Tetramethylrhodamine Methyl Ester Research Articles

Ischemia‐induced mobilization of lysosomal iron predisposes rat hepatocytes to ischemia‐reperfusion (IR) injury

Reactive oxygen species (ROS) initiate onset of the mitochondrial permeability transition (MPT) and play a key role in IR injury. Iron is a critical catalyst for ROS generation. Accordingly, our aim was to investigate the role of chelatable iron in ROS‐dependent MPT formation and cell death during IR to rat hepatocytes. Cells were incubated anoxically at pH 6.2 for 4 h and re‐oxygenated at pH 7.4 to simulate IR. Chelatable iron, ROS, mitochondrial membrane potential and cell death were monitored by confocal imaging of calcein, chloromethyl dichlorofluorescein, tetramethylrhodamine methyl ester and propidium iodide, respectively. Ischemia caused progressive quenching of cytosolic calcein, indicating an increase of chelatable Fe2+, which was suppressed by desferal and starch‐desferal pretreatment. Quenching of calcein was also blocked by ebselen and ferristatin, inhibitors of lysosomal divalent metal transporter‐1. Ischemia induced quenching of calcein loaded into mitochondria, which was blocked by desferal, starch‐desferal and Ru360. These agents also decreased mitochondrial ROS formation, MPT onset and cell killing after reperfusion. In conclusion, lysosomal iron is mobilized into mitochondria during ischemia. Increased mitochondrial iron then predisposes cells for ROS‐mediated MPT opening and cell killing after reperfusion. (Supported by NIH grants DK073336 and DK37034)

Fluorescence response of human HER2+ cancer- and MCF-12F normal cells to 200 MHz ultrasound microbeam stimulation: A preliminary study of membrane permeability variation

Targeted mechanical cell stimulation has been extensively studied for a better understanding of its effect on cellular mechanotransduction signaling pathways and structures by utilizing a variety of mechanical sources. In this work, an ultrasound-driven single cell stimulation method is thus proposed, and a preliminary study is carried out by comparing the fluorescence intensities representing a change in cell membrane permeability between MDA-MB-435 human HER2+ cancer cells (∼40–50μm in diameter) and MCF-12F normal cells (∼50–60μm) in the presence of ultrasound. A 200MHz single element zinc oxide (ZnO) transducer is employed to generate ultrasound microbeam (UM) whose beamwidth and depth of focus are 9.5 and 60μm, comparable to typical cell size. The cells in tetramethyl rhodamine methyl ester (TMRM) are interrogated with 200MHz sinusoidal bursts. The number of cycles per burst is 5 and the pulse repetition frequency (PRF) is 1kHz. The temporal variation of fluorescence intensity in each cell is measured as a function of input voltage to the transducer (16, 32, and 47V), and its corresponding fluorescence images are obtained via a confocal microscope. A systematic method for visualizing UM’s focus by adding Rhodamine B to the immersion medium is also proposed to enhance the precision in aiming the beam at an individual cell.Both types of cells exhibit a decrease in the intensity upon UM irradiation. In particular, normal cells show more fluorescence reduction (down to 0.7 in normalized intensity) than cancer cells (∼0.9) under the same excitation condition of the transducer. With UM being turned off, the normalized intensity level in normal cells is slowly increased to 1.1. The cell images taken before and after UM exposure indicate that the intensity reduction is more pronounced in those cells after exposure. Hence the results show the potential of UM as a non-invasive in vitro stimulation tool for facilitating targeted drug delivery and gene transfection as well as for studying cellular mechanotransduction.

