Organic hydroperoxides at high concentrations cause energization and activation of ATP synthesis in mitochondria

The addition of high concentrations of cumene or tert-butyl hydroperoxide to previously deenergized mitochondria results in the energization of these mitochondria and activation of ATP synthesis. The energization effect was observed in the presence of 0.5-0.7 mM cumene hydroperoxide or 2.0-2.5 mM tert-butyl hydroperoxide. This energization of mitochondria and activation of oxidative phosphorylation by organic hydroperoxides required the presence of ADP in the mitochondrial matrix and does not depend upon the method of deenergization of the mitochondria.     

Substrate Modulation of Fatty Acid Effects on Energization and Respiration of Kidney Proximal Tubules during Hypoxia/Reoxygenation

Kidney proximal tubules subjected to hypoxia/reoxygenation develop a nonesterified fatty acid-induced energetic deficit characterized by persistent partial mitochondrial deenergization that can be prevented and reversed by citric acid cycle substrates. To further assess the role of competition between fatty acids and substrates on inner membrane substrate carriers in the deenergization and the contribution to deenergization of fatty acid effects on respiratory function, digitonin-permeabilized rabbit and mouse tubules were studied using either addition of exogenous oleate after control normoxic incubation or increases of endogenous fatty acids produced by hypoxia/reoxygenation. The results demonstrated major effects of matrix oxaloacetate accumulation on succinate-supported energization and respiration and their modification by fatty acids. Improvements of energization in the presence of fatty acids by glutamate were shown to result predominantly from lowering matrix oxaloacetate rather than from amelioration of transmembrane cycling of fatty acids and uncoupling. Mouse tubules had 2.5 fold higher rates of succinate utilization, which resulted in stronger effects of oxaloacetate accumulation than rabbit tubules. Hypoxia/reoxygenation induced respiratory inhibition that was more severe for complex I-dependent substrates. Fatty acids themselves did not acutely contribute to this respiratory inhibition, but lowering them during 60 min. reoxygenation to allow recovery of ATP during that period alleviated it. These data clarify the basis for the nonesterified fatty acid-induced mitochondrial energetic deficit in kidney proximal tubules that impairs structural and functional recovery and provide insight into interactions that need to be considered in the design of substrate-based interventions to improve mitochondrial function.     

Stabilising action of carnitine on energy linked processes in rat liver mitochondria

Rat liver mitochondria exposed to stressing conditions - ageing at room temperature, incubation in the presence of t-butyl hydroperoxide or damaging concentrations of Ca2+ and phosphate- undergo a rapid fall in their membrane potential (delta psi) with a concomitant release of endogenous Mg2+ and accumulated Ca2+. Addition of L-carnitine to the incubation medium considerably delays mitochondrial deenergization. A similar, though lower, protection has also been observed in L-carnitine pretreated and subsequently washed rat liver mitochondria. Furthermore mitochondria isolated from livers of starved rats, treated with L-carnitine 30 minutes before death and exposed to the same stressing conditions show similar delay in the decrease of delta psi and concurrent energy linked processes as compared with untreated animals. Both the in vitro and in vivo results strongly indicate that the stabilising action of L-carnitine on liver mitochondria is due to the removal of membrane bound long chain acyl CoA.     

Role of the mitochondria in the conversion of 1-aminocyclopropane 1-carboxylic acid to ethylene in plant tissues

Intact plant mitochondria, isolated from climacteric (Lycopersicon esculentum, Mill., tomato) or non-climacteric (Solanum tuberosum, L., potato) tissues, and purified on Percoll density gradients, were unable to convert 1-aminocyclopropane 1-carboxylic acid (ACC) to ethylene. Energization or sonication did not enhance ethylene production. For both tissues, the low activity of ACC conversion found in crude mitochondrial fractions from both tissues was increased by sonication. After mitochondrial purification, this activity was located on top of the gradient together with the microsomal membrane fraction containing a high lipoxygenase activity. Addition of exogenous lipoxygenase and linoleic acid to isolated tomato or potato mitochondria greatly enhanced ACC conversion (to approx. 300 pmol h⁻¹ mg⁻¹ protein). Direct measurements of ACC uptake by mitochondria indicated that ACC uptake is not dependent on energization.     

Energizing plasmalemma of yeasts is necessary for activation of its H+-ATPase by glucose]

ATPase of yeast plasmalemma is known to be activated during incubation of cells or protoplasts with glucose. It has been shown that the level of ATPase activation is sharply decreased after pretreatment of cells or protoplasts with mercaptoethanol, dinitrophenol, gramicidin D, nigericin, or monensin. It is suggested that deenergization of yeast plasmalemma by monensin, nigericin, and mercaptoethanol as uncoupler plays a crucial role in the prevention of in vivo activation of plasma membrane ATPase by glucose. It is concluded that energization of yeast plasmalemma is necessary for activation of ATPase by glucose.     

