S. M. Korotkov

Mechanism of primary Cd2+-induced rat liver mitochondria dysfunction: discrete modes of Cd2+ action on calcium and thiol-dependent domains

We attempted to discern discrete sites of Cd2+ deleterious action on rat liver mitochondrial function. In particular, EGTA, ADP, and cyclosporin A (potent mitochondrial permeability transition antagonists) affected mainly Cd2+-induced changes in resting state respiration, eliminating its stimulation in KCl medium, while dithiothreitol (DTT, a dithiol reductant) produced its effect both on Cd2+ activation of the basal respiration and Cd2+ depression of uncoupler-stimulated respiration, evoking its restoration. Substantial differences in DTT influence on mitochondrial respiration at low and high [Cd2+] were revealed, namely, an enhanced mitochondrial permeabilization in the presence of saturated [DTT] at high [Cd2+] took place. Besides, DTT only partially reversed Cd2+-induced swelling in NH4NO3 medium when glutamate plus malate or succinate without rotenone was used. Contrarily, DTT produced complete reversal of the swelling of succinate-energized mitochondria when rotenone was present in the medium. In addition, in the presence of rotenone both Cd2+-produced activation of the resting state respiration in KCl medium and Cd2+-induced swelling in sucrose medium of succinate-energized mitochondria were more sensitive to cyclosporin A than the same Cd2+ effects obtained on mitochondria oxidizing succinate (without rotenone) or glutamate plus malate. We have concluded that Cd2+, producing primary mitochondrial dysfunction, acts both as a thiol and Me2+ binding site reagent. Suppositions about possible localization of separate sites of direct Cd2+ effects on mitochondrial function were made.

Cyclosporin A-sensitive permeability transition pore is involved in Cd2+-induced dysfunction of isolated rat liver mitochondria: doubts no more

There is dose-dependent Cd(2+)-evoked swelling of isolated rat liver mitochondria energized by complex I, II, or IV respiratory substrates in sucrose medium in the absence of added Ca(2+) and P(i), which is prevented by Sr(2+). Permeability transition effectors (ADP, CsA, EGTA, RR, DTT, ATR, P(i), and Ca(2+)) affect in a corresponding way Cd(2+)-promoted membrane permeabilization in NH(4)NO(3), KCl, and sucrose media. Maximal depression of Cd(2+)-induced swelling is achieved by simultaneous addition of ADP, Mg(2+), and CsA that produces either synergistic (NH(4)NO(3)) or additive (KCl and sucrose media) action. Sustained activation by low [Cd(2+)] of mitochondrial basal respiration in KCl medium is observed both in the absence and in the presence of rotenone and/or oligomycin but only in the latter case (rotenone+oligomycin) CsA inhibits completely Cd(2+) activation of St 4 respiration and partially reverses DNP-uncoupled respiration depressed by cadmium. Cd(2+) effects are discussed in terms of comparison with those of Zn(2+) and PhAsO.

Influence of Tl + on mitochondrial permeability transition pore in Ca 2+ -loaded rat liver mitochondria

The Tl+-induced opening of the MPTP in Ca2+-loaded rat liver mitochondria energized by respiration on the substrates succinate or glutamate plus malate was recorded as increased swelling and dissipation of mitochondrial membrane potential as well as decreased state 4, or state 3, or 2,4-dinitrophenol-stimulated respiration. These effects of Tl+ increased in nitrate media containing monovalent cations in the order of Li+ < NH4+ ≤ Na+ < K+. They were potentiated by inorganic phosphate and diminished by the MPTP inhibitors (ADP, CsA, Mg2+, Li+, rotenone, EGTA, and ruthenium red) both individually and more potently in their combinations. Maximal swelling of both non-energized and energized Ca2+-loaded mitochondria in rotenone-free media is an indication of Ca2+ uptake driven by respiration on mitochondrial endogenous substrates. It is suggested that Tl+ (distinct from Cd2+, Hg2+, and other heavy metals and regardless of the used respiratory substrates) can stimulate opening of the MPTP only in the presence of Ca2+. We discuss the possible participation of Ca2+-binding sites, located near the respiratory complex I and the adenine nucleotide translocase, in inducing opening of the MPTP.

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.

Tl(+) showed negligible interaction with inner membrane sulfhydryl groups of rat liver mitochondria, but formed complexes with matrix proteins.

The effects of Tl+ on protein sulfhydryl (SH) groups, swelling, and respiration of rat liver mitochondria (RLM) were studied in a medium containing TlNO3 and sucrose, or TlNO3 and KNO3 as well as glutamate plus malate, or succinate plus rotenone. Detected with Ellmans reagent, an increase in the content of the SH groups was found in the inner membrane fraction, and a simultaneous decline was found in the content of the matrix‐soluble fraction for RLM, incubated and frozen in 25–75 mM TlNO3. This increase was greater in the medium containing KNO3 regardless of the presence of Ca2+. It was eliminated completely for RLM injected in the medium containing TlNO3 and then washed and frozen in the medium containing KNO3. Calcium‐loaded RLM showed increased swelling and decreased respiration. These results suggest that a ligand interaction of Tl+ with protein SH groups, regardless of the presence of calcium, may underlie the mechanism of thallium toxicity.

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+.