Metabolic consequences of NDUFS4 gene deletion in immortalized mouse embryonic fibroblasts

Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activities and normal citrate synthase and CV (FoF1-ATPase) activity. Native electrophoresis of KO cell mitochondrial fractions revealed two distinct CI subcomplexes of ~830kDa (enzymatically inactive) and ~200kDa (active). The level of fully-assembled CII–CV was not affected by NDUFS4 gene deletion. KO cells exhibited a moderately reduced maximal and routine O2 consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The aberrant CI assembly and reduced O2 consumption in KO cells were fully normalized by NDUFS4 gene complementation. Cellular [NAD+]/[NADH] ratio, lactate production and mitochondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP+]/[NADPH] ratio, cellular glucose consumption, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen species (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

High-Content Imaging Technology for the Evaluation of Drug-Induced Steatosis Using a Multiparametric Cell-Based Assay

In the present study, we developed a cell-based protocol for the identification of drugs able to induce steatosis. The assay measures multiple markers of toxicity in a 96-well plate format using high-content screening (HCS) technology. After treating HepG2 cells with increasing concentrations of the tested compounds, toxicity parameters were analyzed using fluorescent probes: BODIPY493/503 (lipid content), 2′,7′-dihydrodichlorofluorescein diacetate (reactive oxygen species [ROS] generation), tetramethyl rhodamine methyl ester (mitochondrial membrane potential), propidium iodide (cell viability), and Hoechst 33342 (nuclei staining). A total of 16 drugs previously reported to induce liver steatosis through different mechanisms (positive controls) and six nonsteatotic compounds (negative controls) were included in the study. All the steatosis-positive compounds significantly increased BODIPY493/503 fluorescence in HepG2 cells, whereas none of the negative controls induced lipid accumulation. In addition to effects on fat levels, increased ROS generation was produced by certain compounds, which could be indicative of increased risk of liver damage. Our results suggest that this in vitro approach is a simple, rapid, and sensitive screening tool for steatosis-inducing drugs. This conclusion should be confirmed by testing a larger number of steatosis-positive and -negative inducers.

Pulsing of Membrane Potential in Individual Mitochondria: A Stress-Induced Mechanism to Regulate Respiratory Bioenergetics in Arabidopsis

Mitochondrial ATP synthesis is driven by a membrane potential across the inner mitochondrial membrane; this potential is generated by the proton-pumping electron transport chain. A balance between proton pumping and dissipation of the proton gradient by ATP-synthase is critical to avoid formation of excessive reactive oxygen species due to overreduction of the electron transport chain. Here, we report a mechanism that regulates bioenergetic balance in individual mitochondria: a transient partial depolarization of the inner membrane. Single mitochondria in living Arabidopsis thaliana root cells undergo sporadic rapid cycles of partial dissipation and restoration of membrane potential, as observed by real-time monitoring of the fluorescence of the lipophilic cationic dye tetramethyl rhodamine methyl ester. Pulsing is induced in tissues challenged by high temperature, H(2)O(2), or cadmium. Pulses were coincident with a pronounced transient alkalinization of the matrix and are therefore not caused by uncoupling protein or by the opening of a nonspecific channel, which would lead to matrix acidification. Instead, a pulse is the result of Ca(2+) influx, which was observed coincident with pulsing; moreover, inhibitors of calcium transport reduced pulsing. We propose a role for pulsing as a transient uncoupling mechanism to counteract mitochondrial dysfunction and reactive oxygen species production.

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The setup of an islet isolation facility designed along the rules of good manufacturing practice (GMP) is a technically challenging, cost and time intensive process.1 Consequently, several institutions have decided to perform transplantation of islets isolated at another center with an already standing expertise. Such a solution includes the necessity to transport the isolated islets from the isolation to the transplantation facility. In spite of its importance, an ideal solution for the transport of the isolated human islets has still not been established. Here, we present an islet transport device suited to transport human islet cells under reproducible conditions and minimized cell stress. The transport simulation of the human islets was performed in a transfused “rotary transport system for islets” termed “ROTi.” Besides measuring standard metabolic (LDH, lactate, glucose) and physical parameters (pH, dissolved oxygen and temperature), we used five different live stains in combination with real time live confocal microscopy to document islet quality parameters. As live stains we added tetramethylrhodamine methyl ester, cell permeant acetoxymethylester, propidium iodide, annexin-fitc and fluorescent wheat germ agglutinin, and assessed mitochondrial membrane potentials, calcium levels, cell death, apoptosis or cell morphology, respectively. We compared the viability of human islets after 24 h incubation in the ROTi device to an incubation simulating “standard” shipment of islets in 50 ml tubes. All cell viability parameters studied (mitochondrial membrane potentials, calcium content, apoptosis, cell death as well as cell morphology) documented a significantly better cell viability in the ROTi fraction compared with the simulated “standard” shipment fraction. Besides maintaining islet cell viability, the ROTi bears the advantage of a better reproducibility of islet transport conditions.

