Natalia V. Belosludtseva

Possible mechanism for formation and regulation of the palmitate-induced cyclosporin A-insensitive mitochondrial pore

The mechanism of the palmitate-induced opening of the mitochondrial Ca2+-dependent cyclosporin A (CsA)-insensitive pore was studied, as well as the influence on this process of well-known modulators of the CsA-sensitive Ca2+-dependent pore. Palmitic acid, which can bind Ca2+ with high affinity, induced the CsA-insensitive swelling of mitochondria, whereas palmitoleic and 2-bromopalmitic acids, which have no such affinity for Ca2+, failed to induce the pore opening. The palmitate induced Ca2+-dependent swelling of mitochondria was not affected by a well-known inhibitor of the CsA-sensitive pore (ADP) and an activator of this pore (inorganic phosphate, Pi). However, this swelling was inhibited by physiological concentrations of ATP ([I]50 = 1.3 mM), but 100 µM ATP increased by 30% the rate of mitochondria swelling if Ca2+ had been added earlier. The effects of ATP (inhibition and activation) manifested themselves from different sides of the inner mitochondrial membrane. Mg2+ inhibited the palmitate-induced Ca2+-dependent swelling of mitochondria with [I]50 = 0.8 mM. It is concluded that palmitic acid induces the opening of the CsA-insensitive pore due to its ability for complexing with Ca2+. A possible mechanism of the pore formation and the influence of some modulators on this process are discussed.

Ca2+-Induced Phase Separation in the Membrane of Palmitate-Containing Liposomes and Its Possible Relation to Membrane Permeabilization

A Ca2+-induced phase separation of palmitic acid (PA) in the membrane of azolectin unilamellar liposomes has been demonstrated with the fluorescent membrane probe nonyl acridine orange (NAO). It has been shown that NAO, whose fluorescence in liposomal membranes is quenched in a concentration-dependent way, can be used to monitor changes in the volume of lipid phase. The incorporation of PA into NAO-labeled liposomes increased fluorescence corresponding to the expansion of membrane. After subsequent addition of Ca2+, fluorescence decreased, which indicated separation of PA/Ca2+ complexes into distinct membrane domains. The Ca2+-induced phase separation of PA was further studied in relation to membrane permeabilization caused by Ca2+ in the PA-containing liposomes. A supposition was made that the mechanism of PA/Ca2+-induced membrane permeabilization relates to the initial stage of Ca2+-induced phase separation of PA and can be considered as formation of fast-tightening lipid pores due to chemotropic phase transition in the lipid bilayer.

Physiological aspects of the mitochondrial cyclosporin A-insensitive palmitate/Ca2+-induced pore: tissue specificity, age profile and dependence on the animal's adaptation to hypoxia

Earlier we found that being added to rat liver mitochondria, palmitic acid (Pal) plus Ca2+ opened a cyclosporin A-insensitive pore, which remained open for a short time. Apparently, this pore is involved in the Pal-induced apoptosis and may also take part in the mitochondrial Ca2+ recycling as a Ca2+ efflux system (Belosludtsev et al. J Bioenerg Biomembr 38:113–120, 2006; Mironova et al. J. Bioenerg. Biomembr. 39:167–174, 2007). In this paper, we continue studying physiological and regulatory aspects of the pore. The following observations have been made. (1) Cardiolipin has been found to facilitate the Ca2+-induced formation of pores in the Pal-containing liposomal membranes. (2) The opening of Pal/Ca2+-induced pore is accompanied by the release of apoptosis-induced factor (AIF) from mitochondria. (3) The rate of Pal/Ca2+-induced swelling of rat liver mitochondria increases substantially with the age of animals. (4) Although the Pal/Ca2+-induced pore opens both in the liver and heart mitochondria, the latter require higher Pal concentrations for the pore to open. (5) The pore opening depends on the resistance of animals to hypoxia: in the highly resistant to hypoxia rats, the mitochondrial Pal/Ca2+-induced pore opens easier than in the low resistant animals, this being opposite for the classical, cyclosporin A-sensitive MPT pore. The adaptation of the low resistant rats to oxygen deficiency increases the sensitivity of their mitochondria to PalCaP inductors. The paper also discusses a possible role of the mitochondrial Pal/Ca2+-induced pore in the protection of tissues against hypoxia.

Ca(2+)-dependent permeabilization of mitochondria and liposomes by palmitic and oleic acids: a comparative study.

