L. S. Yaguzhinsky

An attempt to prevent senescence: A mitochondrial approach

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 H(2)O(2)-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 H(2)O(2) 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/kgxday 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.

Mitochondria-Targeted Plastoquinone Derivatives as Tools to Interrupt Execution of the Aging Program. 1. Cationic Plastoquinone Derivatives: Synthesis and in vitro Studies*

Synthesis of cationic plastoquinone derivatives (SkQs) containing positively charged phosphonium or rhodamine moieties connected to plastoquinone by decane or pentane linkers is described. It is shown that SkQs (i) easily penetrate through planar, mitochondrial, and outer cell membranes, (ii) at low (nanomolar) concentrations, posses strong antioxidant activity in aqueous solution, BLM, lipid micelles, liposomes, isolated mitochondria, and cells, (iii) at higher (micromolar) concentrations, show pronounced prooxidant activity, the “window” between anti- and prooxidant concentrations being very much larger than for MitoQ, a cationic ubiquinone derivative showing very much lower antioxidant activity and higher prooxidant activity, (iv) are reduced by the respiratory chain to SkQH2, the rate of oxidation of SkQH2 being lower than the rate of SkQ reduction, and (v) prevent oxidation of mitochondrial cardiolipin by OH·. In HeLa cells and human fibroblasts, SkQs operate as powerful inhibitors of the ROS-induced apoptosis and necrosis. For the two most active SkQs, namely SkQ1 and SkQR1, C1/2 values for inhibition of the H2O2-induced apoptosis in fibroblasts appear to be as low as 1·10−11 and 8·10−13 M, respectively. SkQR1, a fluorescent representative of the SkQ family, specifically stains a single type of organelles in the living cell, i.e. energized mitochondria. Such specificity is explained by the fact that it is the mitochondrial matrix that is the only negatively-charged compartment inside the cell. Assuming that the Δψ values on the outer cell and inner mitochondrial membranes are about 60 and 180 mV, respectively, and taking into account distribution coefficient of SkQ1 between lipid and water (about 13,000: 1), the SkQ1 concentration in the inner leaflet of the inner mitochondrial membrane should be 1.3·108 times higher than in the extracellular space. This explains the very high efficiency of such compounds in experiments on cell cultures. It is concluded that SkQs are rechargeable, mitochondria-targeted antioxidants of very high efficiency and specificity. Therefore, they might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo.

Thread-grain transition of mitochondrial reticulum as a step of mitoptosis and apoptosis

Association of mitochondrial population to a mitochondrial reticulum is typical of many types of the healthy cells. This allows the cell to organize a united intracellular power-transmitting system. However, such an association can create some difficulties for the cell when a part of the reticulum is damaged or when mitochondria should migrate from one cell region to another. It is shown that in these cases decomposition of extended mitochondria to small roundish organelles takes place (the thread-grain transition). As an intermediate step of this process, formation of bead-like mitochondria occurs when several swollen parts of the mitochondrial filament are interconnected with thin thread-like mitochondrial structures. A hypothesis is put forward that the thread-grain transition is used as a mechanism to isolate a damaged part of the mitochondrial system from its intact parts. If the injury is not repaired, spherical mitochondrion originated from the damaged part of the reticulum is assumed to convert to a small ultracondensed and presumably dead mitochondrion (this process is called ‘mitoptosis’). Then the dead mitochondrion is engulfed by an autophagosome. Sometimes, an ultracondensed mitoplast co-exists with a normal mitoplast, both of them being surrounded by a common outer mitochondrial membrane. During apoptosis, massive thread-grain transition is observed which, according to Youle et al. (S. Frank et al., Dev Cell 1: 515, 2002), is mediated by a dynamin-related protein and represents an obligatory step of the mitochondria-mediated apoptosis. We found that there is a lag phase between addition of an apoptogenic agent and the thread-grain transition. When started, the transition occurs very fast. It is also found that this event precedes complete de-energization of mitochondria and cytochrome c release to cytosol. When formed, small mitochondria migrate to (and in certain rare cases even into) the nucleus. It is suggested that small mitochondria may serve as a transportable form of organelles (‘cargo boats’ transporting some apoptotic proteins to their nuclear targets).

