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Presents a study which examined the involvement of ion K+-adenosine triphosphate-dependent channel in the transport of monovalent thallium across the inner membrane of rat liver mitochondria. Information on the function of monovalent thallium; Mechanism of the transportation of ions to the mitochondria; Effect of cation and anions on the mitochondria.

Accumulation of Na+ and K+ ions in oocytes of the river lamprey Lampetra fluviatilis and their transport across the plasma membrane is realized by two main mechanisms--Na,K-pump and Na,K,Cl-cotransport. At the prespawning period from December to May the intracellular Na+ concentration was observed to increase from 10 to 25 mM and the K+ concentration--from 28 to 45 mM. Results obtained on isolated oocytes with aid of 204Tl radioactive label have shown that contributions of the Na,K-pump and Na,K,Cl-cotransport to potassium accumulations were close until March. In spring, the total K+ inflow almost doubled owing to activation of the Na,K-pump, whereas contribution of Na,K,Cl-cotransport did not change. It seems that an increase of the intracellular content of the main inorganic cations in oocytes resulted in parallel activation of the Na,K-pump and probably of Na/H-exchange. The biological significance of activation of these mechanisms of ion transport at the prespawning period might be due to a necessity of accumulation of Na+ and K+ ions in concentrations optimal for subsequent embryonic development.

Mechanisms of transport of monovalent thallium across the membrane of oocyte of the lamprey Lampetra fluviatilis were studied by using 204Tl. Transport of Tl+ in lamprey oocytes has been shown to be realized by at least two pathways: through Na/K-pump and by the mechanism of Na,K,Cl-cotransport. In the standard Ringer solution (mM): 4 KCl, 140 NaCl, 0.5 CaCl2, 5 glucose, 10 Tris-HCl--in the presence of oubain, the coefficient of the 204Tl stationary distribution (cell/medium) was within the range of 2.3-2.5, while the time necessary to reach its 50 % value amounted to 40-5 min at 20 degrees C. In potassium-free media, transport of 204Tl via Na/K-pump was described by simple kinetis with saturation and was characterized by the value V(max) = 520 pmol/(cell x h) and K(M) = 0.3 mM. In the presence of 4 mM K+ and 0.1 mM/l Tl+, the oubain-sensitive Tl+ flow decreased to 75 pmol/(cell x h). At activation of the mechanism of Na,K,Cl-cotransport by the outer Na+ (in Na-NMDG media of different composition) the total inflow of Tl+ reached 193 +/- 20 pmol/(cell x h), while the butamenide-sensitive component--119 +/- 12 pmol/(cell x h) with K(M) for Na+ about 20 mM. In the incubation media with variable concentration of chloride ions (replacement of Cl- by NO3(-)) the total Tl+ flow reached 220 +/- 21, while via the mechanisms of Na,K,Cl-cotransport--87 +/- 8 pmol/(cell x h). Under our experimental conditions, mechanisms of active transport and Na,K,Cl-cotransport accounted for 94% of the Tl+ inflow. The potassium channels that usually are also permeable to monovalent thallium ions were not revealed.

