Exploring mitochondrial metabolism of wild-type and diabetic mice skin explants using the Seahorse technology.

BACKGROUND: Skin wound healing is a complex mechanism which requires a lot of energy, mainly provided by mitochondrial respiration. However, little is known about the mitochondrial bioenergetics of mice skin. We sought to develop a microplate-based assay to directly measure oxygen consumption in whole mice skin with the goal of identifying mitochondrial dysfunction in diabetic skin using an extracellular flux. MATERIALS AND METHODS: Different parameters were optimized to efficiently measure the oxygen consumption rate (OCR). First, the most pertinent skin side of wild-type mice was first determined. Then, concentrations of mitochondrial inhibitors were then optimized to get the best efficacy. Finally, punch sizes were modulated to get the best OCR profile. RESULTS: Dermis had the best metabolic activity side of the skin. Unlike the increased concentrations of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and rotenone/antimycin A, which showed no improvement of these drugs' effects, varying the skin punch size was successful. Finally, type II diabetic (T2D) skin produced less ATP through mitochondrial metabolism and had a greater non-mitochondrial oxygen consumption than wild-type or type I diabetic (T1D) skin. CONCLUSION: Here we designed, for the first time, a reliable protocol to measure mitochondria function in whole mouse skin. Our optimized protocol was valuable in assessing alterations associated with diabetes and could be applied to future studies of pathological human skin metabolism.

[PDCD4 knockdown ameliorates lipopolysaccharide-induced endothelial cell damage by improving mitochondrial dynamics].

OBJECTIVE: To elucidate the role of programmed cell death factor 4 (PDCD4) in mitochondrial dysfunction caused by sepsis-related vascular endothelial damage. METHODS: Cultured human umbilical vein endothelial cells (HUVECs) and mouse vascular endothelial cells (C166 cells) were transfected with a small interfering RNA targeting PDCD4 followed by treatment with lipopolysaccharide (LPS) alone or in combination with carbonyl cyanide 3-chlorophenylhydrazone (FCCP). The proteomic changes in the cells after PDCD4 knockdown were analyzed using LC-MS/MS technique. The mRNA expressions of PDCD4 and the genes associated with cell inflammation and apoptosis were detected with RT-PCR, and the expressions of FIS1, DRP1 and OPA1 proteins key to mitochondrial fission and fusion were determined using Western blotting. JC-1 and MitoSOX fluorescent probes were used to observe the changes in mitochondrial membrane potential and mitochondrial reactive oxygen species levels under by a laser confocal microscope. RESULTS: LPS stimulation of the cells significantly increased the mRNA expressions of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein 1 (MCP1) and enhanced the cellular expression of PDCD4 (P < 0.05). Proteomic analysis suggested a correlation between PDCD4 knockdown and changes in mitochondrial dynamics in the cells. LPS treatment significantly increased the expressions of mitochondrial fission proteins FIS1 and DRP1 and lowered the expression of the fusion protein OPA1 in the cells (P < 0.05), causing also mitochondrial oxidative stress and reduction of the mitochondrial membrane potential (P < 0.05). In HUVECs, treatment with FCCP significantly attenuated the protective effect of PDCD4 knockdown, which inhibited LPS-induced inflammation and oxidative stress and restored the balance between mitochondrial fission and fusion. CONCLUSION: PDCD4 knockdown protects vascular endothelial cells against LPS-induced damages by repressing mitochondrial fission and oxidative stress, promoting mitochondrial fusion, and maintaining normal mitochondrial function.

Increased cytotoxicity of Pb2+ with co-exposures to a mitochondrial uncoupler and mitochondrial calcium uniporter inhibitor.

