Label-Free Imaging of Intracellular Temperature by Using the O-H Stretching Raman Band of Water.

We propose a label-free method for measuring intracellular temperature using a Raman image of a cell in the O-H stretching band. Raman spectra of cultured cells and the medium were first measured at various temperatures using a Raman microscope and the intensity ratio of the two regions of the O-H stretching band was calculated. The intensity ratio varies linearly with temperature in both the medium and cells, and the resulting calibration lines allow simultaneous visualization of both intracellular and extracellular temperatures in a label-free manner. We applied this method to the measurement of temperature changes after the introduction of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) in living cells. We observed a temperature rise in the cytoplasm and succeeded in obtaining an image of the change in intracellular temperature after the FCCP treatment.

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.

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.

Comparison of the binding behavior of FCCP with HSA and HTF as determined by spectroscopic and molecular modeling techniques.

The interaction of carbonylcyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) with human serum albumin (HSA) and human transferrin (HTF) was investigated using multiple spectroscopy, molecular modeling, zeta-potential and conductometry measurements of aqueous solutions at pH 7.4. The fluorescence, UV/vis and polarization fluorescence spectroscopy data disclosed that the drug-protein complex formation occurred through a remarkable static quenching. Based on the fluorescence quenching, two sets of binding sites with distinct affinities for FCCP existed in the two proteins. Steady-state and polarization fluorescence analysis showed that there were more affinities between FCCP and HSA than HTF. Far UV-CD and synchronous fluorescence studies indicated that FCCP induced more structural changes on HSA. The resonance light scattering (RLS) and zeta-potential measurements suggested that HTF had a greater resistance to drug aggregation, whereas conductometry measurements expressed the presence of free ions improving the resistance of HSA to aggregation. Thermodynamic measurements implied that a combination of electrostatic and hydrophobic forces was involved in the interaction between FCCP with both proteins. The phase diagram plots indicated that the presence of second binding site on HSA and HTF was due to the existence of intermediate structures. Site marker competitive experiments demonstrated that FCCP had two distinct binding sites in HSA which were located in sub-domains IIA and IIIA and one binding site in the C-lobe of HTF as confirmed by molecular modeling. The obtained results suggested that both proteins could act as drug carriers, but that the HSA potentially had a higher capacity for delivering FCCP to cancerous tissues.

Structural investigations on the mitochondrial uncouplers niclosamide and FCCP.

There has been renewed interest in using mitochondrial uncoupler compounds such as niclosamide and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) for the treatment of obesity, hepatosteatosis and diseases where oxidative stress plays a role. However, both FCCP and niclosamide have undesirable effects that are not due to mitochondrial uncoupling, such as inhibition of mitochondrial oxygen consumption by FCCP and induction of DNA damage by niclosamide. Through structure-activity analysis, we identified FCCP analogues that do not inhibit mitochondrial oxygen consumption but still provided good, although less potent, uncoupling activity. We also characterized the functional role of the niclosamide 4'-nitro group, the phenolic hydroxy group and the anilide amino group in mediating uncoupling activity. Our structural investigations provide important information that will aid further drug development.

Study of Stem Cells Influence on Cardiac Cells Cultured with a Cyanide-P-Trifluoromethoxyphenylhydrazone in Organ-on-a-Chip System.

Regenerative medicine and stem cells could prove to be an effective solution to the problem of treating heart failure caused by ischemic heart disease. However, further studies on the understanding of the processes which occur during the regeneration of damaged tissue are needed. Microfluidic systems, which provide conditions similar to in vivo, could be useful tools for the development of new therapies using stem cells. We investigated how mesenchymal stem cells (MSCs) affect the metabolic activity of cardiac cells (rat cardiomyoblasts and human cardiomyocytes) incubated with a potent uncoupler of mitochondrial oxidative phosphorylation under microfluidic conditions. A cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was used to mimic disfunctions of mitochondria of cardiac cells. The study was performed in a microfluidic system integrated with nanofiber mats made of poly-l-lactid acid (PLLA) or polyurethane (PU). The microsystem geometry allows four different cell cultures to be conducted under different conditions (which we called: normal, abnormal-as both a mono- and co-culture). Metabolic activity of the cells, based on the bioluminescence assay, was assessed in the culture's performed in the microsystem. It was proved that stem cells increased metabolic activity of cardiac cells maintained with FCCP.

Mitochondrial Diagnostics: A Discovery-Based Biochemical Platform for Phenotyping Human Peripheral Blood Cell Mitochondria.