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.     

Calcium-dependent mitochondrial oxidative damage promoted by 5-aminolevulinic acid

Swelling of isolated rat liver mitochondria is shown to be induced by metal-catalyzed 5-aminolevulinic acid (ALA) aerobic oxidation, a putative endogenous source of reactive species (ROS), at concentrations as low as 50–100 μM. In this concentration range, ALA is estimated to occur in the liver of acute intermittent porhyria patients. Removal of Ca²⁺ (10 μM) from the suspension of isolated rat liver mitochondria by added EGTA abolishes both the ALA-induced transmembrane-potential collapse and mitochondrial swelling. Prevention of the ALA-induced swellling by addition of ruthenium red prior to mitochondrial energization by succinate demonstrates the deleterious involvement of internal Ca²⁺. Addition of MgCl₂ at concentrations higher than 2.5 mM, prevents the ALA-induced mitochondrial swelling, transmembrane potential collapse and Ca²⁺ efflux. This indicates that Mg²⁺ protects against the mitochondrial damage promoted by ALA-generated ROS. The ALA-induced mitochondrial damage might be a key event in the liver mitochondrial damage of acute intermittent porphyria patients reported elsewhere.     

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.     

The role of adenine nucleotide translocation in the energization of the inner membrane of mitochondria isolated from rho + and rho degree strains of Saccharomyces cerevisiae

The energization of rho + and rho degrees isolated mitochondria was measured using a tetraphenylphosphonium selective electrode. In both strains translocase mediated ATP/ADP exchange energization was observed. This energization was more sensitive to uncoupler than that induced by respiration in rho + mitochondria. This observation is in accordance with the hypersensitivity of rho - cell growth to uncoupler.     

Intracellular acidosis protects cultured hepatocytes from the toxic consequences of a loss of mitochondrial energization

Cultured rat hepatocytes were treated with potassium cyanide, an inhibitor of cytochrome oxidase; valinomycin, a K+ ionophore; carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophore; and the ATP synthetase inhibitor oligomycin. The effect of these agents on the viability of the cells was related to changes in ATP content and the deenergization of the mitochondria. The ATP content was reduced by over 90% by each inhibitor. All of the agents except oligomycin killed the cells within 4 h. With the exception of oligomycin, the mitochondrial membrane potential as measured by the distribution of [3H]triphenylmethylphosphonium collapsed with each of the agents. Monensin, a H+/Na+ ionophore, potentiated the toxicity of cyanide and CCCP, whereas the toxicity of valinomycin was reduced. The effect of cyanide and monesin on the cytoplasmic pH of cultured hepatocytes was measured with the fluorescent probe, 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Cyanide promptly acidified the cytosol, and the addition of 10 microM monensin caused a rapid alkalinization of the cytosol. A reduction of pH of the culture medium from 7.4 to 6.6 and 6.0 prevented the cell killing both by cyanide alone and by cyanide in the presence of monensin. However, neither monensin nor extracellular acidosis had any effect on the loss of mitochondrial energization in the presence of cyanide. It is concluded that ATP depletion per se is insufficient to explain the cell killing with cyanide, CCCP, and valinomycin. Rather, cell killing is better correlated with a loss of mitochondrial energization. With cyanide an intracellular acidosis interferes with the mechanism that couples collapse of the mitochondrial membrane potential to lethal cell injury.     

Induction of Ca2+-dependent cyclosporin a-insensitive nonspecific permeability of the inner membrane of liver mitochondria and cytochrome c release by α,ω-hexadecanedioic acid in media of varying ionic strength