Lead (Pb2+) is an important developmental toxicant. The mitochondrial calcium uniporter (MCU) imports calcium ions using the mitochondrial membrane potential (MMP), and also appears to mediate the influx of Pb2+ into the mitochondria. Since our environment contains mixtures of toxic agents, it is important to consider multi-chemical exposures. To begin to develop generalizable, predictive models of interactive toxicity, we developed mechanism-based hypotheses about interactive effects of Pb2+ with other chemicals. To test these hypotheses, we exposed HepG2 (human liver) cells to Pb2+ alone and in mixtures with other mitochondria-damaging chemicals: carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler that reduces MMP, and Ruthenium Red (RuRed), a dye that inhibits the MCU. After 24 hours, Pb2+ alone, the mixture of Pb2+ and RuRed, and the mixture of Pb2+ and FCCP caused no decrease in cell viability. However, the combination of all three exposures led to a significant decrease in cell viability at higher Pb2+ concentrations. After 48 hours, the co-exposure to elevated Pb2+ concentrations and FCCP caused a significant decrease in cell viability, and the mixture of all three showed a clear dose-response curve with significant decreases in cell viability across a range of Pb2+ concentrations. We performed ICP-MS analyses on isolated mitochondrial and cytosolic fractions and found no differences in Pb2+ uptake across exposure groups, ruling out altered cellular uptake as the mechanism for interactive toxicity. We assessed MMP following exposure and observed a decrease in membrane potential that corresponds to loss of cell viability but is likely not sufficient to be the causative mechanistic driver of cell death. This research provides a mechanistically-based framework for understanding Pb2+ toxicity in mixtures with mitochondrial toxicants.

Mitochondrial dysfunction in chromaffin cells from the R6/1 mouse model of Huntington's disease: Impact on exocytosis and calcium current regulation.

From a pathogenic perspective, Huntington's disease (HD) is being considered as a synaptopathy. As such, alterations in brain neurotransmitter release occur. As the activity of the sympathoadrenal axis is centrally controlled, deficits in the exocytotic release of catecholamine release may also occur. In fact, in chromaffin cells (CCs) of the adrenal medulla of the R6/1 model of HD, decrease of secretion and altered kinetics of the exocytotic fusion pore have been reported. Those alterations could be linked to mitochondrial deficits occurring in peripheral CCs, similar to those described in brain mitochondria. Here we have inquired about alterations in mitochondrial structure and function and their impact on exocytosis and calcium channel currents (ICa). We have monitored various parameters linked to those events, in wild type (WT) and the R6/1 mouse model of HD at a pre-disease stage (2 months age, 2 m), and when motor deficits are present (7 months age, 7 m). In isolated CCs from 7 m and in the adrenal medulla of R6/1 mice, we found the following alterations (with respect 7 m WT mice): (i) augmented fragmented mitochondria and oxidative stress with increased oxidized glutathione; (ii) decreased basal and maximal respiration; (iii) diminution of ATP cell levels; (iv) mitochondrial depolarization; (v) drastic decrease of catecholamine release with poorer potentiation by protonophore FCCP; (vi) decreased ICa inhibition by FCCP; and (vii) lesser potentiation by BayK8644 of ICa and smaller prolongation of current deactivation. Of note was the fact several of these alterations were already manifested in CCs from 2 m R6/1 mice at pre-disease stages. Based on those results, a plausible hypothesis can be raised in the sense that altered mitochondrial function seems to be an early primary event in HD pathogenesis. This is in line with an increasing number of mitochondrial, metabolic, and inflammatory alterations being recently reported in various HD peripheral tissues.

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry.

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

Relative Importance of Different Elements of Mitochondrial Oxidative Phosphorylation in Maintaining the Barrier Integrity of Retinal Endothelial Cells: Implications for Vascular-Associated Retinal Diseases.

PURPOSE: Mitochondrial dysfunction is central to breaking the barrier integrity of retinal endothelial cells (RECs) in various blinding eye diseases such as diabetic retinopathy and retinopathy of prematurity. Therefore, we aimed to investigate the role of different mitochondrial constituents, specifically those of oxidative phosphorylation (OxPhos), in maintaining the barrier function of RECs. METHODS: Electric cell-substrate impedance sensing (ECIS) technology was used to assess in real time the role of different mitochondrial components in the total impedance (Z) of human RECs (HRECs) and its components: capacitance (C) and the total resistance (R). HRECs were treated with specific mitochondrial inhibitors that target different steps in OxPhos: rotenone for complex I, oligomycin for complex V (ATP synthase), and FCCP for uncoupling OxPhos. Furthermore, data were modeled to investigate the effects of these inhibitors on the three parameters that govern the total resistance of cells: Cell-cell interactions (Rb), cell-matrix interactions (α), and cell membrane permeability (Cm). RESULTS: Rotenone (1 µM) produced the greatest reduction in Z, followed by FCCP (1 µM), whereas no reduction in Z was observed after oligomycin (1 µM) treatment. We then further deconvoluted the effects of these inhibitors on the Rb, α, and Cm parameters. Rotenone (1 µM) completely abolished the resistance contribution of Rb, as the Rb became zero immediately after the treatment. Secondly, FCCP (1 µM) eliminated the resistance contribution of Rb only after 2.5 h and increased Cm without a significant effect on α. Lastly, of all the inhibitors used, oligomycin had the lowest impact on Rb, as evidenced by the fact that this value became similar to that of the control group at the end of the experiment without noticeable effects on Cm or α. CONCLUSION: Our study demonstrates the differential roles of complex I, complex V, and OxPhos coupling in maintaining the barrier functionality of HRECs. We specifically showed that complex I is the most important component in regulating HREC barrier integrity. These observed differences are significant since they could serve as the basis for future pharmacological and gene expression studies aiming to improve the activity of complex I and thereby provide avenues for therapeutic modalities in endothelial-associated retinal diseases.