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 (C12TPP). 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 (ΔpH) to a membrane potential (Δψ) of the Nernstian value (about 60 mV Δψ at ΔpH = 1). A hydrophobic cation with localized charge (cetyltrimethylammonium) failed to substitute for hydrophobic cations with delocalized charge. In isolated mitochondria, SkQ1 and C12TPP, but not cetyltrimethylammonium, potentiated fatty acid-induced (i) uncoupling of respiration and phosphorylation, and (ii) inhibition of H2O2 formation. In intact yeast cells, C12TPP 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.

Evidence in favor of the existence of a kinetic barrier for proton transfer from a surface of bilayer phospholipid membrane to bulk water

When the hydrogen-ion flux is induced by nigericin across the planar bilayer lipid membrane (BLM) with bulk pH values being equal at the opposite sides of the BLM, formation of a difference in boundary potentials (delta phi b) on the membrane is observed by the method of inner membrane field compensation. pH gradients are titrated routinely by the addition of sodium acetate at one side of the membrane. The increase in buffer concentration (citrate, phosphate, Mes) leads to a decrease in delta phi b. delta phi b forms in the presence of phosphatidylserine in the membrane-forming solution only. It is concluded that the steady-state difference of the hydrogen ion binding to the opposite surfaces of the membrane (HIBD) is created under the conditions of equal pH values near surfaces of the BLM. The model of the processes implies that nigericin transfers proton predominantly from interface to interface while acetate transfers the proton from bulk phase to bulk phase. In the other series of experiments the monensin-mediated formation of the HIBD leads to the formation of an potassium-ion gradient in the presence of nigericin. Thus, a possibility of performing a work due to the formation of HIBD is demonstrated. Owing to these properties the hydrogen-ion binding difference can be interpreted in a first approximation as a difference of surface hydrogen-ion concentration at the opposite sides of the membrane, arising due to the existence of a kinetic barrier for the proton transfer at the membrane interfaces. These findings can be significant for the mechanism of energy transduction in membrane phosphorylation in mitochondria and chloroplasts.

Oxygen-Dependent Auto-Oscillations of Water Luminescence Triggered by the 1264 nm Radiation

A 5-min exposure of air-saturated bidistilled water to low-intensity laser infrared radiation at the wavelength of the electronic transition of dissolved oxygen to the singlet state ((3)∑(g)(-)→ (1)Δ(g)) induces, after a long latent period, auto-oscillations of water luminescence in the blue-green region, which last many hours. Laser irradiation causes the accumulation of hydrogen peroxide, which depends on the concentration of dissolved oxygen. The auto-oscillations do not arise if water is irradiated beyond the oxygen absorption band and if the oxygen is removed from water. The wavelet transform analysis of luminescence records indicates that there are two characteristic periods of pulsations of about 300 and 1150 s. The results obtained suggest that auto-oscillations are triggered by photoinduced singlet oxygen (1)Δ(g), and this phenomenon is closely related to formation of hydrogen peroxide.

Subcellular reorganization of mitochondria producing heavy DNA in aging wheat coleoptiles

Unusual closed membrane vesicles containing one or more mitochondria were isolated from homogenates of aging wheat coleoptiles. Very similar (or the same) bodies were shown to exist in situ in vacuoles of undividing cells in the apical part of intact senescent coleoptiles. Vesicles isolated from coleoptile homogenate free of nuclei by 10 min centrifugation at 1700×g and traditional mitochondria (sedimented at between 4300×g and 17 400×g) are similar in respiration rate, composition and content of cytochromes and sensitivity to respiration inhibitors. However, vesicles contain about 2‐fold more Ca2+ ions than free mitochondria do. The specific feature of vesicles containing mitochondria in aging coleoptiles is an intensive synthesis of heavy (ρ=1.718 g/cm3) mitochondrial DNA (H‐mtDNA). Thus, aging in plants is accompanied by an increased selective H‐mtDNA production and change in subcellular organization of mitochondria.

