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Mitochondria can be a source of reactive oxygen species (ROS) and a target of oxidative damage during oxidative stress. In this connection, the effect of photodynamic treatment (PDT) with Mitotracker Red (MR) as a mitochondria-targeted photosensitizer has been studied in HeLa cells. It is shown that MR produces both singlet oxygen and superoxide anion upon photoactivation and causes photoinactivation of gramicidin channels in a model system (planar lipid bilayer). Mitochondria-targeted antioxidant (MitoQ) inhibits this effect. In living cells, MR-mediated PDT initiates a delayed (“dark”) accumulation of ROS, which is accelerated by inhibitors of the respiratory chain (piericidin, rotenone and myxothiazol) and inhibited by MitoQ and diphenyleneiodonium (an inhibitor of flavin enzymes), indicating that flavin of Complex I is involved in the ROS production. PDT causes necrosis that is prevented by MitoQ. Treatment of the cell with hydrogen peroxide causes accumulation of ROS, and the effects of inhibitors and MitoQ are similar to that described for the PDT model. Apoptosis caused by H2O2 is augmented by the inhibitors of respiration and suppressed by MitoQ. It is concluded that the initial segments of the respiratory chain can be an important source of ROS, which are targeted to mitochondria, determining the fate of the cell subjected to oxidative stress.

In our search for fluorescent uncouplers of oxidative phosphorylation, three esters of fluorescein, n-butyl-, n-octyl-, and n-dodecyl-oxycarbonyl-fluorescein (C4-FL, C8-FL, C12-FL) were synthesized and characterized. With increasing liposomal lipid content, the long-chain alkyl derivatives of fluorescein (C8-FL, C12-FL and commercially available C18-FL), but not C4-FL and unsubstituted fluorescein, exhibited an increase in fluorescence polarization reflecting the dye binding to liposomes. C12-FL induced proton permeability in lipid membranes, while C4-FL was inactive. In contrast to C4-FL and C18-FL, C12-FL and C8-FL increased the respiration rate and decreased the membrane potential of isolated rat liver mitochondria with half-maximal effective concentrations of 700nM and 300nM, respectively. The effect of Cn-FL on the respiration correlated with that on proton permeability of the inner mitochondrial membrane, as measured by induction of mitochondria swelling in the potassium acetate medium. Binding of C8-FL to mitochondria depended on their energization, which was apparently associated with pH gradient generation across the inner mitochondrial membrane in the presence of a respiratory substrate. In wild-type yeast cells, C12-FL localized predominantly in plasma membrane, whereas in AD1-8 mutants lacking MDR pumps, it stained cytoplasmic organelles with some preference for mitochondria. Fluorescent uncouplers can be useful as a tool for determining their localization in a cell or distribution between different tissues in a living animal by fluorescent microscopy.

This paper presents a simple model to describe experimental data on weak acid transport across planar bilayer lipid membrane separating two buffered solutions. The model takes into account multiple proton-transfer reactions occurring in the unstirred layers (ULs) adjacent to the membrane. Differential equations of the model are shown to be reduced to a set of nonlinear algebraic equations. Since the latter equations depend monotonically on unknown variables, they can be easily solved numerically, using bisection method. For the particular system studied experimentally (with acetate as the weak acid and TRIS+MES as the buffer mixture) pH profiles in the ULs are calculated from the model. These results are compared with experimental data obtained using pH microelectrode. The agreement between theoretical and experimental pH profiles is found to be satisfactory. The most pronounced deviations are observed at the UL/bulk solution boundary. To obtain a better correlation between the theoretical and experimental results, two other, less idealized models are considered. They take into account, respectively, (a) the electric field arising in the ULs from ion diffusion and (b) finiteness of the rates of proton-transfer reactions. However, both acetate membrane fluxes and pH profiles in the ULs computed from these models are found to be close to those of the simple model. One can thus conclude that the difference between experimental and theoretical pH profiles is due to the inconsistency of the generally accepted model of the "unstirred layer", assuming the existence of a strict boundary between the regions of "pure diffusion" and "ideal stirring".

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.