In vitro experiments using permeabilized cells and/or isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across multiple scientific fields to shed valuable insight on mitochondrial-linked pathologies. The present chapter is intended to serve as a methodological blueprint for comprehensively phenotyping peripheral blood cell mitochondria. While primarily adapted for peripheral blood cells, the protocols outlined herein could easily be made amenable to most all cell types with minimal modifications.

A Comparative Study of the Action of Protonophore Uncouplers and Decoupling Agents as Inducers of Free Respiration in Mitochondria in States 3 and 4: Theoretical and Experimental Approaches.

Theoretical and experimental studies have revealed that that in the liver mitochondria an increase in the rate of free respiration in state 3 induced by protonophore uncouplers 2,4-dinitrophenol and сarbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone is equal to or slightly greater than the increase in respiration rate in state 4 induced by these uncouplers. In contrast to these protonophore uncouplers, the decoupler α,ω-tetradecanedioic acid, increasing the rate of respiration in state 4, does not significantly affect the rate of free respiration in state 3. We have proposed quantitative indicators that allow determining the constituent part of the rate of respiration in state 4, associated with the decoupling effect of the uncoupler. Using the example of palmitic acid, we have found out the fundamental possibility of the simultaneous functioning of uncouplers by two mechanisms: as protonophores and as decouplers. The data obtained contradict the delocalized version of Mitchell's chemiosmotic theory, but are in complete agreement with its local version. It can be assumed that the F0F1-ATP synthase and nearby respiratory chain complexes form a local zone of coupled respiration and oxidative ATP synthesis (zones of oxidative phosphorylation). The uncoupler-induced stimulation of mitochondrial free respiration of mitochondria in state 3 is mainly due to the return of protons to the matrix in local zones, where the generation of a proton motive force (Δр) by respiratory chain complexes is associated with various transport processes, but not with ATP synthesis (zones of protonophore uncoupling). In contrast, respiratory stimulation in state 4 by decouplers is realized in local zones of oxidative phosphorylation by switching the respiratory chain complexes to the idle mode of operation in the absence of ATP synthesis.

Current mechanistic insights into the CCCP-induced cell survival response.

The ring-substituted derivatives of carbonyl cyanide phenylhydrazone, CCCP and FCCP, are routinely used for the analysis of the mitochondrial function in living cells, tissues, and isolated mitochondrial preparations. CCCP and FCCP are now being increasingly used for investigating the mechanisms of autophagy by inducing mitochondrial degradation through the disruption of the mitochondrial membrane potential (ΔΨm). Sustained perturbation of ΔΨm, which is normally tightly controlled to ensure cell proliferation and survival, triggers various stress pathways as part of the cellular adaptive response, the main components of which are mitophagy and autophagy. We here review current mechanistic insights into the induction of mitophagy and autophagy by CCCP and FCCP. In particular, we analyze the cellular modifications produced by the activation of two major pathways involving the signaling of the nuclear factor erythroid 2-related factor 2 (Nrf2) and the transcription factor EB (TFEB), and discuss the contribution of these pathways to the integrated cellular stress response.

Anacardic acid-mediated changes in membrane potential and pH gradient across liposomal membranes.

We have previously shown that anacardic acid has an uncoupling effect on oxidative phosphorylation in rat liver mitochondria using succinate as a substrate (Life Sci. 66 (2000) 229-234). In the present study, for clarification of the physicochemical characteristics of anacardic acid, we used a cyanine dye (DiS-C3(5)) and 9-aminoacridine (9-AA) to determine changes of membrane potential (DeltaPsi) and pH difference (DeltapH), respectively, in a liposome suspension in response to the addition of anacardic acid to the suspension. The anacardic acid quenched DiS-C3(5) fluorescence at concentrations higher than 300 nM, with the degree of quenching being dependent on the log concentration of the acid. Furthermore, the K(+) diffusion potential generated by the addition of valinomycin to the suspension decreased for each increase in anacardic acid concentration used over 300 nM, but the sum of the anacardic acid- and valinomycin-mediated quenching was additively increasing. This indicates that the anacardic acid-mediated quenching was not due simply to increments in the K(+) permeability of the membrane. Addition of anacardic acid in the micromolar range to the liposomes with DeltaPsi formed by valinomycin-K(+) did not significantly alter 9-AA fluorescence, but unexpectedly dissipated DeltaPsi. The DeltaPsi preformed by valinomycin-K(+) decreased gradually following the addition of increasing concentrations of anacardic acid. The DeltaPsi dissipation rate was dependent on the pre-existing magnitude of DeltaPsi, and was correlated with the logarithmic concentration of anacardic acid. Furthermore, the initial rate of DeltapH dissipation increased with logarithmic increases in anacardic acid concentration. These results provide the evidence for a unique function of anacardic acid, dissimilar to carbonylcyanide p-trifluoromethoxyphenylhydrazone or valinomycin, in that anacardic acid behaves as both an electrogenic (negative) charge carrier driven by DeltaPsi, and a 'proton carrier' that dissipates the transmembrane proton gradient formed.