In liver mitochondria loaded with Ca²⁺ or Sr²⁺, α,ω-hexadecanedioic acid (HDA) can induce nonspecific permeability of the inner membrane (mitochondrial pore) by the mechanism insensitive to cyclosporin A (CsA). In this work we studied the effect of ionic strength of the incubation medium on the kinetics of the processes that accompany Ca²⁺-dependent induction of the mitochondrial pore by fatty acid: organelle swelling, Ca²⁺ release from the matrix, changes in transmembrane potential (Δψ) and rate of oxygen consumption, and the release of cytochrome c from the intermembrane space. Two basic incubation media were used: sucrose medium and isotonic ionic medium containing KCl without sucrose. We found that 200 μM Ca²⁺ and 20 μM HDA in the presence of CsA effectively induce high-amplitude swelling of mitochondria both in the case of sucrose and in the ionic incubation medium. In the presence of CsA, mitochondria can rapidly absorb Ca²⁺ and retain it in the matrix for a while without reducing Δψ. Upon incubation in the ionic medium, mitochondria retain most of the added Ca²⁺ in the matrix for a short time without reducing the Δψ. In both cases the addition of HDA to the mitochondria 2 min after the introduction of Ca²⁺ leads to the rapid release of these ions from the matrix and total drop in Δψ. The mitochondrial swelling induced by Ca²⁺ and HDA in non-ionic medium is accompanied by almost maximal stimulation of respiration. Under the same conditions, but during incubation of mitochondria in the ionic medium, it is necessary to add cytochrome c for significant stimulation of respiration. The mitochondrial swelling induced by Ca²⁺ and HDA leads to the release of cytochrome c in a larger amount in the case of ionic medium than for the sucrose medium. We conclude that high ionic strength of the incubation medium determines the massive release of cytochrome c from mitochondria and liberates it from the respiratory chain, which leads to blockade of electron transport along the respiratory chain and consequently to disruption of the energy functions of the organelles.     

MORPHOLOGY OF ISOLATED RABBIT HEART MUSCLE MITOCHONDRIA AND THE OXIDATION OF EXTRAMITOCHONDRIAL REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE

The morphology of rabbit heart muscle mitochondria isolated in several media has been compared by electron microscopy. The internal structure of isolated mitochondria differs from that of in situ mitochondria, with the type and degree of alteration depending on the isolation medium. Examination of the isolated mitochondria after incubation revealed that additional morphological changes occurred during incubation, but these changes were less pronounced when the incubation was conducted in a complete medium containing substrate. The isolated mitochondria have been shown to be capable of catalyzing a slow aerobic oxidation of extramitochondrial reduced diphosphopyridine nucleotide. The rate of DPNH oxidation observed is sufficient to account for the ability of the mitochondria to oxidize lactate in the presence of catalytic amounts of DPNH. The suspensions used were essentially free of mitochondrial fragments, which are known to oxidize DPNH. Possible relationships of these findings to metabolism in situ are discussed. The results indicate the desirability of correlating biochemical activities with the morphology of isolated mitochondria.     

Physiological modulators of cyclosporin A-insensitive nonspecific permeability of the inner membrane of liver mitochondria induced by calcium ions and α,ω-hexadecanedioic acid

Long-chain saturated α,ω-dioic acids can induce nonspecific permeability of the inner membrane (pore opening) of liver mitochondria loaded with Ca²⁺ or Sr²⁺ by the mechanism insensitive to cyclosporin A (CsA). In this work we found that 200 μM Ca²⁺ and 20 μM α,ω-hexadecanedioic acid (HDA) in the presence of 1 μM CsA induced high-amplitude swelling of liver mitochondria (pore opening) only in the presence of succinate as oxidation substrate. Under these conditions protonophore uncoupler of oxidative phosphorylation 2,4-dinitrophenol at the concentration of 75 μM, which is optimal for its uncoupling activity, inhibited mitochondrial swelling induced by Ca²⁺ and HDA, despite the presence of succinate in the incubation medium. Natural uncouplers of oxidative phosphorylation, oleic and linoleic acids, produced a similar effect. These data suggest that energization of organelles, which promotes Ca²⁺ transport into the matrix, is one of the basic requirements of pore opening in liver mitochondria induced by Ca²⁺ and HDA. It is shown that ATP at the physiological concentration of 2 mM inhibits HDA-induced high-amplitude swelling of mitochondria by reducing free Ca²⁺ concentration in the medium. ADP at the same concentration had a similar effect. This modulating effect of nucleotides apparently is attributable to their ability to chelate calcium ions. Polycation spermine, which is known as an inhibitor of the classical CsA-sensitive pore, at the physiological concentration of 1 mM inhibited CsA-insensitive swelling of liver mitochondria induced by sequential addition of Ca²⁺ and HDA. It is assumed that such action of spermine is due to its ability to shield the negative surface charges on the inner membrane of mitochondria. Bovine serum albumin (BSA), which is able to bind free fatty acids and thus prevent the induction of Ca²⁺-dependent pore, inhibited HDA-induced swelling of mitochondria. However, at the same BSA/fatty acid molar ratio inhibitory effect of BSA was much less pronounced if HDA was used as the pore inducer instead of palmitic acid. Apparently, this can be accounted by the fact that BSA binds α,ω-dioic acids weaker than their monocarboxylic analogues.     