Quantitative auto-fluorescence quenching of free and bound NADH in HeLa cell line model with Carbonyl cyanide-p-Trifluoromethoxy phenylhydrazone (FCCP) as quenching agent.

The autofluorescence of endogenous biomolecules (Nicotinamide adenine dinucleotide (NAD, its reduced form NADH and the phosphorylated form NAD(P)H take part in cellular metabolic pathways and has vital importance for in vivo and ex vivo photo diagnostic applications of biological tissues. We present a detailed quenching analysis of Carbonyl cyanide-p-Trifluoromethoxy phenylhydrazone (FCCP) 50-1000 µM and analyzed the fluorescence signal from NADH/ NAD(P)H in vitro (in solution) and in vivo (HeLa cell suspension).The in vitro samples of pure NADH/ NAD(P)H were excited at λ=340±1 nm while the fluorescence signal was collected in the range of 400-550 nm. The quenching process was characterized using excitation emission matrix (EEM) fluorescence spectroscopy and Stern- Volmer plots. The experimental results illustrated maximum fluorescence emission for the control NADH samples (i.e., no FCCP), while the fluorescence signal from the solution progressively decreased with the increasing concentration of the FCCP, until it reaches the base line (i.e., no fluorescence signal) at 1000 µM of FCCP. In vitro study shows that the fluorescence quenching of free NADH was found to be lower than the bound NAD(P)H with similar diminishing trend. The quenching of bound NAD(P)H in cells is attenuated compared to solution quenching possibly due to a contribution from the metabolic/antioxidant response in cells and fluorescence exponential decay curve lies between plated and suspended HeLa cells. A two-fold increase in the fluorescence intensity of NAD(P)H was observed after the bond formation with L-Malate Dehydrogenase (L-MDH, Sigma Aldrich #10127248001) protein This work has applications for sharp tumor demarcation during sensitive surgical procedures as well as to enhance fluorescence based diagnosis of biological tissues.

Mitochondrial uncouplers induce proton leak by activating AAC and UCP1.

Mitochondria generate heat due to H+ leak (IH) across their inner membrane1. IH results from the action of long-chain fatty acids on uncoupling protein 1 (UCP1) in brown fat2-6 and ADP/ATP carrier (AAC) in other tissues1,7-9, but the underlying mechanism is poorly understood. As evidence of pharmacological activators of IH through UCP1 and AAC is lacking, IH is induced by protonophores such as 2,4-dinitrophenol (DNP) and cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP)10,11. Although protonophores show potential in combating obesity, diabetes and fatty liver in animal models12-14, their clinical potential for treating human disease is limited due to indiscriminately increasing H+ conductance across all biological membranes10,11 and adverse side effects15. Here we report the direct measurement of IH induced by DNP, FCCP and other common protonophores and find that it is dependent on AAC and UCP1. Using molecular structures of AAC, we perform a computational analysis to determine the binding sites for protonophores and long-chain fatty acids, and find that they overlap with the putative ADP/ATP-binding site. We also develop a mathematical model that proposes a mechanism of uncoupler-dependent IH through AAC. Thus, common protonophoric uncouplers are synthetic activators of IH through AAC and UCP1, paving the way for the development of new and more specific activators of these two central mediators of mitochondrial bioenergetics.

Contributing role of mitochondrial energy metabolism on platelet adhesion, activation and thrombus formation under blood flow conditions.