Loss of mitochondrial transmembrane potential and glutathione depletion are not sufficient to account for induction of apoptosis by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone in human leukemia K562 cells.

Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), an uncoupler of mitochondrial oxidative phosphorylation, inhibits cell proliferation and induces cell death with apoptotic features. It was reported that the cytotoxic effects of FCCP are preceded by a rapid glutathione (GSH) depletion with a subsequent loss of mitochondrial transmembrane potential (ΔΨ). The GSH depletion was suggested as the cause of apoptosis in FCCP treated cells. This conclusion was further supported by the finding that all adverse effects of FCCP including cell death can be prevented by N-acetylcysteine (NAC) a precursor of GSH synthesis (Han and Park, 2011). Here, we argue that neither loss of ΔΨ nor GSH depletion is sufficient to account for induction of apoptosis in FCCP treated leukemia K562 cells. Indeed, the lowest concentration of FCCP that brings about the permanent loss of ΔΨ and the extensive decrease in GSH level induces cell death in minor population of cells. Only much higher concentrations of FCCP, that exceed the range to achieve permanent collapse of ΔΨ, induce extensive apoptosis. The low proapoptotic activity of FCCP could be explained by hyperactivation of protein kinase B/Akt. A detailed LC/MS/MS analysis of cell extracts revealed extensive formation of FCCP adducts with GSH. This effect could explain the mechanism of GSH depletion, which is currently unknown. Although NAC induces an increase in the GSH pool, this effect is not crucial for abrogation of FCCP cytotoxicity. Indeed, the presence of NAC in the growth medium causes a rapid clearance of FCCP due to its quantitative conversion into the FCCP-NAC adduct, which is the real cause of abrogated FCCP cytotoxicity.

Simultaneous imaging of protonated and deprotonated carbonylcyanide p-trifluoromethoxyphenylhydrazone in live cells by Raman microscopy.

We report the simultaneous imaging of protonated and deprotonated forms of carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP) molecules in live cells by Raman microscopy. Nitriles, structure-sensitive Raman tags, are used to detect the two distinct molecular structures, demonstrating the potential of Raman microscopy for structure-based imaging of bioactive small molecules.

The P2X7 receptor is a key modulator of aerobic glycolysis.

Ability to adapt to conditions of limited nutrient supply requires a reorganization of the metabolic pathways to balance energy generation and production of biosynthetic intermediates. Several fast-growing cells overexpress the P2X7 receptor (P2X7R) for extracellular ATP. A feature of this receptor is to allow growth in the absence of serum. We show here that transfection of P2X7R allows proliferation of P2X7R-transfected HEK293 (HEK293-P2X7) cells not only in the absence of serum but also in low (4 mM) glucose, and increases lactate output compared with mock-transfected HEK293 (HEK293-mock) cells. In HEK293-P2X7, lactate output is further stimulated upon addition of exogenous ATP or the mitochondrial uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). In the human neuroblastoma cell line ACN, lactate output is also dependent on P2X7R function. P2X7R-expressing cells upregulate (a) the glucose transporter Glut1, (b) the glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), (c) phosphofructokinase (PFK), (d) pyruvate kinase M2 (PKM2) and (e) pyruvate dehydrogenase kinase 1 (PDHK1); furthermore, P2X7R expression (a) inhibits pyruvate dehydrogenase (PDH) activity, (b) increases phosphorylated Akt/PKB and hypoxia-inducible factor 1α (HIF-1α) expression and (c) enhances intracellular glycogen stores. In HEK293-P2X7 cells, glucose deprivation increases lactate production, expression of glycolytic enzymes and ph-Akt/PKB level. These data show that the P2X7R has an intrinsic ability to reprogram cell metabolism to meet the needs imposed by adverse environmental conditions.

Mitochondrial dysfunction in Caenorhabditis elegans causes metabolic restructuring, but this is not linked to longevity.

