Analysis of the membrane potential of rat- and mouse-liver mitochondria by flow cytometry and possible applications

Washed and purified rat- or mouse-liver mitochondria exhibiting high membrane integrity and metabolic activity were studied by flow cytometry. The electrophoretic accumulation/redistribution of cationic lipophilic probes, rhodamine 123, safranine O and a cyanine derivative, 3,3'-dihexyloxadicarbocyanine iodide, during the energization process was studied and was consistent with the generation of a negative internal membrane potential. An exception to this was nonylacridine orange which spontaneously bound to the mitochondrial membrane by hydrophobic interactions via its hydrocarbon chain. Energized purified mitochondria stained with potentiometric dyes exhibited both higher fluorescence and population homogeneity than the non-energized or deenergized (nigericin plus valinomycin) mitochondria. By contrast, under non-energized or deenergized conditions, the mitochondrial population exhibited fluorescence intensity heterogeneity related to the residual membrane potential; two subpopulations were evident, one of low fluorescence which may be related to the autofluorescence of the mitochondria (plus non-specific dye binding) and a second population which exhibited high fluorescence. Flow cytometry of the unpurified, simply washed, rat-liver mitochondria stained with rhodamine 123, a classically used dye, provided evidence of their heterogeneity in terms of light-scattering properties and membrane-potential-related fluorescence. One third of the washed mitochondria were found to be non-functional by such assays. The fluorescence of purified rat-liver mitochondria due to the membrane potential built up by endogenous substrates indicates heterogeneity of the mitochondrial population with respect to levels of endogenous substrates. The low-angle light scattering increases upon energization and provides some original information about the shape and modification of the inner mitochondrial conformation accompanying the energization. The heterogeneity of the rat liver mitochondrial population, from a structural, metabolic (existence of endogenous substrates) and functional (active and non-active mitochondrial population dispersion) point of view could thus be demonstrated by flow-cytometry analysis. Two animal models were examined with regard to the alteration of the mitochondrial membrane potential under the effects of drugs (rat-liver mitochondria), and the effects of ammonium toxicity (mouse-liver mitochondria). These results are promising and open new perspectives in the study of mitochondriopathies.     

Peak intensity analysis as a method for estimation of fluorescent probe binding to artificial and natural nanoparticles: Tetramethylrhodamine uptake by isolated mitochondria

A modified version of fluorescence correlation spectroscopy (FCS) closely related to the photon counting histogram (PCH) method, which is used in the case of a mixture of molecules with similar diffusion coefficients, was applied here for analyzing the binding of the potential-sensitive dye tetramethylrhodamine ethyl ester, TMRE, to isolated mitochondria both in energized and deenergized states. Fluorescence time traces of suspensions of TMRE-doped mitochondria representing sequences of peaks of different intensity appeared to be similar to those of fluorescent beads and TMRE-doped latex particles. The experimental data were obtained under stirring conditions which increased the number of events by about three orders of magnitude thus substantially enhancing the resolution of the method. The statistics of the brightness of identical fluorescent particles reflecting their random walk through the confocal volume was described by a simple analytical equation which enabled us to perform the peak intensity analysis (PIA) of TMRE-doped mitochondria. The validity of PIA was tested with fluorescent beads of different sizes and TMRE-doped latex particles. Mitochondrial energization in the presence of TMRE led to the increase in the number and the intensity of the peaks in fluorescence time traces, the PIA of which allowed us to determine mitochondrial membrane potential and additionally a number of mitochondrial particles per ml of the suspension. The value of the membrane potential on a single mitochondrion was estimated to be about 180 mV in agreement with the data related to mitochondrial suspensions. Importantly, the PIA method required less than 1 microgram of mitochondrial protein per measurement.     

Intramitochondrial accumulation of cationic Atto520-biotin proceeds via voltage-dependent slow permeation through lipid membrane

Conjugation to penetrating cations is a general approach for intramitochondrial delivery of physiologically active compounds, supported by a high membrane potential of mitochondria having negative sign on the matrix side. By using fluorescence correlation spectroscopy, we found here that Atto520-biotin, a conjugate of a fluorescent cationic rhodamine-based dye with the membrane-impermeable vitamin biotin, accumulated in energized mitochondria in contrast to biotin-rhodamine 110. The energy-dependent uptake of Atto520-biotin by mitochondria, being slower than that of the conventional mitochondrial dye tetramethyl-rhodamine ethyl ester, was enhanced by the hydrophobic anion tetraphenylborate (TPB). Atto520-biotin also exhibited accumulation in liposomes driven by membrane potential resulting from potassium ion gradient in the presence valinomycin. The induction of electrical current across planar bilayer lipid membrane by Atto520-biotin proved the ability of the compound to permeate through lipid membrane in a cationic form. Atto520-biotin stained mitochondria in a culture of L929 cells, and the staining was enhanced in the presence of TPB. Therefore, the fluorescent Atto520 moiety can serve as a vehicle for intramitochondrial delivery of hydrophilic drugs. Of importance for biotin-streptavidin technology, binding of Atto520-biotin to streptavidin was found to cause quenching of its fluorescence similar to the case of fluorescein-4-biotin.     