Effects of inorganic phosphate on the light dependent thylakoid energization of intact spinach chloroplasts

The light dependent energization of the thylakoid membrane was analyzed in isolated intact spinach (Spinacia oleracea L.) chloroplasts incubated with different concentrations of inorganic phosphate (Pi). Two independent methods were used: (a) the accumulation of [¹⁴C]5,5-dimethyl-2,4-oxazolidinedione and [¹⁴C] methylamine; (b) the energy dependent chlorophyll fluorescence quenching. The inhibition of CO₂ fixation by superoptimal medium Pi or by adding glyceraldehyde—an inhibitor of the Calvin cycle—leads to an increased energization of the thylakoid membrane; however, the membrane energization decreases when chloroplasts are inhibited by suboptimal Pi. This specific `low phosphate' effect could be partially reversed by adding oxaloacetate, which regenerates the electron acceptor NADP<sup>+</sup> and stimulates linear electron transport. The energization seen in low Pi is, however, always lower than in superoptimal Pi, even in the presence of oxaloacetate. Energization recovers in the presence of low amounts of N,N′-dicyclohexylcarbodiimide, which reacts with proton channels including the coupling factor 1 ATP synthase. N,N′-Dicyclohexylcarbodiimide has no effect on energization of chloroplasts in superoptimal Pi. These results suggest there is a specific `low phosphate' proton leak in the thylakoids, and its origin is discussed.     

Induction of permeability of the inner membrane of yeast mitochondria

The current view on apoptosis is given, with a special emphasis placed on apoptosis in yeasts. Induction of a non-specific permeability transition pore (mPTP) in mammalian and yeast mitochondria is described, particularly in mitochon-dria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, which are aerobes possessing the fully competent respiratory chain with all three points of energy conservation and well-structured mitochondria. They were examined for their ability to induce an elevated permeability transition of the inner mitochondrial membrane, being subjected to virtually all conditions known to induce the mPTP in animal mitochondria. Yeast mitochondria do not form Ca²⁺-dependent pores, neither the classical Ca²⁺/P_i-dependent, cyclosporin A-sensitive pore even under deenergization of mitochondria or depletion of the intramitochondrial nucleotide pools, nor a pore induced in mammalian mitochondria upon concerted action of moderate Ca²⁺ concentrations (in the presence of the Ca²⁺ ionophore ETH129) and saturated fatty acids. No pore formation was found in yeast mitochondria in the presence of elevated phosphate concentrations at acidic pH values. It is concluded that the permeability transition in yeast mitochondria is not coupled with Ca²⁺ uptake and is differently regulated compared to the mPTP of animal mitochondria.     

Mitochondria from Dipodascus (Endomyces) magnusii and Yarrowia lipolytica yeasts did not undergo a Ca2+-dependent permeability transition even under anaerobic conditions

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts. The two yeast strains are good alternatives to Saccharomyces cerevisiae, being aerobes containing well-structured mitochondria (thus ensuring less structural limitation to observe their appreciable swelling) and fully competent respiratory chain with three invariantly functioning energy conservation points, including Complex I, that can be involved in induction of the canonical Ca²⁺/P_i-dependent mitochondrial permeability transition (mPTP pore) with an increased open probability when electron flux increases (Fontaine et al. J Biol Chem 273:25734–25740, 1998; Bernardi et al. FEBS J 273:2077–2099, 2006). High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating pore opening. Previously (Kovaleva et al. J Bioenerg Biomembr 41:239–249, 2009; Kovaleva et al. Biochemistry (Moscow) 75:297–303, 2010) we have shown that mitochondria from Y. lipolytica and D. magnusii were very resistant to the Ca²⁺ overload combined with varying concentrations of P_i, palmitic acid, SH-reagents, carboxyatractyloside (an inhibitor of ADP/ATP translocator), as well as depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high [P_i]. Here we subjected yeast mitochondria to other conditions known to induce an mPTP in animal and plant mitochondria, namely to Ca²⁺ overload under hypoxic conditions (anaerobiosis). We were unable to observe Ca²⁺-induced high permeability of the inner membrane of D. magnusii and Y. lipolytica yeast mitochondria under anaerobic conditions, thus suggesting that an mPTP-like pore, if it ever occurs in yeast mitochondria, is not coupled with the Ca²⁺ uptake. The results provide the first demonstration of ATP-dependent energization of yeast mitochondria under conditions of anaerobiosis.     