Platelets have an active energy metabolism mediated by mitochondria. However, the role of mitochondria in platelet adhesion, activation, and thrombus formation under blood flow conditions remains to be elucidated. Blood specimens were obtained from healthy adult volunteers. The consumption of glucose molecules by platelets was measured after 24 hours. Platelet adhesion, activation, and thrombus formation on collagen fibrils and immobilized von Willebrand factor (VWF) at a wall shear rate of 1,500 s-1 were detected by fluorescence microscopy with an ultrafast laser confocal unit in the presence or absence of mitochondrial functional inhibitors of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), antimycin A, and oligomycin. Consumption of glucose molecules within the first 24 h of 4.21 × 10-15 ± 4.46 x 10-15 (n = 6) increased to 13.82 × 10-15 ± 3.46 x 10-15 (n = 4) in the presence of FCCP, 12.11 × 10-15 ± 2.33 x 10-15 (n = 4) in the presence of antimycin A, and 11.87 × 10-15 ± 3.56 x 10-15 (n = 4) in the presence of oligomycin (p < .05). These mitochondrial functional blockers did not influence both surface area coverage by platelets and the 3-dimensional size of platelet thrombi formed on the collagen fibrils. However, a rapid increase in the intracellular calcium ion concentration ([Ca2+]i) upon adhering on immobilized VWF decreased significantly from 405.5 ± 86.2 nM in control to 198.0 ± 79.2 nM in the presence of FCCP (p < .005). A similar decrease in the rapid increase in ([Ca2+]i) was observed in the presence of antimycin A and oligomycin. Mitochondrial function is necessary for platelet activation represented by a rapid increase in [Ca2+]i after platelet adhesion on VWF. However, the influence could not be detected as changes in platelet adhesion or 3-dimensional growth of platelet thrombi on collagen fibrils.

Protonophore FCCP provides fitness advantage to PDR-deficient yeast cells.

Pleiotropic drug resistance (PDR) plasma membrane transporters mediate xenobiotic efflux from the cells and thereby help pathogenic microorganisms to withstand antimicrobial therapies. Given that xenobiotic efflux is an energy-consuming process, cells with upregulated PDR can be sensitive to perturbations in cellular energetics. Protonophores dissipate proton gradient across the cellular membranes and thus increase ATP spendings to their maintenance. We hypothesised that chronic exposure of yeast cells to the protonophores can favour the selection of cells with inactive PDR. To test this, we measured growth rates of the wild type Saccharomyces cerevisiae and PDR-deficient Δpdr1Δpdr3 strains in the presence of protonophores carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), pentachlorophenol (PCP) and niclosamide (NCA). Although the protonophore-induced respiration rates of these two strains were similar, the PDR-deficient strain outperformed the control one in the growth rate on non-fermentable carbon source supplemented with low concentrations of FCCP. Thus, active PDR can be deleterious under conditions of partially uncoupled oxidative-phosphorylation. Furthermore, our results suggest that tested anionic protonophores are poor substrates of PDR-transporters. At the same time, protonophores imparted azole tolerance to yeasts, pointing that they are potent PDR inducers. Interestingly, protonophore PCP led to a persistent increase in the levels of a major ABC-transporter Pdr5p, while azole clotrimazole induced only a temporary increase. Together, our data provides an insight into the effects of the protonophores in the eukaryotes at the cellular level and support the idea that cells with activated PDR can be selected out upon conditions of energy limitations.

Heme modulates Trypanosoma cruzi bioenergetics inducing mitochondrial ROS production.

Trypanosoma cruzi is the causative agent of Chagas disease and has a single mitochondrion, an organelle responsible for ATP production and the main site for the formation of reactive oxygen species (ROS). T. cruzi is an obligate intracellular parasite with a complex life cycle that alternates between vertebrate and invertebrate hosts, therefore the development of survival strategies and morphogenetic adaptations to deal with the various environments is mandatory. Over the years our group has been studying the vector-parasite interactions using heme as a physiological oxidant molecule that triggered epimastigote proliferation however, the source of ROS induced by heme remained unknown. In the present study we demonstrate the involvement of heme in the parasite mitochondrial metabolism, decreasing oxygen consumption leading to increased mitochondrial ROS and membrane potential. First, we incubated epimastigotes with carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation, which led to decreased ROS formation and parasite proliferation, even in the presence of heme, correlating mitochondrial ROS and T. cruzi survival. This hypothesis was confirmed after the mitochondria-targeted antioxidant ((2-(2,2,6,6 Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (MitoTEMPO) decreased both heme-induced ROS and epimastigote proliferation. Furthermore, heme increased the percentage of tetramethylrhodamine methyl ester (TMRM) positive parasites tremendously-indicating the hyperpolarization and increase of potential of the mitochondrial membrane (ΔΨm). Assessing the mitochondrial functional metabolism, we observed that in comparison to untreated parasites, heme-treated epimastigotes decreased their oxygen consumption, and increased the complex II-III activity. These changes allowed the electron flow into the electron transport system, even though the complex IV (cytochrome c oxidase) activity decreased significantly, showing that heme-induced mitochondrial ROS appears to be a consequence of the enhanced mitochondrial physiological modulation. Finally, the parasites that were submitted to high concentrations of heme presented no alterations in the ultrastructure. Consequently, our results suggest that heme released by the insect vector after the blood meal, modify epimastigote mitochondrial physiology to increase ROS as a metabolic mechanism to maintain epimastigote survival and proliferation.