Fluorescent cationic probes of mitochondria. Metrics and mechanism of interaction.

Mitochondria strongly accumulate amphiphilic cations. We report here a study of the association of respiring rat liver mitochondria with several fluorescent cationic dyes from differing structural classes. Using gravimetric and fluorometric analysis of dye partition, we find that dyes and mitochondria interact in three ways: (a) uptake with fluorescence quenching, (b) uptake without change in fluorescence intensity, and (c) lack of uptake. For dyes that quench upon uptake, the extent of quenching correlates with the degree of aggregation of the dye to dimers, as predicted by theory (Tomov, T.C. 1986. J. Biochem. Biophys. Methods. 13:29-38). Also predicted is the relationship observed between quenching and the mitochondria concentration when constant dye is titrated with mitochondria. Not predicted is the relationship observed between quenching and dye concentration when constant mitochondria are titrated with dye. Because a limit to dye uptake exists, in this case, the degree of quenching decreases as dye is added. A Langmuir isotherm analysis gives phenomenological parameters that predict quenching when it is observed as a function of dye concentration. By allowing for a decrease in membrane potential, caused by incorporation of cationic dye into the lipid bilayer, a modification of the Tomov theory predicts the dye titration data. We present a model of cationic dye-mitochondria interaction and discuss the use of these as probes of mitochondrial membrane potential.     

Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties

The spectral and metabolic properties of Rhodamine 123, a fluorescent cationic dye used to label mitochondria in living cells, were investigated in suspensions of isolated rat-liver mitochondria. A red shift of Rhodamine 123 absorbance and fluorescence occurred following mitochondrial energization. Fluorescence quenching of as much as 75% also occurred. The red shift and quenching varied linearly with the potassium diffusion potential, but did not respond to delta pH. These energy-linked changes were accompanied by dye uptake into the matrix space. Concentration ratios, in-to-out, approached 4000:1. A large fraction of internalized dye was bound. At concentrations higher than those needed to record these spectral changes, Rhodamine 123 inhibited ADP-stimulated (State 3) respiration of mitochondria (Ki = 12 microM) and ATPase activity of inverted inner membrane vesicles (Ki = 126 microM) and partially purified F1-ATPase (Ki = 177 microM). The smaller Ki for coupled mitochondria was accounted for by energy-dependent Rhodamine 123 uptake into the matrix. Above about 20 nmol/mg protein (10 microM), Rhodamine 123 caused rapid swelling of energized mitochondria. Effects on electron-transfer reactions and coupling were small or negligible even at the highest Rhodamine 123 concentrations employed. delta psi-dependent Rhodamine 123 uptake together with Rhodamine 123 binding account for the intense fluorescent staining of mitochondria in living cells. Inhibition of mitochondria ATPase likely accounts for the cytotoxicity of Rhodamine 123. At concentrations which do not inhibit mitochondrial function, Rhodamine 123 is a sensitive and specific probe of delta psi in isolated mitochondria.     

Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties

The spectral and metabolic properties of Rhodamine 123, a fluorescent cationic dye used to label mitochondria in living cells, were investigated in suspensions of isolated rat-liver mitochondria. A red shift of Rhodamine 123 absorbance and fluorescence occurred following mitochondrial energization. Fluorescence quenching of as much as 75% also occurred. The red shift and quenching varied linearly with the potassium diffusion potential, but did not respond to ΔpH. These energy-linked changes were accompanied by dye uptake into the matrix space. Concentration ratios, in-to-out, approached 4000:1. A large fraction of internalized dye was bound. At concentrations higher than those needed to record these spectral changes, Rhodamine 123 inhibited ADP-stimulated (State 3) respiration of mitochondria (K_i = 12 μM) and ATPase activity of inverted inner membrane vesicles (K_i = 126 μM) and partially purified F₁-ATPase (K_i = 177 μM). The smaller K_i for coupled mitochondria was accounted for by energy-dependent Rhodamine 123 uptake into the matrix. Above about 20 nmol/mg protein (10 μM), Rhodamine 123 caused rapid swelling of energized mitochondria. Effects on electron-transfer reactions and coupling were small or negligible even at the highest Rhodamine 123 concentrations employed. Δψ-dependent Rhodamine 123 uptake together with Rhodamine 123 binding account for the intense fluorescent staining of mitochondria in living cells. Inhibition of mitochondria ATPase likely accounts for the cytotoxicity of Rhodamine 123. At concentrations which do not inhibit mitochondrial function, Rhodamine 123 is a sensitive and specific probe of Δψ in isolated mitochondria.     