Mitochondrial permeability transition in rat hepatocytes after anoxia/reoxygenation: role of Ca2+-dependent mitochondrial formation of reactive oxygen species

Onset of the mitochondrial permeability transition (MPT) is the penultimate event leading to lethal cellular ischemia-reperfusion injury, but the mechanisms precipitating the MPT after reperfusion remain unclear. Here, we investigated the role of mitochondrial free Ca(2+) and reactive oxygen species (ROS) in pH- and MPT-dependent reperfusion injury to hepatocytes. Cultured rat hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 4 h and then reoxygenated at pH 7.4 to simulate ischemia-reperfusion. Some cells were loaded with the Ca(2+) chelators, BAPTA/AM and 2-[(2-bis-[carboxymethyl]aono-5-methoxyphenyl)-methyl-6-methoxy-8-bis[carboxymethyl]aminoquinoline, either by a cold loading protocol for intramitochondrial loading or by warm incubation for cytosolic loading. Cell death was assessed by propidium iodide fluorometry and immunoblotting. Mitochondrial Ca(2+), inner membrane permeability, membrane potential, and ROS formation were monitored with Rhod-2, calcein, tetramethylrhodamine methylester, and dihydrodichlorofluorescein, respectively. Necrotic cell death increased after reoxygenation. Necrosis was blocked by 1 μM cyclosporin A, an MPT inhibitor, and by reoxygenation at pH 6.2. Confocal imaging of Rhod-2, calcein, and dichlorofluorescein revealed that an increase of mitochondrial Ca(2+) and ROS preceded onset of the MPT after reoxygenation. Intramitochondrial Ca(2+) chelation, but not cytosolic Ca(2+) chelation, prevented ROS formation and subsequent necrotic and apoptotic cell death. Reoxygenation with the antioxidants, desferal or diphenylphenylenediamine, also suppressed MPT-mediated cell death. However, inhibition of cytosolic ROS by apocynin or diphenyleneiodonium chloride failed to prevent reoxygenation-induced cell death. In conclusion, Ca(2+)-dependent mitochondrial ROS formation is the molecular signal culminating in onset of the MPT after reoxygenation of anoxic hepatocytes, leading to cell death.

ROS Dependent Modulation of Calcium Sparks in Cardiomyocytes

Mitochondrial regulation of cytosolic calcium ([Ca2+]i) is thought to depend on the mitochondrial inner membrane potential (ΔΨm). ΔΨm arises from activity of the electron transport chain and is thought to play a critical role in all ion movements across the inner membrane. With ΔΨmito ≈ −150 mV to −200 mV, there is clearly a strong electrochemical potential for the movement of Ca2+ from the cytosol (about 100 nM) into the matrix (around 100 nM). If significant rapid Ca2+ influx occurs, then ΔΨmito per se should influence the time-course, frequency, magnitude and other characteristics of Ca2+ sparks and the [Ca2+]i transients. Tetramethylrhodamine methyl ester (TMRM) was used to monitor ΔΨmito in freshly isolated rat cardiomyocytes and photon stress was used to depolarize the mitochondria. In addition to the changes in electrochemical potential for Ca2+ entry, depolarization of mitochondria was associated with an increase in cellular reactive oxygen species (ROS) as measured by DCF (as has been previously reported by several laboratories). Quantitative analysis of these findings permits us to separate the influence of each on the changes in Ca2+ signaling observed. Consistent with findings by Zhou et al., 2011, we report a role for local [ROS] in altering Ca2+ signaling.ReferencesZhou, L., Aon., M.A., Lui, T., O'Rourke, B. Dynamic modulation of Ca2+ sparks by mitochondrial oscillations in isolated guinea pig cardiomyocytes under oxidative stress. JMCC. 2011. 51(5):632–9.