Effect of carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) on the interaction of 1-anilino-8-naphthalene sulfonate (ANS) with phosphatidylcholine liposomes.

The weak hydrophobic acid carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) is a protonophoric uncoupler of oxidative phosphorylation in mitochondria. It dissipates the electrochemical proton gradient (ΔµH (+)) increasing the mitochondrial oxygen consumption. However, at concentrations higher than 1 µM it exhibits additional effects on mitochondrial energy metabolism, which were tentatively related to modifications of electrical properties of the membrane. Here we describe the effect of FCCP on the binding of 1-anilino-8-naphthalene sulfonate (ANS) to 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) unilamellar vesicles. FCCP inhibited the binding of ANS to liposomes either in the gel or in the liquid crystalline phase, by increasing the apparent dissociation constant of ANS. Smaller effect on the dissociation constant was observed at high ionic strength, suggesting that the effect of FCCP is through modification of the electrostatic properties of the membrane interface. In addition, FCCP also decreased (approximately 50 %) the quantum yield and increased the intrinsic dissociation constant of membrane-bound ANS, results that suggest that FCCP makes the environment of the ANS binding sites more polar. On those grounds we postulate that the binding of FCCP: i) increases the density of negative charges in the membrane surface; and ii) distorts the phospholipid bilayer, increasing the mobility of the polar headgroups making the ANS binding site more accessible to water.

Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling.

Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O2 consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca(2+) or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment.

Glutamate-induced alterations in Ca2+ signaling are modulated by mitochondrial Ca2+ handling capacity in brain slices of R6/1 transgenic mice.

Huntington's disease is a neurodegenerative disorder caused by an expansion of CAGs repeats and characterized by alterations in mitochondrial functions. Although changes in Ca(2+) handling have been suggested, the mechanisms involved are not completely understood. The aim of this study was to investigate the possible alterations in Ca(2+) handling capacity and the relationship with mitochondrial dysfunction evaluated by NAD(P)H fluorescence, reactive oxygen species levels, mitochondrial membrane potential (DeltaPsi(m)) measurements and respiration in whole brain slices from R6/1 mice of different ages, evaluated in situ by real-time real-space microscopy. We show that the cortex and striatum of the 9-month-old R6/1 transgenic mice present a significant sustained increase in cytosolic Ca(2+) induced by glutamate (Glu). This difference in Glu response was partially reduced in R6/1 when in the absence of extracellular Ca(2+), indicating that N-methyl-D-aspartate receptors participation in this response is more important in transgenic mice. In addition, Glu also lead to a decrease in NAD(P)H fluorescence, a loss in DeltaPsi(m) and a further increase in respiration, which may have evoked a decrease in mitochondrial Ca(2+) Ca(2+)(m) uptake capacity. Taken together, these results show that alterations in Ca(2+) homeostasis in transgenic mice are associated with a decrease in Ca(2+)(m) uptake mechanism with a diminished Ca(2+) handling ability that ultimately causes dysfunctions and worsening of the neurodegenerative and the disease processes.

Penetrating cation/fatty acid anion pair as a mitochondria-targeted protonophore.