In the present work, we examine and compare the effects of saturated (palmitic) and unsaturated (oleic) fatty acids in relation to their ability to cause the Ca(2+)-dependent membrane permeabilization. The results obtained can be summarized as follows. (1) Oleic acid (OA) permeabilizes liposomal membranes at much higher concentrations of Ca(2+) than palmitic acid (PA): 1mM versus 100μM respectively. (2) The OA/Ca(2+)-induced permeabilization of liposomes is not accompanied by changes in the phase state of lipid bilayer, in contrast to what is observed with PA and Ca(2+). (3) The addition of Ca(2+) to the PA-containing vesicles does not change their size; in the case of OA, it leads to the appearance of larger and smaller vesicles, with larger vesicles dominating. This can be interpreted as a result of fusion and fission of liposomes. (4) Like PA, OA is able to induce a Ca(2+)-dependent high-amplitude swelling of mitochondria, yet it requires higher concentrations of Ca(2+) (30 and 100μM for PA and OA respectively). (5) In contrast to PA, OA is unable to cause the Ca(2+)-dependent high-amplitude swelling of mitoplasts, suggesting that the cause of OA/Ca(2+)-induced permeability transition in mitochondria may be the fusion of the inner and outer mitochondrial membranes. (6) The presence of OA enhances PA/Ca(2+)-induced permeabilization of liposomes and mitochondria. The paper discusses possible mechanisms of PA/Ca(2+)- and OA/Ca(2+)-induced membrane permeabilization, the probability of these mechanisms to be realized in the cell, and their possible physiological role.

Effect of several flavonoid-containing plant preparations on activity of mitochondrial ATP-dependent potassium channel

Flavonoid-containing plant preparations (water soluble extracts of Pentaphylloides fruticosa [Extralife], Emblica officinalis Gaerth [Amla], and Bergenia crassifolia [Bergenia]) produced a dose-dependent and tissue-specific effect on activity of mitochondrial ATP-dependent potassium channel. The effect of these preparations was biphasic (activation and inhibition). The activating effect of Extralife was one order of magnitude higher than that of Amla and Bergenia and was observed in a wider concentration range. The activating effect of preparations was abolished by inhibitors of the mitochondrial ATP-dependent potassium channel, which attested to specificity of their influence on mitochondrial channel. Under in vivo conditions, the antihypoxic effect of Extralife was partially abolished by mitochondrial ATP-dependent potassium channel inhibitor 5-hydroxydecanoate.

Involvement of palmitate/Ca2+(Sr2+)-induced pore in the cycling of ions across the mitochondrial membrane

The palmitate/Ca2+-induced (Pal/Ca2+) pore, which is formed due to the unique feature of long-chain saturated fatty acids to bind Ca2+ with high affinity, has been shown to play an important role in the physiology of mitochondria. The present study demonstrates that the efflux of Ca2+ from rat liver mitochondria induced by ruthenium red, an inhibitor of the energy-dependent Ca2+ influx, seems to be partly due to the opening of Pal/Ca2+ pores. Exogenous Pal stimulates the efflux. Measurements of pH showed that the Ca2+-induced alkalization of the mitochondrial matrix increased in the presence of Pal. The influx of Ca2+ (Sr2+) also induced an outflow of K+ followed by the reuptake of the ion by mitochondria. The outflow was not affected by a K+/H+ exchange blocker, and the reuptake was prevented by an ATP-dependent K+ channel inhibitor. It was also shown that the addition of Sr2+ to mitochondria under hypotonic conditions was accompanied by reversible cyclic changes in the membrane potential, the concentrations of Sr2+ and K+ and the respiratory rate. The cyclic changes were effectively suppressed by the inhibitors of Ca2+-dependent phospholipase A2, and a new Sr2+ cycle could only be initiated after the previous cycle was finished, indicating a refractory period in the mitochondrial sensitivity to Sr2+. All of the Ca2+- and Sr2+-induced effects were observed in the presence of cyclosporin A. This paper discusses a possible role of Pal/Ca2+ pores in the maintenance of cell ion homeostasis.

Effect of surface-potential modulators on the opening of lipid pores in liposomal and mitochondrial inner membranes induced by palmitate and calcium ions.

