The voltage-dependent anion channel as a biological transistor: theoretical considerations.

The voltage-dependent anion channel (VDAC) is a porin of the mitochondrial outer membrane with a bell-shaped permeability-voltage characteristic. This porin restricts the flow of negatively charged metabolites at certain non-zero voltages, and thus might regulate their flux across the mitochondrial outer membrane. Here, we have developed a mathematical model illustrating the possibility of interaction between two steady-state fluxes of negatively charged metabolites circulating across the VDAC in a membrane. The fluxes interact by contributing to generation of the membrane electrical potential with subsequent closure of the VDAC. The model predicts that the VDAC might function as a single-molecule biological transistor and amplifier, because according to the obtained calculations a small change in the flux of one pair of different negatively charged metabolites causes a significant modulation of a more powerful flux of another pair of negatively charged metabolites circulating across the same membrane with the VDAC. Such transistor-like behavior of the VDAC in the mitochondrial outer membrane might be an important principle of the cell energy metabolism regulation under some physiological conditions.

Failure of exogenous NADH and cytochrome c to support energy-dependent swelling of mitochondria.

The possibility of direct oxidation of external NADH in rat liver mitochondria and of the inner membrane potential generation in this process is still not clear. In the present work, the energy-dependent swelling of mitochondria in the medium containing valinomycin and potassium acetate was measured as one of the main criteria of the proton-motive force generation by complex III, complex IV, and both complexes III and IV of the respiratory chain. Mitochondria swelling induced by external NADH oxidation was compared with that induced by succinate or ferrocyanide oxidation, or by electron transport from succinate to ferricyanide. Mitochondria swelling, nearly equal to that promoted by ferrocyanide oxidation, was observed under external NADH oxidation, but only after the outer mitochondrial membrane was ruptured as a result of the swelling-contraction cycle, caused by succinate oxidation and its subsequent inhibition. In this case, significantly accelerated intermembrane electron transport and well-detected inner membrane potential generation, in addition to mitochondria swelling, were also observed. Presented results suggest that exogenous NADH and cytochrome c do not support the inner membrane potential generation in intact rat liver mitochondria, because the external NADH-cytochrome c reductase system, oriented in the outer mitochondrial membrane toward the cytoplasm, is inaccessible for endogenous cytochrome c reduction; as well, the inner membrane cytochrome c oxidase is inaccessible for exogenous cytochrome c oxidation.

Metabolically derived potential on the outer membrane of mitochondria: a computational model.

The outer mitochondrial membrane (OMM) is permeable to various small substances because of the presence of a voltage-dependent anion channel (VDAC). The voltage dependence of VDAC's permeability is puzzling, because the existence of membrane potential on the OMM has never been shown. We propose that steady-state metabolically derived potential (MDP) may be generated on the OMM as the result of the difference in its permeability restriction for various charged metabolites. To demonstrate the possibility of MDP generation, two models were considered: a liposomal model and a simplified cell model with a creatine kinase energy channeling system. Quantitative computational analysis of the simplified cell model shows that a MDP of up to -5 mV, in addition to the Donnan potential, may be generated at high workloads, even if the OMM is highly permeable to small inorganic ions, including potassium. Calculations show that MDP and DeltapH, generated on the OMM, depend on the cytoplasmic pH and energy demand rate. Computational modeling suggests that MDP may be important for cell energy metabolism regulation in multiple ways, including VDAC's permeability modulation and the effect of electrodynamic compartmentation. The osmotic pressure difference between the mitochondrial intermembrane space and the cytoplasm, as related to the electrodynamic compartmentation effects, might explain the morphological changes in mitochondria under intense workloads.

Mg(2+) induces intermembrane electron transport by cytochrome c desorption in mitochondria with the ruptured outer membrane.