A unique phenomenon of mitochondria-targeted protonophores is described. It consists in a transmembrane H(+)-conducting fatty acid cycling mediated by penetrating cations such as 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) or dodecyltriphenylphosphonium (C(12)TPP). The phenomenon has been modeled by molecular dynamics and directly proved by experiments on bilayer planar phospholipid membrane, liposomes, isolated mitochondria, and yeast cells. In bilayer planar phospholipid membrane, the concerted action of penetrating cations and fatty acids is found to result in conversion of a pH gradient (DeltapH) to a membrane potential (Deltapsi) of the Nernstian value (about 60 mV Deltapsi at DeltapH = 1). A hydrophobic cation with localized charge (cetyltrimethylammonium) failed to substitute for hydrophobic cations with delocalized charge. In isolated mitochondria, SkQ1 and C(12)TPP, but not cetyltrimethylammonium, potentiated fatty acid-induced (i) uncoupling of respiration and phosphorylation, and (ii) inhibition of H(2)O(2) formation. In intact yeast cells, C(12)TPP stimulated respiration regardless of the extracellular pH value, whereas a nontargeted protonophorous uncoupler (trifluoromethoxycarbonylcyanide phenylhydrazone) stimulated respiration at pH 5 but not at pH 3. Hydrophobic penetrating cations might be promising to treat obesity, senescence, and some kinds of cancer that require mitochondrial hyperpolarization.

Altered myoplasmic Ca(2+) handling in rat fast-twitch skeletal muscle fibres during disuse atrophy.

Calcium-dependent signalling pathways are believed to play an important role in skeletal muscle atrophy, but whether intracellular Ca(2+) homeostasis is affected in that situation remains obscure. We show here that there is a 20% atrophy of the fast-type flexor digitorum brevis (FDB) muscle in rats hind limb unloaded (HU) for 2 weeks, with no change in fibre type distribution. In voltage-clamp experiments, the amplitude of the slow Ca(2+) current was found similar in fibres from control and HU animals. In fibres loaded with the Ca(2+) dye indo-1, the value for the rate of [Ca(2+)] decay after the end of 5-100-ms-long voltage-clamp depolarisations from -80 to +10 mV was found to be 30-50% lower in fibres from HU animals. This effect was consistent with a reduced contribution of both saturable and non-saturable components of myoplasmic Ca(2+) removal. However, there was no change in the relative amount of parvalbumin, and type 1 sarco-endoplasmic reticulum Ca(2+)-ATPase was increased by a factor of three in the atrophied muscles. Confocal imaging of mitochondrial membrane potential showed that atrophied FDB fibres had significantly depolarized mitochondria as compared to control fibres. Depolarization of mitochondria in control fibres with carbonyl cyanide-p-trifluoromethoxyphenylhydrazone induced a slowing of the decay of [Ca(2+)] transients accompanied by an increase in resting [Ca(2+)] and a reduction of the peak amplitude of the transients. Overall results provide the first functional evidence for severely altered intracellular Ca(2+) removal capabilities in atrophied fast-type muscle fibres and highlight the possible contribution of reduced mitochondrial polarisation.

Features of mitochondrial energetics in living unicellular eukaryote Tetrahymena pyriformis. A model for study of mammalian intracellular adaptation.

Tetrahymena pyriformis is used in diverse studies as a non-mammalian alternative due to their resemblance in many main metabolic cycles. However, such basic features of mitochondrial energetics as Delta psi (electrical potential difference across the inner mitochondrial membrane) or maximal stimulation of respiration by uncouplers with different mechanisms of uncoupling, such as DNP (2,4-dinitrophenol) and FCCP (p-trifluoromethoxycarbonylcyanide phenylhydrazone), have not been studied in living ciliates. Tetrahymena pyriformis GL cells during stationary growth phase after incubation under selected conditions were used in this study. Maximal stimulation of cellular respiration by FCCP was about six-fold, thus the proton motive force was high. The DNP uncoupling effect was significantly lower. This suggests low activity of the ATP/ADP-antiporter, which performs not only exchange of intramitochondrial ATP to extramitochondrial ADP, but also helps in the uncoupling process. It participates by a similar mechanism in electrophoretic transport from matrix to cytosol of ATP(4-) and DNP anion, but not FCCP anion. Thus, in contrast with mammalian mitochondria, T. pyriformis mitochondria cannot rapidly supply the cytosol with ATP; possibly the cells need high intramitochondrial ATP. The difference between DNP and FCCP is hypothetically explained by low Delta psi value and/or an increase in concentration of long-chain acyl-CoAs, inhibitors of the ATP/ADP-antiporter. The first suggestion is confirmed by absence of mitochondria with bright fluorescence in T. pyriformis stained with the Delta psi-sensitive probe MitoTracker Red. These data suggest that T. pyriformis cells are useful as a model for study of mitochondrial role in adaptation at the intracellular level.