Mitochondria from rat uterine smooth muscle possess ATP-sensitive potassium channel

The objective of this study was to detect ATP-sensitive K⁺ uptake in rat uterine smooth muscle mitochondria and to determine possible effects of its activation on mitochondrial physiology. By means of fluorescent technique with usage of K⁺-sensitive fluorescent probe PBFI (potassium-binding benzofuran isophthalate) we showed that accumulation of K ions in isolated mitochondria from rat myometrium is sensitive to effectors of K_ATP-channel (ATP-sensitive K⁺-channel) – ATP, diazoxide, glibenclamide and 5HD (5-hydroxydecanoate). Our data demonstrates that K⁺ uptake in isolated myometrium mitochondria results in a slight decrease in membrane potential, enhancement of generation of ROS (reactive oxygen species) and mitochondrial swelling. Particularly, the addition of ATP into incubation medium led to a decrease in mitochondrial swelling and ROS production, and an increase in membrane potential. These effects were eliminated by diazoxide. If blockers of K_ATP-channel were added along with diazoxide, the effects of diazoxide were removed. So, we postulate the existence of K_ATP-channels in rat uterus mitochondria and assume that their functioning may regulate physiological conditions of mitochondria, such as matrix volume, ROS generation and polarization of mitochondrial membrane.     

Changes in mitochondrial protein composition during testicular differentiation in mouse and bull

The morphology of testicular mitochondria changes markedly during spermatogenesis from a form normally seen in somatic cells to a “germ cell” form in which the matrix is diffuse and vacuolated and finally to a form with a condensed matrix seen in spermatozoa. Colloidal silica gel gradients and high-resolution, two-dimensional gel electrophoresis were used to define the changes in density and polypeptide composition that occur in testicular mitochondria during spermatogenesis. Similar densities were observed for mitochondria isolated from the same bovine or murine tissue, but mitochondria from different tissues usually had different densities. Mitochondria from testis of calf, bull, or sexually mature mouse had densities of 1.06 gm/cm³ while liver mitochondria were more dense, having a density of 1.09 gm/cm³. “Somatic-type” testicular mitochondria from calf and “germ cell-type” mitochondria from sexually mature mouse or bull had similar densities, 1.06 gm/cm³, while the density of mitochondria from ejaculated spermatozoa differed, ρ = 1.08 gm/cm³. Analysis of polypeptide composition of somatic and germ cell mitochondria from testes of prepuberal and sexually mature animals and from highly enriched populations of pachytene primary spermatocytes and round spermatids revealed a staining pattern of mitochondrial proteins that was markedly constant throughout development with most polypeptides being conserved and a few specific spots changing in abundance. Marked differences were detected, however, when mitochondria from ejaculated spermatozoa were compared with those from testis with many minor and major polypeptides missing and several new polypeptides present at high concentration.     

Role of Carboxylic Groups in the Control of Nonspecific Permeability of Mitochondrial Membranes

Ionized COOH groups are present in molecular structures involved in the process of formation of mitochondrial permeability transition pores (MPTPs), in particular, in the ADP/ATP antiporter and/or voltage-dependent anion channels. In experiments on preparations of isolated mitochondria obtained from rat hepatocytes, we found that, in the case of induction of nonspecific permeability through mitochondrial membranes under the action of Cа²⁺ in a relatively low concentration (15 μM), modulation of the activity of COOH groups with the use of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1 mM) led to unidirectional effects, namely to acceleration of the processes of formation of MPTPs and transport of incubation solution and Са²⁺ through these megachannels, prolongation of the open state of the latter, as well as to increases in the final volume (swelling) of the mitochondria and to a rise in the amount of Са²⁺ released from these organelles. In contrast, when calcium was used in a high concentration (100 μM), the directions of the above processes were dissimilar. Slowing down of the flow of incubation solution through MPTPs and the process of their formation was observed; at the same time, Са²⁺ release from the mitochondria was accelerated. However, the final volume of the mitochondria and the amount of Са²⁺ released from these cellular structures increased. Differences between the effects of the used modulator of the activity of COOH groups on the nonspecific permeability of the mitochondria induced by calcium applied in low and high concentrations are perhaps determined by the following. The process of swelling of the mitochondria is saturable, while Са²⁺ release from these organelles shows an unlimited pattern. The latter process (Са²⁺ release) probably undergoes calcium-initiated inactivation. The mechanisms of induction of nonspecific permeability of the mitochondrial membranes under the action of low and high calcium concentrations differ from each other. The calcium uniporter in the mitochondria is sensitive to the modulator of the activity of COOH groups. Diffusion of water through the inner mitochondrial membrane and/or other systems provides some contribution to the studied processes; this can lead to changes in the transport of liquids in these organelles.