Voltage Dependent Anion Channel-3 (VDAC3) is the Major Isoform Contributing to Mitochondrial Metabolism in HepG2 Cells and is Regulated by Free Tubulin and Erastin

BACKGROUND: VDAC controls the flux of hydrophilic metabolites into mitochondria. In vitro, tubulin closes VDAC and in situ free tubulin dynamically modulates ΔΨ. Erastin, a ligand for VDAC, increases ΔΨ in HepG2 cells. Here, we hypothesize that VDAC regulates mitochondrial metabolism and that free tubulin decreases and erastin increases VDAC conductance. Our AIM was to determine ΔΨ, ATP, NADH, and response to free tubulin and erastin after VDAC knockdown (KD). METHODS: HepG2 cells were transfected with siRNAs (5 nM, Ambion) against VDAC1/2/3. At 48 h after transfection, ΔΨ was assessed by fluorescence of tetramethylrhodamine methylester (TMRM) and NADH by autofluorescence using confocal/multiphoton microscopy. Fluorescent beads were fiduciary markers. Adenine nucleotides were determined by HPLC. RESULTS: siRNA decreased mRNA and protein for each VDAC isoform by ∼90%. Double KD of VDAC1/2, VDAC1/3 and VDAC2/3 decreased TMRM fluorescence by ∼20, 55 and 73%, respectively, compared to 100% for non-target siRNA. In reconstituted bilayers, VDAC from HepG2 formed typical anion selective and voltage-gated channels reversibly blocked by dimeric tubulin. Nocodazole decreased ΔΨ by ∼61% in non-target cells and 43, 14 and 17% after KD of VDAC1/2, VDAC1/3 and VDAC2/3. VDAC3 KD decreased ATP by ∼48%, total adenine nucleotides by ∼45%, NADH by ∼33% and the NADH/NAD ratio by ∼60%. Erastin increased ΔΨ by ∼46-48% in non-target and VDAC1/2 KD cells but failed to return ΔΨ to baseline levels after VDAC1/3 and VDAC2/3 KD. Erastin also blocked and reversed depolarization induced by nocodazole. CONCLUSION: VDAC3, the least abundant isoform, contributes most to maintenance of mitochondrial metabolism in HepG2 cells. Erastin antagonizes the inhibitory effect of free tubulin. These results indicate that VDAC, especially VDAC3, regulates mitochondrial metabolism in hepatoma cells.

12 ERK activation is necessary for temperature preconditioning in isolated adult rat ventricular myocytes

In the present study we investigated the molecular effects of temperature preconditioning (TP) in freshly isolated adult rat cardiac myocytes. TP consisted of incubating myocytes at 16°C for 2 min,...

Estrogen Receptor Alpha Expression in Podocytes Mediates Protection against Apoptosis In-Vitro and In-Vivo

Context/ObjectiveEpidemiological studies have demonstrated that women have a significantly better prognosis in chronic renal diseases compared to men. This suggests critical influences of gender hormones on glomerular structure and function. We examined potential direct protective effects of estradiol on podocytes.MethodsExpression of estrogen receptor alpha (ERα) was examined in podocytes in vitro and in vivo. Receptor localization was shown using Western blot of separated nuclear and cytoplasmatic protein fractions. Podocytes were treated with Puromycin aminonucleoside (PAN, apoptosis induction), estradiol, or both in combination. Apoptotic cells were detected with Hoechst nuclear staining and Annexin-FITC flow cytometry. To visualize mitochondrial membrane potential depolarization as an indicator for apoptosis, cells were stained with tetramethyl rhodamine methylester (TMRM). Estradiol-induced phosphorylation of ERK1/2 and p38 MAPK was examined by Western blot. Glomeruli of ERα knock-out mice and wild-type controls were analysed by histomorphometry and immunohistochemistry.ResultsERα was consistently expressed in human and murine podocytes. Estradiol stimulated ERα protein expression, reduced PAN-induced apoptosis in vitro by 26.5±24.6% or 56.6±5.9% (flow cytometry or Hoechst-staining, respectively; both p<0.05), and restored PAN-induced mitochondrial membrane potential depolarization. Estradiol enhanced ERK1/2 phosphorylation. In ERα knockout mice, podocyte number was reduced compared to controls (female/male: 80/86 vs. 132/135 podocytes per glomerulus, p<0.05). Podocyte volume was enhanced in ERα knockout mice (female/male: 429/371 µm3 vs. 264/223 µm3 in controls, p<0.05). Tgfβ1 and collagen type IV expression were increased in knockout mice, indicating glomerular damage.ConclusionsPodocytes express ERα, whose activation leads to a significant protection against experimentally induced apoptosis. Possible underlying mechanisms include stabilization of mitochondrial membrane potential and activation of MAPK signalling. Characteristic morphological changes indicating glomerulopathy in ERα knock-out mice support the in vivo relevance of the ERα for podocyte viability and function. Thus, our findings provide a novel model for the protective influence of female gender on chronic glomerular diseases.

Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

Mitochondrial calcium plays a crucial role in mitochondrial metabolism, cell calcium handling, and cell death. However, some mechanisms concerning mitochondrial calcium regulation are still unknown, especially how mitochondrial calcium couples with cytosolic calcium. In this work, we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation. Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester, a mitochondrial membrane potential indicator. The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2. The apparent K(d) of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes. Furthermore, we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria. In HeLa cells, we found that mitochondrial calcium ([Ca(2+)](mito)) responded to the changes of cytosolic calcium ([Ca(2+)](cyto)) induced by histamine or thapasigargin. Moreover, external Ca(2+) (100 μmol/L) directly induced an increase of [Ca(2+)](mito) in permeabilized HeLa cells. However, in rat cardiomyocytes [Ca(2+)](mito) did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine. In permeabilized cardiomyocytes, 600 nmol/L free Ca(2+) repeatedly increased the fluorescent signals of mito-GCaMP2, which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria. These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.

Dehydroepiandrosterone inhibits the Src/STAT3 constitutive activation in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy characterized by enhanced pulmonary artery smooth muscle cell (PASMC) proliferation and suppressed apoptosis. This phenotype is sustained by the activation of the Src/signal transducer and activator of transcription 3 (STAT3) axis, maintained by a positive feedback loop involving miR-204 and followed by an aberrant expression/activation of its downstream targets such as Pim1 and nuclear factor of activated T-cells (NFATc2). Dehydroepiandrosterone (DHEA) is a steroid hormone shown to reverse vascular remodeling in systemic vessels. Since STAT3 has been described as modulated by DHEA, we hypothesized that DHEA reverses human pulmonary hypertension by inhibiting Src/STAT3 constitutive activation. Using PASMCs isolated from patients with PAH (n = 3), we demonstrated that DHEA decreases both Src and STAT3 activation (Western blot and nuclear translocation assay), resulting in a significant reduction of Pim1, NFATc2 expression/activation (quantitative RT-PCR and Western blot), as well as Survivin and upregulation of bone morphogenetic protein receptor 2 (BMPR2) and miR-204. Src/STAT3 axis inhibition by DHEA is associated with 1) mitochondrial membrane potential (tetramethylrhodamine methyl-ester perchlorate; n = 150; P < 0.05) depolarization increasing apoptosis by 25% (terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling; n = 150; P < 0.05); and 2) decreased intracellular Ca(2+) concentration (fluo-3 AM; n = 150; P < 0.05) and proliferation by 30% (PCNA). Finally, in vivo similarly to STAT3 inhibition DHEA improves experimental PAH (monocrotaline rats) by decreasing mean PA pressure and right ventricle hypertrophy. These effects were associated with the inhibition of Src, STAT3, Pim1, NFATc2, and Survivin and the upregulation of BMPR2 and miR-204. We demonstrated that DHEA reverses pulmonary hypertension in part by inhibiting the Src/STAT3.