Closure of mitochondrial potassium channels favors opening of the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

It is known that a closure of ATP sensitive (mitoKATP) or BK-type Ca2+ activated (mitoKCa) potassium channels triggers opening of the mitochondrial permeability transition pore (MPTP) in cells and isolated mitochondria. We found earlier that the Tl+-induced MPTP opening in Ca2+-loaded rat liver mitochondria was accompanied by a decrease of 2,4-dinitrophenol-uncoupled respiration and increase of mitochondrial swelling and ΔΨmito dissipation in the medium containing TlNO3 and KNO3. On the other hand, our study showed that the mitoKATP inhibitor, 5-hydroxydecanoate favored the Tl+-induced MPTP opening in the inner membrane of Ca2+-loaded rat heart mitochondria (Korotkov et al. 2013). Here we showed that 5-hydroxydecanoate increased the Tl+-induced MPTP opening in the membrane of rat liver mitochondria regardless of the presence of mitoKATP modulators (diazoxide and pinacidil). This manifested in more pronounced decrease in the uncoupled respiration and acceleration of both the swelling and the ΔΨmito dissipation in isolated rat liver mitochondria, incubated in the medium containing TlNO3, KNO3, and Ca2+. A slight delay in Ca2+-induced swelling of the mitochondria exposed to diazoxide could be result of an inhibition of succinate oxidation by the mitoKATP modulator. Mitochondrial calcium retention capacity (CRC) was markedly decreased in the presence of the mitoKATP inhibitor (5-hydroxydecanoate) or the mitoKCa inhibitor (paxilline). We suggest that the closure of mitoKATP or mitoKCa in calcium loaded mitochondria favors opening of the Tl+-induced MPTP in the inner mitochondrial membrane.

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).

Action of La3+ on the systems providing contractility of vertebrate myocardium

The inotropic action of La3+ on frog myocardium was studied with taking into account its effect on mitochondria of cardiomyocytes (CM). It has been established that in the range of studied concentrations (0.2–6.0 mM), La3+ decreases dose-dependently the force of cardiac contractions (by 3.3–92.2%). In parallel experiments on isolated rat heart mitochondria (RHM), La3+ at a concentration of 25 μM has been shown to cause swelling of non-energized and energized mitochondria incubated in isotonic medium with 125 mM NH4NO3 and in hypotonic medium with 25 mM CH3COOK. The study of oxidative processes in mitochondria with aid of polarographic method of measurement of oxygen concentration has shown that La3+ at concentrations of 50 and 100 μM increases the oxygen consumption rate by mitochondria in the state 2. However, La3+ does not decrease the respiration rate of isolated mitochondria in the state 3, as this takes place in the case of use of Cd2+ or at the Ca2+-overloading of mitochondria. The rate of endogenous respiration of isolated mitochondria in the medium with La3+ was higher than in control, which suggests its effect on ion permeability of the inner membrane. The data obtained in this work indicate that the La3+-produced decrease of contractility of cardiac muscle is not only due to the direct blocking effect on the potential-controlled Ca2+-channels, but is also mediated by its unspecific action on the CM mitochondria. This action is manifested as an acceleration of the energy-dependent K+ transport in matrix and as an increase of ion permeability of the inner mitochondrial membrane (IMM).

Effects of pinacidil and calcium on isolated rat heart mitochondria

113 It was shown in [1–3] that pinacidil, a nonselective modulator of mitochondrial K ATP dependent chann nels (mitoK ATP), had a pronounced protective effect on the contractile parameters of an isolated rat heart after exposure to ischemia and subsequent reperfuu sion. This protective effect of pinacidil and diazoxide, a selective modulator of these channels, was termed pharmacological preconditioning [3]. To date, there is no single point of view on the nature of this phenomee non [2, 4–8]. Some researchers believe that activation of mitochondrial K ATP dependent channels by pinacii dil triggers a complex cascade mechanism of preconn ditioning involving protein kinase C. Others believe that this protective effect of pinacidil is determined by its ability for mild uncoupling of respiration of mitoo chondria. This, in turn, facilitates their swelling, stimm ulates reduction of electrochemical potential (ΔΨ mito) on the inner mitochondrial membrane (IMM), and ultimately reduces the voltageedependent accumulaa tion of calcium in the matrix after exposure of the heart to ischemia/reperfusion. Pinacidil was shown to activate respiration of isoo lated mitochondria in states 2 and 4 [1, 3, 9–13]. However, mitochondrial respiration in state 3 and the ATP synthesis rate are changed only slightly [9, 13]. Pinacidil decreases calcium transport to the matrix and stimulates swelling of mitochondria and the proo ton permeability of their inner membrane [1, 3, 8, 12]. It is known that mitochondrial calcium load followed by reduction of ΔΨ mito may lead to the opening of the mitochondrial permeability transition pore (MPTP) in the inner mitochondrial membrane [7, 14]. In recent years, the relationship between the structural elements that form mitoK ATP and MPTP is widely diss cussed [6, 7]. It was shown that pinacidil prevents the opening of the MPTP in cardiomyocytes loaded with tetramethylrhodamine methyl ester (TMRM) [15]. Given the above, it is clear that the relationship of the effects of pinacidil with the MPTP induction in the inner membrane of mitochondria under conditions of calcium load has almost not been studied. For this reaa son, we investigated the combined effect of pinacidil (a mild uncoupling agent) and calcium on the swelll ing, respiration, and membrane potential of isolated rat heart mitochondria. Experiments were performed with male Wistar rats weighing 200–250 g. Rat heart mitochondria (RHM) were isolated by the method developed by us earlier [4] and suspended in 3 mL of a medium containing 300 mM sucrose and 10 mM …