By monitoring the concentration distribution of several solutes that are diffusing at the same time under given mixing conditions, it was established that the unstirred layer (USL) has no clearly defined boundary. For the cases of solute permeation and water movement across planar bilayer lipid membranes, respectively, experiments carried out with double-barreled microelectrodes have shown that the thickness of the USL depends on which species is diffusing. Small molecules with a larger diffusion coefficient encounter an apparently thicker USL than larger molecules with a smaller diffusion coefficient. The ratio of the USL thicknesses of two different substances is equal to the third root of the ratio of the respective diffusion coefficients. This experimental finding is in good agreement with theoretical predictions from the theory of physicochemical hydrodynamics.

Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. The search for cationic uncouplers is promising as their protonophorous effect is self-limiting because these uncouplers lower membrane potential which is the driving force for their accumulation in mitochondria. In this work, the penetrating cation Rhodamine 19 butyl ester (C 4 R1) was found to decrease membrane potential and to stimulate respiration of mitochondria, appearing to be a stronger uncoupler than its more hydrophobic analog Rhodamine 19 dodecyl ester (C 12 R1). Surprisingly, C 12 R1 increased H + conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C 4 R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C 4 R1. In experiments with HeLa cells, C 4 R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C 4 R1 was effective in preventing oxidative stress induced by brain ischemia and reperfusion in rats: it suppressed stroke-induced brain swelling and prevented the decline in neurological status more effectively than C 12 R1. Thus, C 4 R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress.

Antioxidants specifically addressed to mitochondria have been studied to determine if they can decelerate senescence of organisms. For this purpose, a project has been established with participation of several research groups from Russia and some other countries. This paper summarizes the first results of the project. A new type of compounds (SkQs) comprising plastoquinone (an antioxidant moiety), a penetrating cation, and a decane or pentane linker has been synthesized. Using planar bilayer phospholipid membrane (BLM), we selected SkQ derivatives with the highest permeability, namely plastoquinonyl-decyl-triphenylphosphonium (SkQ1), plastoquinonyl-decyl-rhodamine 19 (SkQR1), and methylplastoquinonyldecyltriphenylphosphonium (SkQ3). Anti- and prooxidant properties of these substances and also of ubiquinonyl-decyl-triphenylphosphonium (MitoQ) were tested in aqueous solution, detergent micelles, liposomes, BLM, isolated mitochondria, and cell cultures. In mitochondria, micromolar cationic quinone derivatives were found to be prooxidants, but at lower (sub-micromolar) concentrations they displayed antioxidant activity that decreases in the series SkQ1 = SkQR1 > SkQ3 > MitoQ. SkQ1 was reduced by mitochondrial respiratory chain, i.e. it is a rechargeable antioxidant. Nanomolar SkQ1 specifically prevented oxidation of mitochondrial cardiolipin. In cell cultures, SkQR1, a fluorescent SkQ derivative, stained only one type of organelles, namely mitochondria. Extremely low concentrations of SkQ1 or SkQR1 arrested H2O2-induced apoptosis in human fibroblasts and HeLa cells. Higher concentrations of SkQ are required to block necrosis initiated by reactive oxygen species (ROS). In the fungus Podospora anserina, the crustacean Ceriodaphnia affinis, Drosophila, and mice, SkQ1 prolonged lifespan, being especially effective at early and middle stages of aging. In mammals, the effect of SkQs on aging was accompanied by inhibition of development of such age-related diseases and traits as cataract, retinopathy, glaucoma, balding, canities, osteoporosis, involution of the thymus, hypothermia, torpor, peroxidation of lipids and proteins, etc. SkQ1 manifested a strong therapeutic action on some already pronounced retinopathies, in particular, congenital retinal dysplasia. With drops containing 250 nM SkQ1, vision was restored to 67 of 89 animals (dogs, cats, and horses) that became blind because of a retinopathy. Instillation of SkQ1-containing drops prevented the loss of sight in rabbits with experimental uveitis and restored vision to animals that had already become blind. A favorable effect of the same drops was also achieved in experimental glaucoma in rabbits. Moreover, the SkQ1 pretreatment of rats significantly decreased the H2O2 or ischemia-induced arrhythmia of the isolated heart. SkQs strongly reduced the damaged area in myocardial infarction or stroke and prevented the death of animals from kidney ischemia. In p53−/− mice, 5 nmol/kg × day SkQ1 decreased the ROS level in the spleen and inhibited appearance of lymphomas to the same degree as million-fold higher concentration of conventional antioxidant NAC. Thus, SkQs look promising as potential tools for treatment of senescence and age-related diseases.