Role of oxidative stress and the mitochondrial permeability transition in methylmercury cytotoxicity

Oxidative stress has been implicated in the pathogenesis of methylmercury (MeHg) neurotoxicity. Studies of mature neurons suggest that the mitochondrion may be a major source of MeHg-induced reactive oxygen species and a critical mediator of MeHg-induced neuronal death, likely by activation of apoptotic pathways. It is unclear, however, whether the mitochondria of developing and mature neurons are equally susceptible to MeHg. Murine embryonal carcinoma (EC) cells, which differentiate into neurons following exposure to retinoic acid, were used to compare the differentiation-dependent effects of MeHg on ROS production and mitochondrial depolarization. EC cells and their neuronal derivatives were pre-incubated with the ROS indicator 2′,7′-dichlorofluoroscein diacetate or tetramethylrhodamine methyl ester, an indicator of mitochondrial membrane potential, with or without cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore opening, and examined by laser scanning confocal microscopy in the presence of 1.5μM MeHg. To examine consequences of mitochondrial perturbation, immunohistochemical localization of cytochrome c (cyt c) was determined after incubation of cells in MeHg for 4h. MeHg treatment induced earlier and significantly higher levels of ROS production and more extensive mitochondrial depolarization in neurons than in undifferentiated EC cells. CsA completely inhibited mitochondrial depolarization by MeHg in EC cells but only delayed this response in the neurons. In contrast, CsA significantly inhibited MeHg-induced neuronal ROS production. Cyt c release was also more extensive in neurons, with less protection afforded by CsA. These data indicate that neuronal differentiation state influences mitochondrial transition pore dynamics and MeHg-stimulated production of ROS.

Labeling Mitochondria with MitoTracker Dyes

INTRODUCTION Membrane-potential-dependent dyes such as Rhodamine 123, tetramethylrhodamine methyl ester (TMRM), and tetramethylrhodamine ethyl ester (TMRE) are useful as long as the mitochondrion maintains its negative membrane potential. MitoTracker is a commercially available fluorescent dye (Invitrogen/Molecular Probes) that, like the aforementioned dyes, labels mitochondria within live cells utilizing the mitochondrial membrane potential. However, MitoTracker is chemically reactive, linking to thiol groups in the mitochondria. The dye becomes permanently bound to the mitochondria, and thus remains after the cell dies or is fixed. In addition, it can be used in experiments in which multiple labeling diminishes mitochondrial function. This protocol describes the labeling of mitochondria in live and fixed cells with MitoTracker dyes.

Effects of FK506 and cyclosporin a on calcium ionophore‐induced mitochondrial depolarization and cytosolic calcium in astrocytes and neurons

This study tested the hypothesis that sensitivity to the Ca(2+) -induced loss of mitochondrial membrane potential (ΔΨ(m)) and the sensitivity of the loss of ΔΨ to mitochondrial permeability transition pore (PTP) inhibitors are different for neurons and astrocytes. Primary cultures of rat cortical neurons and astrocytes were exposed to the Ca(2+) ionophore 4-Br-A23187, and ΔΨ(m) was monitored with the fluorescent probe tetramethylrhodamine methyl ester (TMRM). Ca(2+) ionophore caused a decline in ΔΨ(m) in both cell types that was partially inhibited by cyclosporin A (CsA) in astrocytes but not in neurons. Another PTP inhibitor, 2-aminoethoxy-diphenylborate, was ineffective at protecting against mitochondrial depolarization, but depolarization was inhibited by FK506, an immunosuppressant drug similar to CsA that does not inhibit the PTP. CsA and FK506 both significantly reduced the ionophore-induced rise in [Ca(2+) ](i) in both neurons and astrocytes. We conclude that the protective effects of CsA and FK506 against Ca(2+) ionophore-induced mitochondrial membrane depolarization in intact astrocytes is not due to PTP inhibition but possibly is a consequence of inhibiting the rise in [Ca(2+) ](i).

Labeling Mitochondria with TMRM or TMRE: Figure 1.

INTRODUCTIONTMRM (tetramethylrhodamine methyl ester) or the related TMRE (tetramethylrhodamine ethyl ester) is extensively used for labeling and measuring the membrane potential (and the function) of mitochondria in living cells. Because of its optical and chemical properties, it has been one of the main dyes used for mitochondria studied with confocal microscopy to carry out time-resolved and spatially resolved analyses. Quantitative observation of mitochondrial membrane potential should be performed with confocal microscopy or spot photometry-based microscopy. This protocol describes labeling of mitochondria with TMRM or TMRE.