Intracellular distribution of the fluorescent dye nonyl acridine orange responds to the mitochondrial membrane potential: implications for assays of cardiolipin and mitochondrial mass

Cardiolipin, a polyunsaturated acidic phospholipid, is found exclusively in bacterial and mitochondrial membranes where it is intimately associated with the enzyme complexes of the respiratory chain. Cardiolipin structure and concentration are central to the function of these enzyme complexes and damage to the phospholipid may have consequences for mitochondrial function. The fluorescent dye, 10 nonyl acridine orange (NAO), has been shown to bind cardiolipin in vitro and is frequently used as a stain in living cells to assay cardiolipin content. Additionally, NAO staining has been used to measure the mitochondrial content of cells as dye binding to mitochondria is reportedly independent of the membrane potential. We used confocal microscopy to examine the properties of NAO in cortical astrocytes, neonatal cardiomyocytes and in isolated brain mitochondria. We show that NAO, a lipophilic cation, stained mitochondria selectively. However, the accumulation of the dye was clearly dependent upon the mitochondrial membrane potential and depolarisation of mitochondria induced a redistribution of dye. Moreover, depolarisation of mitochondria prior to NAO staining also resulted in a reduced NAO signal. These observations demonstrate that loading and retention of NAO is dependant upon membrane potential, and that the dye cannot be used as an assay of either cardiolipin or mitochondrial mass in living cells.     

Undesirable feature of safranine as a probe for mitochondrial membrane potential

This communication describes experiments showing that safranine, at the concentrations usually employed as a probe of mitochondrial membrane potential, causes significant undesirable side effects on Ca2+ transport by liver mitochondria. The major observations are: (i) safranine potentiates the spontaneous Ca2+ release from liver mitochondria induced by phosphate or acetoacetate. This is paralelled by potentiation of the release of state-4 respiration and of the rate of mitochondrial swelling, indicating a generalized effect of the dye on the mitochondrial membrane; (ii) the efflux of mitochondrial Ca2+ stimulated by hydroperoxide is irreversible in the presence of safranine even if membrane stabilizers such as Mg2+ and ATP are present. It is concluded that the use of safranine to monitor the changes in membrane potential during Ca2+ transport by mitochondria should be avoided or special care be taken.     

Use of the fluorescent probe, 1-anilino-8-naphthalene sulfonate, to monitor the interaction of pesticide chemicals with mitochondrial membranes

1. The interaction of mitochondrial and submitochondrial membranes with DDT, methoxychlor and kelthane as monitored by fluorescence changes of ANS was studied. 2. The three organochlorine pesticide chemicals reduced the succinate- and ATP-dependent quenching of ANS fluorescence of mitochondria in a concentration-dependent manner. The results suggest that they blocked the supply of energy to the inner membrane. 3. The reversal of the substrate-induced quenching of ANS by uncouplers was abolished by DDT and kelthane probably as a consequence of the inhibition of protonophoric activity of the uncoupler. 4. Both DDT and kelthane were relatively ineffective inhibitors of substrate-induced quenching of ANS fluorescence of submitochondrial particles. The results suggest an asymmetry of the inner membrane with respect to DDT-inhibition of substrate-induced energization. 5. The possible basis of this asymmetry as well as of inhibition of energy supply to the membrane is discussed.     