The mechanism of external NADH oxidation in rat liver mitochondria is not clear yet, but it evidently includes the intermembrane electron transport (IMET). We showed that Mg(2+) significantly stimulated IMET insensitive to rotenone-antimycin A-myxothiazol in mitochondria with the hypotonically damaged outer membrane, even in the absence of exogenous cytochrome c. This effect was more specific than simply ionic strength increase. Direct evidence for cytochrome c desorption from mitochondrial membranes under 4 mM Mg(2+) influence was obtained to explain the stimulatory effect of Mg(2+) on external NADH oxidation that was coupled to the inner membrane potential generation. Obtained data suggest that cytochrome b(5) is normally oriented towards the cytosol in the outer membrane, and can be accessible for endogenous cytochrome c reduction only through the outer membrane rupture or permeabilization, to activate external NADH oxidation.

[Mathematical modeling of energy coupling in mitochondria in frames of proton-chemical hypothesis]. / Matematicheskoe modelirovanie energeticheskogo sopriazheniia v mitokhondriiakh v ramkakh protonno-chimicheskoi gipotesy.

The dependence of ATP-synthesis and mitochondria respiration rates on membrane potential was analyzed in frames of proton-chemical hypothesis of oxidative phosphorylation. The mathematical and electrical models of the unit of energy coupling are proposed. Computer analysis of the mathematical model allowed to obtain the theoretical dependence of the rates of ATP synthesis and of the respiration on the membrane potential. Both of all are in good qualitative agreement with experimental data, contradicting chemiosmotic hypothesis.

[Irreversible changes in cytochrome P-420 differential spectrum amplitude in microsomal membranes. New properties of Cu(II)-EDTA complex]. / Neobratimye izmeneniia amplitudy differentsial'nogo spektra tsitokhromaP-420 v membranakh mikrosom. Novye svolstva kompleksa Cu(II)-'EDTA.

The influence of EDTA on the CO-differential spectra of cytochrome P-420 formed in the rat liver microsomes under the effect of 1 mM CuCl2 in the presence of dithionite has been studied. It was shown that the level of revealed cytochrome P-420 was diminished two-fold following 10 mM EDTA addition if dithionite concentration initially added to the medium was about 2 mM. Cytochrome b5 had no changes. Cytochrome P-420, formed under the effect of desoxycholate, was insensitive to EDTA, but its amount decreased after addition of CuCl2. It is supposed to proceed under chelatation of heme Fe(II).

[Effect of paracetamol on the microsomal oxidation, oxidative phosphorylation and liver mitochondria swelling in rats of various ages]. / Vliianie paratsetamola na mikrosomal'noe okislenie, okislitel'noe fosforilirovanie i nabukhanie mitokhondrii pecheni krys raznogo vozrasta.

The effect of hepatotoxic dose of paracetamol (800 mg per kg, intraperitoneally, once a day during two days) on the system of microsomal oxidation, respiration, oxidative phosphorylation and high amplitude swelling of liver mitochondria was studied on 1-, 4- and 30-months old Wistar male rats. It has been shown, that paracetamol injection leads to the decrease of content of cytochrome P-450, to disorders of the function of monooxygenase system (the aminopyrine-N-demethylase and aniline hydroxylase activities were diminished), mitochondria macrostructure (the mitochondria high amplitude swelling time was decreased) and function (the respiratory control was decreased). These alterations have been observed to manifest to more extent in the liver of young rats as compared with old ones.

[Intermembrane electron transport in the dynamics of high-amplitude swelling of rat liver mitochondria]. / Mezhmembrannyi perenos élektronov v dinamike vysokoamplitudnogo nabukhaniia mitokhondrii pecheni krys.

