较人皮肤成纤维细胞（humandermalfibroblasts,HDFs）与小鼠胚胎成纤维细胞（Mouseembryonicfibroblasts,MEFs）的增殖能力及研究人皮肤成纤维细胞作为饲养层支持人胚胎干细胞（humanembryonicstemcells,hESCs）未分化生长的能力。方法：利用组织贴壁法从人皮肤中分离出HDFs,通过细胞形态的观察和生长曲线的绘制比较HDFs与MEFs的体外增殖能力。将HDFs作为饲养层细胞与hESCs共培养,传代12代后,检测hESCs碱性磷酸酶（AKP）、表面特异性标志及胚胎干细胞特异性转录因子。结果：HDFs可连续传代培养15代以上,10代以下的HDFs增殖迅速,而MEFs自第4代起,增殖能力就明显下降;hESCs在HDFs饲养层上可传代培养12代以上,克隆边界清晰,细胞排列紧密,碱性磷酸酶、表面标志物检测均呈阳性,表达了hESCs特异性转录因子。结论：HDFs比MEFs具有更强的增殖能力;HDFs可作为培养hEscs的饲养层细胞。