Berberine derivatives as cationic fluorescent probes for the investigation of the energized state of mitochondria

The interaction of rat liver and bovine heart mitochondria with a series of fluorescent, cationic berberine derivatives varying in the length of alkyl chain has been investigated. An increase in the hydrophobicity of the derivative was accompanied by a larger value of the partition coefficient and by binding to a more hydrophobic region of the inner mitochondrial membrane. It was found that berberines could be used as sensitive indicators of processes which take place on the outer surface of the mitochondrial membrane; the greatest (15-fold) increase in fluorescence was obtained with 13-methylberberine in the energized state of mitochondria. The fluorescence increase was due to the increase in fluorescence quantum yield although a small increase in the amount of bound derivative could also be detected upon energization. The fluorescence was linearly dependent on the magnitude of the membrane potential. In parallel with an observed fluorescence enhancement a considerable decrease in rotational mobility was found. We suggest that berberines move in the inner membrane according to the polarity of the membrane potential; consequently, deeper immersion in the less polar region in the energized state brings about a larger fluorescence increase. More hydrophobic derivatives inhibited NAD-linked respiration in rat liver mitochondria but exerted no effect on succinate oxidation up to 10 μM concentration.     

Does the energy state of mitochondria influence the surface potential of the inner mitochondrial membrane? A critical appraisal

Binding of 8-anilino-1-naphthalene sulphonate (ANS) to rat liver mitochondria and submitochondrial inside-out particles was measured under energized and de-energized conditions. In mitochondria, energization/de-energization changed the binding capacity for ANS extrapolated for its infinitely high concentration, whereas the apparent Kd value remained unchanged. In submitochondrial particles apparent Kd was changed but the extrapolated maximum binding was not altered. These results are compatible with theoretical considerations assuming a free permeability of mitochondrial membranes to ANS and its distribution according to the transmembrane potential. The spin-labelled cationic amphiphile, 4-(dodecyl dimethyl ammonium)-1-oxyl-2,2,6,6-tetramethyl piperidine bromide (CAT12), was trapped by de-energized mitochondria in such a way that about half of the bound probe became inaccessible to reduction by externally added ascorbate. This inaccessible fraction was increased by energization. This indicates that this cationic probe can penetrate through the inner mitochondrial membrane. De-energization produced a parallel shift of the Lineweaver-Burk plots for the oxidation of external ferrocytochrome c by mitoplasts and of succinate by submitochondrial particles. A similar shift was obtained by a partial inhibition of succinate oxidation by antimycin A. Thus, the observed changes of the kinetics of the two membrane-bound enzyme systems on de-energization can be interpreted as reflecting changes of the control points of mitochondrial respiration rather than changes of the surface potential. It is concluded that neither the fluorescent probe ANS, the spin-labelled amphiphilic cation CAT12, nor the kinetics of some respiratory enzyme systems provide a sufficient proof for changes of the surface potential of the inner mitochondrial membrane upon energization.     

Use of oxonol V as a probe of membrane potential in proteoliposomes containing cytochrome oxidase in the submitochondrial orientation

Absorbance changes in the anionic dye bis[3-phenyl-5-oxoisoxazol-4-yl]pentamethineoxonol (oxonol V) can be used to monitor the membrane potential of liposomes and cytochrome c containing cytochrome oxidase proteoliposomes (c-loaded COV). Diffusion potentials (positive inside the vesicles) cause an increase in the dye extinction, with a maximum at 640 nm. A similar increase is seen upon energization of internally facing cytochrome oxidase molecules in c-loaded COV. Both "passive" and "active" responses are only seen when the dye is fully bound to the vesicle membrane. Calibration curves using potassium or n-butyltriphenylphosphonium ion (BTPP+) diffusion potentials are linear up to 100 mV and pass through the origin. Diffusion potentials (positive inside) also cause an increase and red shift in the oxonol V fluorescence emission spectrum. However, potentials of the same sign induced by cytochrome oxidase turnover induce a large fluorescence quenching in c-loaded COV. A similar anomaly has been observed with submitochondrial particles [Smith, J. C., Russ, P., Cooperman, B. S., & Chance, B. (1976) Biochemistry 15, 5094-5105]. A model is proposed consistent with these responses. It is suggested that the dye molecules move further into the membrane phase upon energization, causing the absorbance increase. In the presence of active enzyme, anionic dye molecules are attracted to a positive dipole on each enzyme molecule, causing self-quenching of the fluorescence.     

On measurement of mitochondrial transmembrane potential with fluorescent probes

It is commonly thought that rhodamine, cyanine, and some other fluorescent dyes are specific potential-dependent ones and that they allow quantitatively measuring the transmembrane potential in mitochondria and cells. However, a critical analysis of the experimental data shows that this statement is only a supposition. In reality, widely used fluorescent probes, such as merocyanine 540, Dis-C3-(5), safranin O, or 8-anilino-1-naphthalene sulfonate, poorly bind to the native mitochondria and weakly react to their energization or uncoupling. It can be concluded that calculations of the magnitude of the transmembrane potential of the inner mitochondrial membrane in response to addition of succinate, ATP, or dinitrophenol from the change in fluorescence of these probes are incorrect.     

