Larisa V. Emelyanova

Mitochondrial Electron Transport Chain in Heavy Metal-Induced Neurotoxicity: Effects of Cadmium, Mercury, and Copper

To clarify the role of mitochondrial electron transport chain (mtETC) in heavy-metal-induced neurotoxicity, we studied action of Cd2+, Hg2+, and Cu2+ on cell viability, intracellular reactive oxygen species formation, respiratory function, and mitochondrial membrane potential of rat cell line PC12. As found, the metals produced, although in a different way, dose- and time-dependent changes of all these parameters. Importantly, Cd2+ beginning from 10 [mu]M and already at short incubation time (3 h) significantly inhibited the FCCP-uncoupled cell respiration; besides, practically the complete inhibition of the respiration was reached after 3 h incubation with 50 [mu]M Hg2+ or 500 [mu]M Cd2+, whereas even after 48 h exposure with 500 [mu]M Cu2+, only a 50% inhibition of the respiration occurred. Against the Cd2+-induced cell injury, not only different antioxidants and mitochondrial permeability transition pore inhibitors were protective but also such mtETC effectors as FCCP and stigmatellin (complex III inhibitor). However, all mtETC effectors used did not protect against the Hg2+- or Cu2+-induced cell damage. Notably, stigmatellin was shown to be one of the strongest protectors against the Cd2+-induced cell damage, producing a 15–20% increase in the cell viability. The mechanisms of the mtETC involvement in the heavy-metal-induced mitochondrial membrane permeabilization and cell death are discussed.

Tl+ induces the permeability transition pore in Ca2+-loaded rat liver mitochondria energized by glutamate and malate

It is known that Ca2+ and heavy metals more actively induce MPTP opening in mitochondria, energized by the I complex substrates. Thus, a rise in a Tl+-induced MPTP was proposed in experiments on isolated rat liver mitochondria energized by the complex I substrate (glutamate and malate). Expose of the mitochondria to Ca2+ into a medium containing TlNO3, glutamate, and malate as well as sucrose or KNO3 resulted in a decrease in state 3, state 4, or DNP-stimulated respiration as well as an increase of both mitochondrial swelling and ΔΨmito dissipation. The MPTP inhibitors, CsA and ADP, almost completely eliminated the effect of Ca2+, which was more pronounced in the presence of the complex I substrates than the complex II substrate (succinate) and rotenone (Korotkov and Saris, 2011). The present study concludes that Tl+-induced MPTP opening is more appreciable in mitochondria energized by glutamate and malate but not succinate in the presence of rotenone. We assume that the Tl+-induced MPTP opening along with followed swelling and possible structural deformations of the complex I in Ca2+-loaded mitochondria may be a part of the thallium toxicity mechanism on mitochondria in living organisms. At the same time, oxidation of Tl+ to Tl3+ by mitochondrial oxygen reactive species is proposed for the mechanism.

Inorganic Phosphate Stimulates the Toxic Effects of Tl + in Rat Liver Mitochondria

We studied action of inorganic phosphate (Pi) on toxic effects of Tl+ in isolated rat liver mitochondria. This is a convenient model to study the toxicity of heavy metals. Pi markedly retarded contraction of energized mitochondria swollen in the TlNO3 medium and even stronger stimulated swelling and state 4 of succinate‐energized mitochondria in the TlNO3 medium. A valinomycin‐induced decrease of K+‐diffusion potential was also accelerated by Tl+ in the presence of Pi. The mitochondrial permeability transition pore in the medium containing Ca2+, TlNO3, and nitrates of univalent cations was distinctly stimulated by Pi. However, Pi did not affect both the Tl+‐stimulated swelling of nonenergized mitochondria in the TlNO3 medium and swelling of energized mitochondria in the Tl acetate medium. Respiration stimulated by 2,4‐dinitrophenol and monoamine oxidase activity of energized mitochondria were not affected by Tl+ regardless of the presence of Pi. We suggested that stimulation by Pi of toxic action of Tl+ in mitochondria and cells could be due to even greater enhancement of uncoupling of mitochondria as shown by an additional increase of swelling and state 4, and in the greater probability of opening of MPTP in the presence of Pi and Ca2+.

Y3 +, La3 +, and some bivalent metals inhibited the opening of the Tl+-induced permeability transition pore in Ca2 +-loaded rat liver mitochondria