Functional Mutation of SMAC/DIABLO, Encoding a Mitochondrial Proapoptotic Protein, Causes Human Progressive Hearing Loss DFNA64

SMAC/DIABLO is a mitochondrial proapoptotic protein that is released from mitochondria during apoptosis and counters the inhibitory activities of inhibitor of apoptosis proteins, IAPs. By linkage analysis and candidate screening, we identified a heterozygous SMAC/DIABLO mutation, c.377C>T (p.Ser126Leu, refers to p.Ser71Leu in the mature protein) in a six-generation Chinese kindred characterized by dominant progressive nonsyndromic hearing loss, designated as DFNA64. SMAC/DIABLO is highly expressed in human embryonic ears and is enriched in the developing mouse inner-ear hair cells, suggesting it has a role in the development and homeostasis of hair cells. We used a functional study to demonstrate that the SMAC/DIABLO(S71L) mutant, while retaining the proapoptotic function, triggers significant degradation of both wild-type and mutant SMAC/DIABLO and renders host mitochondria susceptible to calcium-induced loss of the membrane potential. Our work identifies DFNA64 as the human genetic disorder associated with SMAC/DIABLO malfunction and suggests that mutant SMAC/DIABLO(S71L) might cause mitochondrial dysfunction.

Acute hyperglycemia induces Cardiac Mitochondrial Depolarization

Mitochondrial ATP generation depends on the enormous potential across the inner mitochondrial membrane (ΔΨmito ~ −180 mV), among many other factors. Oxidative phosphorylation requires the large ΔΨmito and when the mitochondria are depolarized, oxidative phosphorylation becomes uncoupled and ATP production falls. Here we investigate the effect of hyperglycemia (30 mM versus the physiological glucose of 5 mM) on mitochondrial ΔΨmito under photonic stress in rat ventricular myocytes. Using tetra-methyl rhodamine methyl ester (TMRM) as a fluorescence monitor of ΔΨmito we examined how the time-dependent depolarization of mitochondria may be altered by hyperglycemia. Using a normal Tyrode's solution on isolated ventricular myocytes, changing the [glucose] has dramatic effect on the rate of ΔΨmito depolarization. After 3hours of hyperglycemic exposure, the rate of depolarization is reduced nearly 30% compared to controls. Antioxidant treatment protects cardiac myocytes from this hyperglycemic-induced effect while selectively inhibiting complex I causes an abrogation of the effect. These data elucidate the biophysical effects of acute hyperglycemia on cardiac myocytes.

Proteomic identification of carbonylated proteins in 1,3-dinitrobenzene neurotoxicity

This study demonstrated that 1,3-dinitrobenzene-induced (1,3-DNB) oxidative stress led to the oxidative carbonlyation of specific protein targets in DI TNC1 cells. 1,3-DNB-induced mitochondrial dysfunction, as indicated by loss of tetramethyl rhodamine methyl ester (TMRM) fluorescence, was initially observed at 5h and coincided with peak reactive oxygen species (ROS) production. ROS production was inhibited in cells pre-treated with the mitochondrial permeability transition (MPT) inhibitor, bonkrekic acid (BkA). Pre-incubation with the antioxidant deferoxamine inhibited loss of TMRM fluorescence until 24h after initial exposure to 1,3-DNB. Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and subsequent Oxyblot analysis were used to determine if 1,3-DNB exposure led to the formation of protein carbonyls. Exposing DI TNC1 cells to 1,3-DNB led to marked protein carbonylation 45min following initial exposure. Pre-treatment with deferoxamine or Trolox reduced the intensity of protein carbonylation in DI TNC1 cells exposed to 1mM 1,3-DNB. Tandem MS/MS performed on protein samples isolated from 1,3-DNB-treated cells revealed that specific proteins within the mitochondria, endoplasmic reticulum (ER), and cytosol are targets of protein carbonylation. The results presented in this study are the first to suggest that the molecular mechanism of 1,3-DNB neurotoxicity may occur through selective carbonylation of protein targets found within specific intracellular compartments of susceptible cells.