Plant mitochondrial electrical potential monitored by fluorescence quenching of rhodamine 123

The suitability of the fluorescent dye rhodamine 123 for qualitative and quantitative determinations of the electrical potential difference (ΔΨ) in isolated pea (Pisum sativum L.) stem mitochondria was evaluated. A fluorescence quenching of rhodamine 123, as a consequence of dye uptake, occurred following mitochondria energization by both external and internal substrates. This quenching was associated to the generation of ΔΨ, because it was completely released by uncouplers and respiratory inhibitors. The conversion of the proton gradient (ΔpH) into ΔΨ, induced by nigericin or a permeant weak acid (phosphate), increased the quenching. The uptake of the probe was accompanied by 40 % of unspecific binding in coupled, but not in uncoupled, mitochondria. Rhodamine 123 quenching varied linearly with a K+-diffusion potential. ADP induced a transient and cyclic change of fluorescence which was associated to ATP synthesis. Consequently, rhodamine 123 did not influence oxygen consumption by mitochondria in both state 4 and 3, thus indicating that, at the concentrations assayed, the probe was not toxic. It is concluded that rhodamine 123, followed by fluorescence quenching, is a suitable probe to study the energetics of isolated plant mitochondria. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hexokinase inhibits flux of fluorescently labeled ATP through mitochondrial outer membrane porin

Mitochondrial function requires maintaining metabolite fluxes across the mitochondrial outer membrane, which is mediated primarily by the voltage dependent anion channel (VDAC). We applied fluorescence correlation spectroscopy (FCS) to study regulation of the VDAC functional state by monitoring distribution of fluorescently labeled ATP (BODIPY-FL-ATP) in isolated intact rat liver and heart mitochondria. Addition of mitochondria to BODIPY-FL-ATP solution resulted in accumulation of the fluorescent probe in these organelles. The addition of hexokinase II (HKII) isolated from rat heart led to a decrease in the BODIPY-FL-ATP accumulation, while a 15-residue peptide corresponding to the N-terminal domain of hexokinase did not produce this effect. Therefore, the hexokinase-induced inhibition of the ATP flow mediated by VDAC was revealed in isolated mitochondria.     

Cyanine and safranine dyes as membrane potential probes in cytochrome c oxidase reconstituted proteoliposomes

Safranine and the cyanine dye, 3',3'-dipropylthiadicarbocyanine (diSC3-5), were examined as membrane potential probes in cytochrome c oxidase vesicles. The spectra of the vesicle-associated dyes resemble those of the same dyes in organic solvents, indicating that safranine and diSC3-5 probably dissolve in a hydrophobic region of the proteoliposomal membrane. This binding of safranine to proteoliposomes occurs with a dye-membrane dissociation constant in the micromolar range. The binding of safranine and of diSC3-5 to liposomes or proteoliposomes is accompanied by fluorescence enhancement. This enhanced fluorescence is quenched by respiration or by the establishment of a K+ diffusion potential by valinomycin (negative interior). An optimal dye/lipid ratio was required to secure maximum fluorescence quenching of the dyes, whether that quenching was active or passive. Calibrations of both the safranine and the diSC3-5 responses with K+ diffusion potentials were also affected by the dye/lipid ratio. At lower dye/lipid ratios, the calibration curve was linear at higher potentials but deviated from linearity at lower potentials. The converse was true at higher dye/lipid ratios. The non-linearity of the calibration curve at higher potential was attributed to a 'saturation' effect; it may also involve increased permeability of proteoliposomal membrane to protons. Destacking of dye at the lower dye/lipid ratio was probably responsible for the non-linearity of the calibration curves at lower potentials. When all these effects are taken into account, the steady-state value of delta psi generated during maximal proteoliposomal respiration was calculated to be between 140 and 160 mV (interior negative) when measured with either safranine or diSC3-5. We conclude that quantitative estimates of delta psi values can be made using these probes in cytochrome c oxidase reconstituted proteoliposomes provided that appropriate precautions are taken.     

Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F(0) during ATP synthesis

Rhodamine 123 (RH-123) was used to monitor the membrane potential of mitochondria isolated from rat liver. Mitochondrial energization induces quenching of RH-123 fluorescence and the rate of fluorescence decay is proportional to the mitochondrial membrane potential. Exploiting the kinetics of RH-123 fluorescence quenching in the presence of succinate and ADP, when protons are both pumped out of the matrix driven by the respiratory chain complexes and allowed to diffuse back into the matrix through ATP synthase during ATP synthesis, we could obtain an overall quenching rate proportional to the steady-state membrane potential under state 3 condition. We measured the kinetics of fluorescence quenching by adding succinate and ADP in the absence and presence of oligomycin, which abolishes the ADP-driven potential decrease due to the back-flow of protons through the ATP synthase channel, F(0). As expected, the initial rate of quenching was significantly increased in the presence of oligomycin, and conversely preincubation with subsaturating concentrations of the uncoupler carbonyl cyanide p-trifluoro-metoxyphenilhydrazone (FCCP) induced a decreased rate of quenching. N,N'-dicyclohexylcarbodiimide (DCCD) behaved similarly to oligomycin in increasing the rate of quenching. These findings indicate that RH-123 fluorescence quenching kinetics give reliable and sensitive evaluation of mitochondrial membrane potential, complementing steady-state fluorescence measurements, and provide a mean to study proton flow from the mitochondrial intermembrane space to the matrix through the F(0) channel.     

Interaction of fluorescent berberine alkyl derivatives with respiratory chain of rat liver mitochondria

The cationic fluorescent dyes, berberines, have been observed to inhibit NAD-linked respiration in rat liver mitochondria. Low concentrations inhibit electron transport in the NAD-ubiquinone span after penetration into mitochondria. More hydrophobic alkyl derivatives proved to be stronger inhibitors showing more rapid onset of inhibition. The inhibition was totally dependent on the energization of the membrane; however, the addition of a hydrophobic anion stimulated the inhibition effects in uncoupled mitochondria. Substantially higher concentrations of berberines are needed for the inhibition of the oxidation of succinate. The excess of dye interacting with surface dipoles in the energized state can inhibit the energy transduction through the complexbc ₁. On the basis of the difference in the rate of fluorescence response when berberines are added to coupled mitochondria and the corresponding inhibition effects, the presence minimally of two binding sites was suggested. The dye bound on the outer surface is highly fluorescent and inhibits the energy transduction if added in excess. The remaining dye interacting with NADH dehydrogenase does not fluoresce. The accumulation of alkylberberine in mitochondria results in additional effects in the region of cytochromeb the nature of which is not fully understood.     

Quantitative analysis of spontaneous mitochondrial depolarizations

Spontaneous transient depolarizations in mitochondrial membrane potential (DeltaPsi(m)), mitochondrial flickers, have been observed in isolated mitochondria and intact cells using the fluorescent probe, tetramethylrhodamine ethyl ester (TMRE). In theory, the ratio of [TMRE] in cytosol and mitochondrion allows DeltaPsi(m) to be calculated with the Nernst equation, but this has proven difficult in practice due to fluorescence quenching and binding of dye to mitochondrial membranes. We developed a new method to determine the amplitude of flickers in terms of millivolts of depolarization. TMRE fluorescence was monitored using high-speed, high-sensitivity three-dimensional imaging to track individual mitochondria in freshly dissociated smooth muscle cells. Resting mitochondrial fluorescence, an exponential function of resting DeltaPsi(m), varied among mitochondria and was approximately normally distributed. Spontaneous changes in mitochondrial fluorescence, indicating depolarizations and repolarizations in DeltaPsi(m), were observed. The depolarizations were reversible and did not result in permanent depolarization of the mitochondria. The magnitude of the flickers ranged from <10 mV to >100 mV with a mean of 17.6 +/- 1.0 mV (n = 360) and a distribution skewed to smaller values. Nearly all mitochondria flickered, and they did so independently of one another, indicating that mitochondria function as independent units in the myocytes employed here.     

Changes in mitochondrial function in the cells and cytoplasts of a pig embryo kidney culture as affected by the action of actinomycin D

Vital staining of PE kidney cells by fluorescent cationic dye, ethyl rhodamine, (accumulated inside mitochondria by the membrane potential and hence reflecting their functional state) shows rather close level of the fluorescence intensity both in cells and in their cytoplasts for 10 hours of culture survival in the standard medium. In cells and cytoplasts cultivated in the medium with 0.2 mg/ml of actinomycin D inhibiting RNA synthesis, fluorescence intensity of mitochondria sharply decreases after 10 hours as against the control patterns. It is concluded that mitochondria possess a significant degree of autonomy of the nucleus and it is supposed that a considerable part of mitochondrial RNA is under the nucleus control.