Effect of the alkyl chain length of monocarboxylic acid on the permeation through bilayer lipid membranes

Electrically silent hydrogen ion fluxes across a planar bilayer lipid membrane (BLM) induced by an addition of monocarboxylic acid at one side of BLM were studied by measuring pH changes in the unstirred layers near the BLM surface. The pH changes were assayed by recording protonophore-dependent potentials as well as by direct measurements of pH shifts in he unstirred layers close to the membrane by the pH microelectrode. It was shown that the mechanism of the acid transport changed qualitatively upon the increase of the hydrophobic chain length of the acid. In the case of short-chain acids at pH < pKa, the total transport was limited by diffusion of the anionic form of the acid across the unstirred layers, while at the alkaline pH (pH>>pKa) the transport was limited by diffusion of the neutral form across the membrane. In the alkaline pH range the pH shifts induced by short-chain acids were sensitive to the presence of cholesterol in the BLM as well as to the stirring conditions in the cell. However, in the case of long chain acids (more than 8 carbonic atoms) the transport was limited by diffusion of the anionic form of the acid in the whole range of pH studied. In the latter case, pH changes in the unstirred layers did not depend on the presence of cholesterol in the membrane, and moreover pH shifts were not dependent on the thickness of the unstirred layer. It was proposed that the peculiarities of the long-chain acid-induced proton transport were associated with the formation of micelles of the acid in bathing solutions.

Necessity of superoxide production for development of etiolated wheat seedlings.

It was found that production of superoxide (O2– ·) is crucial for normal morphogenesis of etiolated wheat seedlings in the early stages of plant development. The development of etiolated wheat seedlings was shown to be accompanied with cyclic changes in the rate of O2– · production both in the entire intact seedling and in its separated organs (leaf, coleoptile). First increase in the rate of O2– · production was clearly observed in the period from two to four days of seedling development, then the rate of O2– · production decreased to the initial level, and then it increased again for two days to a new maximum. An increase in O2– · production in the period of the first four days of seedling development correlates with an increase in DNA and protein contents in the coleoptile. The second peak of increased rate of O2– · production observed on the sixth or seventh day of seedling development coincides with a decrease in DNA and protein contents and apoptotic internucleosomal nuclear DNA fragmentation in the coleoptile. Incubation of seedlings in the presence of the antioxidant BHT (ionol) strongly affects their development but it does not influence the increase in DNA and protein contents for the initial four days of seedling life, and it slows down the subsequent age-dependent decrease in protein content and fully prevents the age-dependent decrease in DNA content in the coleoptile. A decrease in the O2– · amount induced by BHT distorts the seedling development. BHT retards seedling growth, presumably by suppression of cell elongation, and it increases the life span of the coleoptile. It seems that O2– · controls plant growth by cell elongation at the early stages of seedling development but later O2– · controls (induces) apoptotic DNA fragmentation and protein disintegration.

Kinetic properties of cation/H+-exchange : calcimycin (A23187)-mediated Ca2+/2H+-exchange on the bilayer lipid membrane

The calcimycin (A23187)-mediated electrically silent flux of hydrogen ions coupled with a counter transport of calcium or magnesium ions was measured by the method of local pH changes recording in the unstirred layers near the planar bilayer lipid membrane (BLM). It was shown that: (1) the pH dependence of calcimycin-mediated Ca2+/2H+ exchange had a maximum at pH 7; (2) the apparent Michaelis constant for the alkali earth cations were higher at acidic pH than the corresponding values at alkaline pH; (3) the apparent Michaelis constant for calcium was similar to that for magnesium ions in agreement with calcimycine cation binding constants; (4) the ratio of calcium and magnesium fluxes was independent of pH in the pH range from 5 to 8. (5) the flux was proportional to the calcimycin concentration at pH greater than 6.3 and proportional to the square of the carrier concentration at pH less than 5; (6) the addition of calcium ion chelator EDTA increased the flux significantly. These data were discussed in terms of the model of cation/H(+)-exchange and it was concluded that the dissociation of the cation-carrier complex at the membrane/water interface played an important role in the process of calcimycine operation. The comparison of the kinetic properties of calcimycin with the previously described kinetics of nigericin (Antonenko and Yaguzhinsky (1988) Biol. Membr. (Russian) 5, 718-728) revealed much similarity. On the other hand, a significant difference was found between the mechanism of the nigericin K/Na selectivity and calcimycin Ca/Mg selectivity.