The effect of surface-potential modulators on palmitate/Ca2+-induced formation of lipid pores was studied in liposomal and inner mitochondrial membranes. Pore formation was monitored by sulforhodamine B release from liposomes and swelling of mitochondria. ζ-potential in liposomes was determined from electrophoretic mobility. Replacement of sucrose as the osmotic agent with KCl decreased negative ζ-potential in liposomes and increased resistance of both mitochondria and liposomes to the pore inducers, palmitic acid, and Ca2+. Micromolar Mg2+ also inhibited palmitate/Ca2+-induced permeabilization of liposomes. The rate of palmitate/Ca2+-induced, cyclosporin A-insensitive swelling of mitochondria increased 22% upon increasing pH from 7.0 to 7.8. At below the critical micelle concentration, the cationic detergent cetyltrimethylammonium bromide (10 μM) and the anionic surfactant sodium dodecylsulfate (10-50 μM) made the ζ-potential less and more negative, respectively, and inhibited and stimulated opening of mitochondrial palmitate/Ca2+-induced lipid pores. Taken together, the findings indicate that surface potential regulates palmitate/Ca2+-induced lipid pore opening.

[The influence of spermine on Ca(2+)-dependent permeability transition in mitochondria and liposomes induced by palmitic and α,Ω-hexadecanedioic acids].

The effect of spermine on Ca2+-dependent permeability transition in mitochondria and liposomes induced by palmitic and α,ω-hexadecanedioic acid was studied. It has been shown that spermine inhibited the cyclosporin A-insensitive mitochondrial swelling induced by palmitic acid and Ca2+ and α,ω-hexadecanedioic acid and Ca2+. 100 μM spermine did not influence the mitochondrial respiration in state V2 and the respiration stimulated by palmitic acid, α,ω-hexadecanedioic acid and Ca2+. Preincubation of liposomes with 100 μM spermine resulted in inhibition of the palmitic acid/Ca2+- and α,ω-hexadecanedioic acid/Ca2+-induced release of a fluorescent dye sulforhodamine B from liposomes. At the same time, spermine added to fatty acid-containing liposomes stimulated Ca2+-dependent release of sulforhodamine B from liposomes. Addition of spermine to liposomes resulted in a significant increase in the ζ -potential of liposomal membranes (from −39.8 to −18.6 mV). A possible mechanism of spermine influence on palmitic acid/Ca2+- and α,ω-hexadecanedioic acid/Ca2+-induced permeability transition in mitochondria and liposomes is discussed.

Oxidative phosphorylation and ion transport in the mitochondria of two strains of rats varying in their resistance to stress and hypoxia

The role of mitochondria in the inherited or ontogenetically acquired reactions of organism to stress is not studied enough. In the present work, we examined the functional state of the coupled respiratory chain, potassium and calcium transport and rate of hydrogen peroxide production on two rat lines: August and Wistar—which possess different resistance to emotional stress and hypoxia. It was established that the respiration rate and efficiency of oxidative phosphorylation were higher in August rats than in Wistar ones. In August rats, the rate of potassium transport and ATP-dependent mitochondrial swelling as well as the concentration of the ion in the mitochondrial matrix were almost twice as higher comparatively to those parameters in Wistar rats. The rate of H2O2 production was found to be decreased in the mitochondria of August rats. It was also demonstrated that the two rat lines differed by their resistance to the opening of the palmitate/Ca2+-induced pore and by their ability to retain calcium within mitochondria. The paper discusses the involvement of the mitochondrial ATP-dependent potassium channel in the adaptation of animals to adverse effects.

Mitochondrial lipid pore in the mechanism of glutamate-induced calcium deregulation of brain neurons

The work examines the mechanism of central nerve cell death upon stimulation of brain NMDA receptors with the stimulatory mediator glutamate. A prolonged stimulation of neurons with glutamate is known to result in the disorder of Ca2+ homeostasis and severe mitochondrial depolarization followed by cell death. It has been shown that the overload of mitochondria with Sr2+ leads to the release of the cation, medium alkalization, decrease of membrane potential and mitochondrial swelling, indicating a nonspecific permeabilization of the mitochondrial membrane. The permeabilization, in our opinion, is caused by the activation of Ca2+/Sr2+-dependent phospholipase A2 (PLA2), resulting in the formation of free palmitic and stearic acids in the mitochondrial membrane. These fatty acids bind Ca2+ with high affinity and the process of binding is accompanied by the formation of a transient lipid pore—a phenomenon demonstrated earlier on both artificial and mitochondrial membranes. The inhibitors of PLA2 have been shown to suppress permeabilization of mitochondrial membranes. In the culture of granular cerebellum neurons, the PLA2 inhibitors prolonged the lag of the delayed Sr2+ deregulation and membrane depolarization. On the basis of data obtained on isolated mitochondria and neurons we suppose that the initial stages of glutamate-induced Ca2+ deregulation of neurons are underlain by the opening of lipid pores in brain mitochondria.