The water conductivity of desformylgramicidin exceeds the permeability of gramicidin A by two orders of magnitude. With respect to its single channel hydraulic permeability coefficient of 1.1·10 −12 cm 3 s −1 , desformylgramicidin may serve as a model for extremely permeable aquaporin water channel proteins (AQP4 and AQPZ). This osmotic permeability exceeds the conductivity that is predicted by the theory of single-file transport. It was derived from the concentration distributions of both pore-impermeable and -permeable cations that were simultaneously measured by double barreled microelectrodes in the immediate vicinity of a planar bilayer. From solvent drag experiments, approximately five water molecules were found to be transported by a single-file process along with one ion through the channel. The single channel proton, potassium, and sodium conductivities were determined to be equal to 17pS (pH 2.5), 7 and 3pS, respectively. Under any conditions, the desformyl-channel remains at least 10 times longer in its open state than gramicidin A.

According to recent data, gramicidin A analogues having positively charged amino acid sequences at the C-termini exhibit two types of channel activity in lipid membranes: classical cation-selective channels and large unselective pores. The induction of unselective pores was shown here to strongly depend on the redox state of the membrane-bathing solution, if the gramicidin analogue contained a cysteine residue in the sequence GSGPKKKRKVC attached to the C-terminus. In particular, the addition of H2O2 led to an increase in the transmembrane current and the loss of cationic selectivity on planar bilayer lipid membranes and an increase in the carboxyfluorescein leakage of liposomes. The effect was observed at high concentration of the peptide while was absent at the single-channel level. It was concluded that oxidation led to possible formation of dimers of the peptide, which promoted the formation of large unselective pores.

Novel penetrating cations were used for the design of mitochondria-targeted compounds and tested in model lipid membranes, in isolated mitochondria and in living human cells in culture. Rhodamine-19, berberine and palmatine were conjugated by aliphatic linkers with plastoquinone possessing antioxidant activity. These conjugates (SkQR1,SkQBerb, SkQPalm) and their analogs lacking plastoquinol moiety (C12R1,C10Berb and C10Palm) penetrated bilayer phospholipid membrane in their cationic forms and accumulated in isolated mitochondria or in mitochondria of living cells due to membrane potential negative inside. Reduced forms of SkQR1, SkQBerb and SkQPalm inhibited lipid peroxidation in isolated mitochondria at nanomolar concentrations. In human fibroblasts SkQR1, SkQBerb and SkQPalm prevented fragmentation of mitochondria and apoptosis induced by hydrogen peroxide. SkQR1 was effective at subnanomolar concentrations while SkQberb, SkQPalm and SkQ1 (prototypic conjugate of plastoquinone with dodecyltriphenylphosphonium) were effective at 10-times higher concentrations. The aliphatic conjugates of berberine and palmatine (as well as the conjugates of triphenylphosphonium) induced proton transport mediated by free fatty acids (FA) both in the model and mitochondrial membrane. In mitochondria this process was facilitated by the adenine nucleotide carrier. In contrast to the other cationic conjugates, SkQR1 and C12R1 induced FA-independent proton conductivity due to protonation/deprotonation of the rhodamine residue. This property in combination with the antioxidant activity probably makes rhodamine conjugates highly effective in protection against oxidative stress. The novel cationic conjugates described here are promising candidates for drugs against various pathologies and aging as mitochondria-targeted antioxidants and selective mild uncouplers.

Photosensitized damage to liposome membranes was studied by using different dye-leakage assays based on fluorescence dequenching of a series of dyes upon their release from liposomes. Irradiation of liposomes with red light in the presence of a photosensitizer, trisulfonated aluminum phthalocyanine (AlPcS3), resulted in the pronounced leakage of carboxyfluorescein, but rather weak leakage of sulforhodamin B and almost negligible leakage of calcein from the corresponding dye-loaded liposomes. The photosensitized liposome permeabilization was apparently associated with oxidation of lipid double bonds by singlet oxygen as evidenced by the requirement of unsaturated lipids in the membrane composition for the photosensitized liposome leakage to occur and the sensitivity of the latter to sodium azide. The fluorescence correlation spectroscopy measurements revealed marked permeability of photodynamically induced pores in liposome membranes for such photosensitizers as AlPcS3 and AlPcS4. It was proposed that the difference in permeability of these pores to carboxyfluorescein and calcein was associated with size restriction. Verification of this hypothesis by studying the effect of PEGs of different molecular weights is under way.