Lifespan in Caenorhabditis elegans, Drosophila, and mice can be extended by a decrease in mitochondrial electron transport chain (ETC) function, but the mechanism behind this extension is unknown. In the present study, we combine detailed metabolic analysis with lifespan determination following suppression of individual genes encoding respiratory complexes I-IV. We report that reduced complexes I, III, and IV activity extend lifespan but that complex II disruption does not. However, disruption to all four complexes affected metabolism in a similar manner suggesting that metabolic effects induced by ETC disruption are separable from lifespan extension. We found that suppression of ETC components induces a starvation-like metabolic response via the nuclear hormone receptor NHR-49. This includes induction of genes for mitochondrial fatty-acid ß-oxidation (acs-2), the glyoxylate cycle (gei-7), gluconeogensis (PEPCK), and glycolysis (gpd-3). Interestingly, a null mutation of nhr-49 attenuated induction of these metabolic pathways, but did not affect the lifespan extension associated with decreases in complexes I, III, and IV function. Together, our results suggest that restructuring of metabolism via NHR-49 in C. elegans with mitochondrial dysfunction does not cause lifespan extension.

Tamoxifen and estradiol interact with the flavin mononucleotide site of complex I leading to mitochondrial failure.

This study evaluated the action of tamoxifen and estradiol on the function of isolated liver mitochondria. We observed that although tamoxifen and estradiol per se did not affect mitochondrial complexes II, III, or IV, complex I is affected, this effect being more drastic (except for state 4 of respiration) when mitochondria were coincubated with both drugs. Furthermore, using two respiratory chain inhibitors, rotenone and diphenyliodonium chloride, we identified the flavin mononucleotide site of complex I as the target of tamoxifen and/or estradiol action(s). Tamoxifen (25 microm) per se induced a significant increase in hydrogen peroxide production and state 4 of respiration. Additionally, a significant decrease in respiratory control ratio, transmembrane, and depolarization potentials were observed. Estradiol per se decreased carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)-stimulated respiration, state 3 of respiration, and respiratory control ratio and increased lag phase of repolarization. With the exception of state 4 of respiration whose increase induced by tamoxifen was reversed by the presence of estradiol, the effects of tamoxifen were highly exacerbated when estradiol was present. We observed that 10 microm tamoxifen in the presence of estradiol affected mitochondria significantly by decreasing FCCP-stimulated respiration, state 3 of respiration, respiratory control ratio, and ADP depolarization and increasing the lag phase of repolarization. All of the deleterious effects induced by 25 microm tamoxifen were highly exacerbated in the presence of estradiol. Furthermore, we observed that the effects of both compounds were independent of estrogen receptors because the pure estrogen antagonist ICI 182,780 did not interfere with tamoxifen and/or estradiol detrimental effects. Altogether, our data provide a mechanistic explanation for the multiple cytotoxic effects of tamoxifen including its capacity to destroy tamoxifen-resistant breast cancer cells in the presence of estradiol. This new piece of information provides a basis for the development of new and promising anticancer therapeutic strategies.

[Membrane ATPase of Vibrio alginolyticus. Ion transport activity and homology with F0F1-ATPase from E. coli]. / Membrannaia ATPaza Vibrio alginolyticus. Ion-transportnaia aktivnost' i gomologiia s F0F1-ATPazoi E. coli.

F0F1-ATPase has been isolated from the marine alkali-resistant bacterium Vibrio alginolyticus. The enzyme subunits cross-reacted with antibodies against subunits alpha, beta, gamma, epsilon, and b of E. coli ATPase. The purified ATPase was reconstituted into liposomes effecting an ATP-dependent uptake of H+. Proton transport was inhibited by the ATPase blockers DCCD, triphenyltin, and venturicidin. Na+ ions had no effect on ATP-dependent proton transport. No ATP-dependent transport of Na+ was detected in proteoliposomes.

Calcium binding to the cytosol and calcium extrusion mechanisms in intact synaptosomes and their alterations with aging.

A simple method to measure cytosolic calcium binding in intact presynaptic nerve terminals (synaptosomes) from rat brain, which is based on the simultaneous determination of [Ca2+]i and total [45Ca2+] in quin2-loaded synaptosomes undergoing a switch from high- to low-calcium containing medium, is presented. Binding to the cytosolic compartment alone was obtained following depletion of calcium storing organelles in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone/oligomycin plus caffeine. Synaptosomes, as compared to various cells types, have a high calcium binding capacity to the cytosolic compartment; maximum binding, Ca.Bmax, was 4.76 mM and calculated s0.5 was 218 nM. Calcium binding to the cytosolic compartment as a function of aging was also determined; Ca.Bmax was reduced to 1.84 mM and s0.5 increased to 492 nM in 30-month-old rats, indicating that the buffering of high calcium loads is impaired in old animals. The results obtained for binding of calcium to mitochondria and caffeine-sensitive calcium stores are consistent with an age-dependent reduction in calcium bound to mitochondria, whereas caffeine-sensitive calcium stores were unaffected. Finally, we have estimated the net rates of calcium extrusion in intact synaptosomes, and found that calcium efflux through the Na/Ca exchanger and Ca(2+)-ATPase was markedly reduced in old rats.