The rate of rotenone insensitive NADH oxidation depending on the extent of rat liver mitochondria swelling in isotonic sucrose medium has been studied. The rate of exogenous NADH oxidation was determined fluorimetrically in the presence of 4 microM cytochrome c in the medium after the inhibition of high-amplitude swelling at its different stages by means of rotenone +Mg2+ + trifluoromethoxyphenylhydrasonecarbonylcyanide (FCCP) addition. Activation of external pathway of NADH oxidation in phosphate containing medium was conditioned by quick energy and phosphate-dependent mitochondria swelling that leads to the outer membrane disruption. An increase in the rate of NADH oxidation as a result of the outer membrane disruption during high amplitude swelling in isotonic sucrose medium has been shown to be nearly proportional to the extent of the decrease in optical density of mitochondria suspension. Similar decrease in optical density of homogenates incubated 25-30 min in isotonic sucrose medium was also accompanied by a significant increase in Mg(2+)-cytochrome c-dependent rotenone insensitive NADH oxidation. A decrease in optical density of liver homogenates was prevented by EGTA+2+, rotenone+Mg2++FCCP, rotenone or FCCP alone and was nearly completely inhibited at any stages of changes by means of rotenone+Mg2++FCCP addition that usually inhibit mitochondrial swelling. That means that the rate of Mg(2+)-cytochrome-c-dependent rotenone insensitive NADH oxidation in homogenates may be used as the simple method to evaluate the outer mitochondrial membrane disruption with the purpose to analyse the liver bioptate to study the effect of hormonal, xenobiotic and physical factors on mitochondrial macrostructure.

Hypotonic fragility of outer membrane and activation of external pathway of NADH oxidation in rat liver mitochondria are increased with age.

Great importance is attached to structural and functional deterioration of mitochondria as a reason for ageing of an organism; the attention of many scientists has been concentrated on such questions as age changes in the system of oxidative phosphorylation, damage of mitochondrial DNA by free radicals generated in the respiratory chain and inclusion of some fragments of mitochondrial DNA into the nuclear genome. Mitochondrial high amplitude swelling in a cell under some extreme conditions can possibly play a very important role in mechanisms of deterioration of energy transformation function, in activation of lipid peroxidation and mitochondrial DNA damage as a result of outer membrane disruption and release of enzymes from the intermembrane space (e.g. superoxide dismutase amd adenylate kinase). In this work the age changes of the hypotonic fragility of the outer membrane of rat liver mitochondria and the activation of the external, rotenone-insensitive pathway of NADH oxidation have been examined. It is shown that the obligatory condition for activation of rotenone-insensitive NADH oxidation is a break in the outer membrane and that the rate of NADH oxidation substantially increases in the presence of physiological concentrations of Mg2+ which cause a multiple increase in the affinity of the inner membrane to cytochrome c. Research on the rate of rotenone-insensitive NADH oxidation with respect to the osmotic pressure, the ionic strength of the medium, the presence of Mg2+ ions and cytochrome c in the medium has demonstrated a considerable increase in the hypotonic fragility of the outer membrane of liver mitochondria with age in male rats. In female rats the age changes were insignificant. It is supposed that the damage to the outer membrane of mitochondria in cells can serve as one of the possible explanations of both decrease in the reliability of an aged organism under extreme conditions and sex differences of life-span.

Decrease of phosphorylating oxidation and increase of heat producing NADH oxidation in rat liver mitochondria during life-span prolongation of rats by calorie-restricted diet.

The influence of calorie-restricted diet, initiated at weaning, on some of the oxidative processes in liver homogenates and isolated mitochondria of 2-, 3-, 4-, 24-, 35- and 45-month-old male Wistar rats was studied in comparison with control ad libitum-fed 1-2 day-old rats and 0.5-, 1-, 2-, 3-, 4- and 24-month-old rats. It was shown that a calorie-restricted diet (at 37% of the ad libitum calorific level) did not change the rate of succinate oxidation coupled with oxidative phosphorylation in homogenates, but resulted in a decrease of succinate, glutamate plus malate and beta-hydroxybutyrate oxidation and cytochrome c-oxidase activity in isolated mitochondria without any uncoupling of oxidative phosphorylation or change in cytochrome content in the mitochondria. On the other hand, a significant increase in mitochondrial rotenone-insensitive NADH oxidation and a higher liver mass/body mass ratio in rats under the calorie-restricted diet was established. It may be considered that the activation of a heat-producing mechanism is a very important physiological function in such a condition.

[The effect of osmotic pressure on oligomycin-sensitive ATPase activity and the liberation of Mg2+ from liver mitochondria of rats of various ages]. / Vliianie osmoticheskogo davleniia na oligomitsinchuvstvitel'nuiu ATP- aznuiu aktivnost' i osvobozhdenie Mg2+ iz mitokhondrii pecheni krys raznogo vozrasta.