Amiodarone increases the accumulation of DEA in a human alveolar epithelium-derived cell line.

Amiodarone (AMD)-induced pulmonary toxicity (AIPT) is the most life-threatening side-effect of AMD treatment. N-Monodesethylamiodarone (DEA), an active metabolite of AMD, also exhibits cytotoxicity and tends to accumulate in the lung more intensively than AMD. In this study, we characterized the mechanism of DEA accumulation using A549 cells as a model of the alveolar epithelium. Typical ATP-depletion compounds caused an approximately 30% increase in the accumulation of DEA in A549 cells, although these effects were less than those in Caco-2 cells. Triiodothyronine (T(3)), which exhibited an inhibitory effect on DEA efflux in Caco-2 cells, did not affect the accumulation of DEA in A549 cells. On the other hand, 100 microM AMD caused an approximately 200% increase in DEA content in A549 cells, although AMD accumulation was not affected by 100 microM DEA. Since the reducing effect of AMD on cellular ATP levels and that of FCCP were similar, the mechanism by which DEA accumulation is increased by AMD might be different from the ATP-dependent DEA efflux mechanism. The decrease in cell viability by DEA in the presence of AMD (IC(50) value of DEA for A549 cell viability: 25.4+/-2.4 microM) was more pronounced than that by DEA alone (IC(50) value: 11.5+/-3.0 microM). This further DEA accumulation by AMD might be a factor responsible for the greater accumulation of DEA than that of AMD in the lung in long-term AMD-treated patients.

Roles of mitochondria and temperature in the control of intracellular calcium in adult rat sensory neurons.

We recorded Ca2+ current and intracellular Ca2+ ([Ca2+](i)) in isolated adult rat dorsal root ganglion (DRG) neurons at 20 and 30 degrees C. In neurons bathed in tetraethylammonium and dialyzed with cesium, warming reduced resting [Ca2+](i) from 87 to 49 nM and the time constant of the decay of [Ca2+](i) transients (tau(r)) from 1.3 to 0.99s (Q(10)=1.4). The Buffer Index, the ratio between Ca2+ influx and Delta[Ca2+](i) (f I(ca)d(t)/Delta[Ca2+]i) , increased two- to threefold with warming. Neither inhibition of the plasma membrane Ca2+ -ATPase by intracellular sodium orthovanadate nor inhibition of Ca2+ uptake by the endoplasmic reticulum by thapsigargin plus ryanodine were necessary for the effects of warming on these parameters. In contrast, inhibition of the mitochondrial Ca2+ uniporter by intracellular ruthenium red largely reversed the effects of warming. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 500 nM) increased resting [Ca2+](i) at 30 degrees C. Ten millimolar intracellular sodium prolonged the recovery of [Ca2+](i) transients to 10-40s. This effect was reversed by an inhibitor of mitochondrial Na(+)/Ca2+ -exchange (CGP 37157, 10 microM). Thus, mitochondrial Ca2+ uptake is necessary for the temperature-dependent increase in Ca2+ buffering and mitochondrial Ca2+ fluxes contribute to the control of [Ca2+](i) between 50 and 150 nM at 30 degrees C.

Targeting the hypoxia inducible factor pathway with mitochondrial uncouplers.

Hypoxia inducible factor-1 (HIF-1) is central to most adaptation responses of tumors to hypoxia, and consists of a hypoxia inducible HIF-1alpha or -2alpha subunit, and a constitutively expressed HIF-1beta subunit. Previously, mitochondrial uncouplers, rottlerin and FCCP, were shown to increase the rate of cellular O(2 )consumption. In this study, we determined that mitochondrial uncouplers, rottlerin and FCCP, significantly decreased hypoxic as well as normoxic HIF-1 transcriptional activity which was in part mediated by down-regulation of the oxygen labile HIF-1alpha and HIF-2alpha protein levels in PC-3 and DU-145 prostate cancer cells. Our results also revealed that mitochondrial uncouplers decreased the expression of HIF target genes, VEGF and VEGF receptor-2. Taken together, our results indicate that functional mitochondria are important in HIF-1alpha and HIF-2alpha protein stability and transcriptional activity during normoxia as well as in hypoxia, and that mitochondrial uncouplers may be useful in the inhibition of HIF pathway in tumors.