We showed earlier that diminution of 2,4-dinitrophenol (DNP)-stimulated respiration and increase of both mitochondrial swelling and electrochemical potential (ΔΨmito) dissipation in medium containing TlNO3 and KNO3 were caused by opening of Tl(+)-induced mitochondrial permeability transition pore (MPTP) in the inner membrane of Ca(2+)-loaded rat liver mitochondria. The MPTP opening was studied in the presence of bivalent metal ions (Sr(2+), Ba(2+), Mn(2+), Co(2+) and Ni(2+)), trivalent metal ions (Y(3+) and La(3+)), and ruthenium red. We found that these metal ions (except Ba(2+) and Co(2+)) as well as ruthenium red inhibited to the MPTP opening that manifested in preventing both diminution of the DNP-stimulated respiration and increase of the swelling and of the ΔΨmito dissipation in medium containing TlNO3, KNO3, and Ca(2+). Inhibition of the MPTP opening by Sr(2+) and Mn(2+) is suggested because of their interaction with high affinity Ca(2+) sites, facing the matrix side and participating in the MPTP opening. The inhibitory effects of metal ions (Y(3+), La(3+), and Ni(2+)), and ruthenium red are accordingly discussed in regard to competitive and noncompetitive inhibition of the mitochondrial Ca(2+)-uniporter. High concentrations (50μM) of Y(3+) and La(3+) favored of MPTP opening in the inner membrane of rat liver mitochondria in Ca(2+) free medium containing TlNO3. The latter MPTP opening was markedly eliminated by MPTP inhibitors (cyclosporine A and ADP).

On the Mechanism(s) of Membrane Permeability Transition in Liver Mitochondria of Lamprey, Lampetra fluviatilis L.: Insights from Cadmium

Previously we have shown that opening of the mitochondrial permeability transition pore in its low conductance state is the case in hepatocytes of the Baltic lamprey (Lampetra fluviatilis L.) during reversible metabolic depression taking place in the period of its prespawning migration when the exogenous feeding is switched off. The depression is observed in the last year of the lamprey life cycle and is conditioned by reversible mitochondrial dysfunction (mitochondrial uncoupling in winter and coupling in spring). To further elucidate the mechanism(s) of induction of the mitochondrial permeability transition pore in the lamprey liver, we used Cd2+ and Ca2+ plus Pi as the pore inducers. We found that Ca2+ plus Pi induced the high-amplitude swelling of the isolated “winter” mitochondria both in isotonic sucrose and ammonium nitrate medium while both low and high Cd2+ did not produce the mitochondrial swelling in these media. Low Cd2+ enhanced the inhibition of basal respiration rate of the “winter” mitochondria energized by NAD-dependent substrates whereas the same concentrations of the heavy metal evoked its partial stimulation on FAD-dependent substrates. The above changes produced by Cd2+ or Ca2+ plus Pi in the “winter” mitochondria were only weakly (if so) sensitive to cyclosporine A (a potent pharmacological desensitizer of the nonselective pore) added alone and they were not sensitive to dithiothreitol (a dithiol reducing agent). Under monitoring of the transmembrane potential of the “spring” lamprey liver mitochondria, we revealed that Cd2+ produced its decrease on both types of the respiratory substrates used that was strongly hampered by cyclosporine A, and the membrane potential was partially restored by dithiothreitol. The effects of different membrane permeability modulators on the lamprey liver mitochondria function and the seasonal changes in their action are discussed.

Bioenergetics of the lower vertebrates. Molecular mechanisms of adaptations to anoxia and hypoxia

In this review, causes of tremendous differences in respiration and oxidative metabolism between the homoiothermal and poikilothermal vertebrates are discussed. Tissues of the latter contain no lower, and sometimes even higher amount of mitochondria with the powerful potential of oxidative enzymes, as compared with the homoiothermal vertebrates; only 3–4% of this potential are realized due to a low rate of oxygen transport because of peculiarities of the cardiovascular system of the lower vertebrates. Limitation of the ATP synthesis provided a specific structure of plasma membranes with the much lower amount of channels for the passive ion leak as compared with the mammalian plasma membranes. Examples are presented of the homoiothermal animal cells (the frog olfactory receptor cell) that contact environment without participation of the blood circulation system and have the energy metabolism level comparable with that of mammalian cells. Examples of energetic aromorphosis are tunas with heat exchangers in several organs and with a powerful system of oxygen delivery to cells; owing to this, intensity of their oxidative metabolism could reach that of the higher terrestrial vertebrates. The closing part of this paper describes the molecular mechanisms that allow some representatives of reptiles, amphibians, and fish to survive long periods of hypoxia and anoxia.

Participation of ADP/ATP- and Aspartate/Glutamate Antiporters in the Uncoupling Action of Fatty Acids in Liver Mitochondria of the Frog Rana temporaria

Data are presented on molecular mechanisms of uncoupling of oxidative phosphorylation by fatty acids (laurate) in liver mitochondria of one of the poikilothermal animals, the frog Rana temporaria. It has been shown that the uncoupling action of laurate in frog liver mitochondria, like in those of mammals, occurs with participation of protein carriers of anions of the inner mitochondrial membrane, ADP/ATP- and aspartate/glutamate antiporters. At the same time, in frog liver mitochondria the uncoupling activity of laurate is lower than in liver mitochondria of mammals (white mice). Seasonal differences in the laurate uncoupling activity in frog liver mitochondria are revealed: it is much lower in April, than in January, the season of metabolic depression. This difference is due to that in January the degree of participation of the aspartate/glutamate antiporter in the uncoupling is considerably decreased.