The influence of osmotic pressure of the incubating medium (25-500 mM sucrose) on oligomycin--sensitive, 2,4-dinitrophenyl-stimulated ATP-ase-activity, Mg2+ release and swelling of the liver mitochondria in 1-, 3-, 12-, 24-months Wistar rats is, investigated to determine age changes of structurally functional state of mitochondria. An increase in the sucrose concentration in the medium from 150 to 500 mM causes almost equal and practically absolute inhibition of ATP-ase-activity in different-age groups of rats, regardless of the presence or absence of Mg2+ ions in the medium A fall of the sucrose concentration to 150-25 mM induces a decrease in mitochondria ATP-ase-activity in Mg2+ free medium in 12- and 24-months rats (to 30 and 22%, respectively). No changes are observed in 1- and 3-months animals. Differences in rates of exogenous NADH oxidation by mitochondria of 1- and 12-months rats as a reflection of inner membrane damage degree are not observed under these conditions. Relative changes in ATP-ase-activity in a Mg2+ free medium with sucrose concentration of 25 mM (compared with 150 mM) correlate (r = 0.82) with those of optical density of mitochondria, measured at light wave length of 520 nm. It is obvious that the liver mitochondria of young and old rats sufficiently differ in spontaneous swelling rate in the media with different osmotic pressure: mitochondria of 1-month rats swell much faster than those of old rats. Considerable age differences of osmotic dependence of Mg2+ output from mitochondria are observed. They depend also on peculiarities of spontaneous organelle swelling dynamics.(ABSTRACT TRUNCATED AT 250 WORDS)

[Age-related differences in the effect of osmotic pressure on liver mitochondrial respiration in rats]. / Vozrastnye razlichiia vliianiia osmoticheskogo davleniia na dykhanie mitokhondrii pecheni krys.

The subject under investigation is the influence of osmotic pressure of incubation medium (25-500 mM of sucrose) upon the respiration and the respiration control (RC) of mitochondria of the liver of rats aged 1, 3, 12 and 24 months when oxidizing succinate. In a medium with 0.3 M of sucrose the respiration rate under condition 3 (V3) and RC increased from the age of 1 to 12 months and decreased by 24 months. In a medium with 0.15 M of sucrose the age differences have not been observed. In a uncoupling state the osmotic dependence of the respiration of mitochondria of 1- and 12-month-old rats did not vary. It is assumed that with age there is a change in the rate of structural coupling of the carrier of adenine nucleotides with H(+)-ATP synthetase complex and (or) the viscosity of the matrix.

[A simple method for estimation of phospholipids in biological objects]. / Prostoi metod opredeleniia fosfolipidov v biologicheskikh ob"ektakh.

A simple method to estimate phospholipids is elaborated. This method is based on determination of optical density of phospholipid-molybdate complexes in chloroform. The method is used for the quantitative determination of phospholipids in biomembranes, liposomes and blood serum without their preliminary extraction, as well as in chloroform, methanol and chloroform-methanol solutions. It is also modified for the phospholipids determination in chromatographic fractions on silufol plates.

[The effect of tetracycline and silibore on lipid peroxidation in the liver od rats of various age]. / Vliianie tetratsiklina i silibora na perekisnoe okislenie lipidov pecheni krys raznogo vozrasta.

As shown by accumulation of malonic dialdehyde and chemiluminescence rate monitoring tetracycline activated both NADPH- and ascorbate-dependent lipid peroxidation in liver tissue of 1, 3, 12 and 24 months old rats. The most distinct induction of lipid peroxidation was observed in old animals, which appears to occur due to a decrease in efficiency of NADPH-GSH-dependent enzymatic antioxidant system. Silibore prevented the stimulating effect of tetracycline on enzymatic and nonenzymatic lipid peroxidation, being apparently involved in hepatoprotective effect.

[Features of ischemic injuries to membranes and activation of lipid peroxidation in the myocardium of rats of various ages]. / Osobennosti ishemicheskogo povrazhdeniia membran i aktivatsiia perekisnogo okisleniia lipidov v miokarde krys raznogo vozrasta.