We have studied Cd2+-induced effects on mitochondrial respiration and swelling in various media as a function of the [Cd2+] in the presence or absence of different bivalent metal ions or ruthenium red (RR). It was confirmed by monitoring oxygen consumption by isolated rat liver mitochondria that, beginning from 5 microM, Cd2+ decreased both ADP and uncoupler-stimulated respiration and increased their basal respiration when succinate was used as respiratory substrate. At concentrations higher than 5 microM, Cd2+ stimulated ion permeability of the inner mitochondrial membrane, which was monitored in this study by swelling of both nonenergized mitochondria in 125 mM KNO3 or NH4NO3 medium and succinate-energized mitochondria incubated in a medium containing 25 mM K-acetate and 100 mM sucrose. We have found substantial changes in the above-mentioned Cd2+ effects on mitochondria treated in sequence with 100 microM of Ca2+, Sr2+, Mn2+ or Ba2+(Me2+) and 7.5 microM RR, as well as the alterations in Cd2+ action on the uptake of 137Cs+ by succinate-energized mitochondria in the presence or absence of valinomycin in acetate medium (50 mM Tris-acetate and 140 mM sucrose) with or without Ca2+ or RR. The evidence obtained indicate that Ca2+ exhibits a synergestic action on all Cd2+ effects examined, whereas Sr2+ and Mn2+, conversely, are antagonistic. In the presence of RR, the Cd2+ effects on respiration [stimulation of State 4 respiration and inhibition of 2,4-dinitrophenol (DNP)-uncoupled respiration] still exist, but are observed at concentrations of cadmium more than one order higher; the inhibition of State 3 respiration by Cd2+ conversely, takes place under even lower cadmium concentrations than those determined without RR in the medium. In addition, RR added simultaneously with cadmium in the incubation medium prevents any swelling in the nitrate media, but induces an increment both in Cd2+-stimulated swelling and 137Cs+ (analog of K+) uptake in the acetate media. For the first time, we have shown that Cd2+-induced swelling in all media under study is susceptible to cyclosporin A (CSA), a high-potency inhibitor of the mitochondrial permeability transition (PT) pore. The observations are interpreted in terms of a dual effect of cadmium on respiratory chain activity and permeability transition.

Effects of Cd2+ and two complexes of bivalent cadmium with 1,3-bis(4-chlorbenzylidenamino)-guanidine and anabasine on ion permeability of the inner membrane and respiration of isolated rat liver mitochondria were studied. Starting from 5 microM, Cd2+ decreased state 3 and DNP-stimulated respiration of mitochondria and increased their state 4 respiration. At 30 microM, Cd2+ decreased state 4 respiration. The complexes, particularly complex of Cd2+ with 1,3-bis(4-chlorbenzylidenamino)-guanidine, inhibited the mitochondrial respiration at lower concentration of Cd2+. Nonenergized mitochondria incubated in media containing 125 mM of NH4NO3 or KNO3 showed more pronounced swelling in experiments with 10 microM of the complexes than with Cd2+. The complexes produced swelling of the mitochondria energized by 5 mM of succinate and incubated in medium containing 25 mM K-acetate and 100 mM sucrose. Uptake of 137-Cs by succinate-energized mitochondria in the presence of 10(-8) M of valinomycin was substantially decreased in experiments with 10 microM of the complexes than with Cd2+. Ruthenium red (7.5 microM) prevented this effect with 10 microM of complex of Cd2+ with 1,3-bis(4-chlorbenzylidenamino)-guanidine and especially complex of Cd2+ with anabasine and Cd2+. These results indicate that the cadmium organic complexes affect respiration and perturb ion permeability significantly stronger than Cd2+.

Ion permeability of internal membrane and a respiration in isolated rat liver mitochondria, further related to as "sodium ones", were studied following replacement of K+ ions for Na+ ones in the mitochondrial matrix. As compared with the control ("potassium mitochondria"), state 4 respiration in the sodium mitochondria, energized by succinate, was shown to be enhanced in KCl or sucrose media. Oxygen consumption rates in the sodium mitochondria, being in state 3 or stimulated by 2,4-dinitrophenol, were lower than rates for the control mitochondria. This effect was much pronounced in the sucrose medium. The coefficients, characterizing the distribution of 137Cs between mitochondria and the medium, were lower for the sodium mitochondria than for the control in the presence of 2.5 mM succinate and 10(-8) M valinomycin. In comparison with the control, a more extensive swelling for the sodium mitochondria was found, first, in the medium containing 25 mM K-acetate and 100 mM sucrose for succinate-energized mitochondria, and second, in the medium containing 125 mM NH4NO3 without mitochondrial energization. Changes disclosed in respiration, swelling and coefficients of 137Cs distribution for the sodium mitochondria are supposed to be caused by non-uniform effects of Na+ and K+ ions on the water structure of mitochondrial matrix, ion permeability of internal membrane, and the activity in oxidative phosphorylation enzymes.