The model of interrupted ischemia of the isolated heart has been used to show that the yield of lactate dehydrogenase into perfusate in experiments on old rats is described by more sharply pronounced S-like dependence than that in young immature animals. The activation of lipid peroxidation precedes the sarcolemma damage and is relatively more manifested in rats of middle and old age. The respiratory control of mitochondria (in homogenates) decreases considerably already 20 min after ischemia, however mitochondria retain their ability to synthesize ATP 3 h later as well. The causal-effect relationship between the activation of lipid peroxidation and partial disturbance of the mitochondrial function remains unclear.

[Mg2+ release from heart mitochondria in ischemia: is it the defense mechanism or damage?]. / Vybros Mg2+ iz mitokhondrii serdtsa pri ishemii--mekhanizm zashchity ili povrezhdeniia?

It is established that at the early stage of total heart ischemia in rats (5-10 min) the oligomycin-sensitive ATPase activity of mitochondria measured in heart homogenates in the medium and containing no Mg2+ decreases considerably, whereas the activity of respiratory chain, the value of respiratory control and Mg2+-ATPase activity are practically unchanged. The hypothetic trigger mechanism is suggested for Mg2+ ejection from mitochondria under conditions of respiration cessation. It is supposed to be a defence reaction of cells aimed to prevent ATP hydrolysis by mitochondria under ischemia.

[Damage to the Ca2+-transport function and the lipid component of the sarcoplasmic reticulum membrane in total ischemia of the rat myocardium]. / Narushenie Ca2+-transportiruiushchei funktsii i lipidnogo komponenta membran sarkoplazmaticheskogo retikuluma pri total'noi ishemii miokarda krys.

The Ca2+-transporting activity, lipoperoxide chemiluminescence and phospholipid spectrum of sarcoplasmic reticular membranes were studied in ischemic rats. It was shown that a substantial reduction in Ca2+ uptake rate by the sarcoplasmic reticulum occurred within the first 30 minutes and correlated with the increase in chemiluminescence intensity and accumulation of lysophosphatidylcholine. It has been suggested that free radical lipid peroxidation and phospholipase activation are directly related to the reduction of Ca2+-transporting rate by sarcoplasmic reticulum in myocardial ischemia.

Ion permeability induction by the SH cross-linking reagents in rat liver mitochondria is inhibited by the free radical scavenger, butylhydroxytoluene.

The hydrophobic, potentially SH cross-linking reagent, phenylarsine oxide (PhAsO), was found to induce K+ and Ca2+ effluxes from mitochondria and to accelerate the respiration rate in state 4. The hydrophobic monofunctional electrophilic agent, N-ethylmaleimide, does not exhibit this effect but prevents the action of PhAsO. The polar potentially SH cross-linking regents (arsenite, diamide) induce ion fluxes only in the presence of Pi. Ion fluxes induced by the SH reagents are inhibited by butylhydroxytoluene (an inhibitor of free radical reactions), and N,N'-dicyclohexylcarbodiimide, not by oligomycin. It is inferred that the induction of ion fluxes in mitochondria caused by cross-linking of two juxtaposed SH groups is related to the development of free radical reactions.

[Peroxidation of biomembrane lipids and its enzymatic regulation during aging in the rat]. / Perekisnoe okislenie lipidov biomembran i ego fermentativnaia reguliatsiia pri starenii krys.

Measurement of the intensity of chemiluminescence and accumulation of the peroxidation products detected in the reaction with thiobarbituric acid (TBA-active products) has shown that with the ageing of rats the intensity of peroxidation of biomembranes' lipids gets lower in the liver and heart homogenates and tends to decrease in the brain homogenates. A strict correlation is established to exist between the level of TBA-active products and the chemiluminescence intensity independent of the age, organ and the method of initiation of lipids' peroxidation and its intensity. Considerable age changes in the rate of lipids' peroxidation in the liver and heart homogenates may be associated with an increase in the activity of a number of such antioxidant enzymes as superoxide dismutase, glutathione peroxidase, glutathione transferase and catalase in the postnatal ontogenesis of rats.