Cadmium inhibits many mitochondrial enzymes participating in respiratory and oxidative phosphorylation processes, with reactive SH-groups of some of these enzymes being localized in hydrophobic domains of mitochondrial membrane. A comparative investigation was made of the action of Cd2+ and two its hydrophobic complexes (a more hydrophobic one with 1, 3-bis(4-chlorbenzilidenamino) guanidine [Cd(CBG)], and a less hydrophobic one with anabazin [Cd(ANB)] on ion permeability of the mitochondrial membrane and on respiration of rat liver mitochondria in vitro. Cd2+ ions in concentration of more than 2.5 microM and in the presence of DNP decreased mitochondrial respiration in state 3 and activated in in state 4; however, in concentration higher than 20 microM Cd2+ the activation acquired a short-term character and the state 4 respiration was seen to decrease. The experiments on mitochondrial swelling in the medium containing 125 mM NH4NO3 revealed that the complex and CBG, like Cd2+, increased proton permeability of the internal mitochondrial membrane. 10 microM Cd(ANB) and Cd(CBG) had more effect on mitochondrial respiration than Cd2+ CBG (in DNP-stimulated respiration), and ANB had no effect on mitochondrial respiration. The toxicity of Cd(CBG) was higher than that of Cd2+ and Cd(ANB), due to a higher hydrophobicity of Cd(CBG). A higher activity of Cd(CBG) was suggested to be due to its hydrophobicity. In the experiments with 137Cs in succinate-energized mitochondria at 20 degrees C and 10(-8) valinomycine, the susceptibility of toxic effects caused by the complex was lower than that caused by Cd2+ in the presence of ruthenium red, which is a blocker of Ca2+ transport. A higher Cd(CBG) effect in experiments with 137Cs and RR is accounted for by the fact that these agents are transported into mitochondria via different pathways. On the contrary, the effect of RR in similar experiments with Cd(ANB) was stronger than that of Cd2+. It is assumed that transport of Cd(ANB) in mitochondria, opposite to that of Cd(CBG), is not carried out across hydrophobic domains of the mitochondrial membrane.

Studies have been made on the valinomycin-induced transport of Cs+ at 0 degree C in rat liver mitochondria energized with succinate. Under these conditions, mitochondria preserve their ability to accumulate Cs+ for 6 hours of incubation or even longer. The rate of both the inward and outward movement of Cs+ at 0 degree C was approximately an order lower than at room temperature, although the stationary distribution of Cs+ remained at a high level. Electron microscopic observations revealed no significant changes in the structure of mitochondria in spite of their prolonged incubation. These properties of mitochondria make it possible to determine the kinetic parameters of ion transport and in this way to evaluate functional condition of mitochondria at low temperatures.

The influence of Cd2+ on the function and structure of liver mitochondria of rats and lamprey (Lampetra fluviatilis L.) has been studied in vitro. It is shown that Cd2+ can penetrate into the mitochondrial matrix due to Ca2+-transport mechanism. Being stored in the mitochondria, Cd2+ inhibits respiration and an energy dependent transport of penetrating cations (Cs+-valinomycin), and disturbs passive permeability of the inner mitochondrial membrane for monovalent cations and H+. The effect of Cd2+ on the lamprey liver mitochondria is more pronounced than in the case of rats.

Inhibiton of respiration at 0 degrees C believed to decrease the membrane potential in mitochondria did not lead to a substantial acceleration of the efflux of penetrating cations (triphenylmethylphosphonium, Cs- valinomycin). On the contrary the energy-dependent influx of the cations was rapidly inhibited indicating that there was no prolonged delay in the membrane deenergization. Low sensitivity of the cation efflux to respiration inhibition suggests under the conditions studied the metabolic component of the membrane potential to be rather small.

Energy-dependent accumulation of triphenylmethylphosphonium (TPMP+) in rat liver mitochondria at 0 degree C is only 30-50% less than that observed at room temperature, while the rate of TPMP+ penetration drastically decreases enabling accurate studying of transport kinetics. Potassium diffusion potential accelerates the influx of TPMP+ and decreases the efflux rate in agreement with the expected behaviour of penetrating cations diffusing along or against an electric field. On the contrary, energization of mitochondria supposed to generate the metabolic membrane potential increases only the influx rate of TPMP+ without affecting the efflux of the cation. The data are clearly inconsistent with the presence of a metabolically dependent membrane potential. Energy-dependent accumulation of TPMP+ appears to be an active process transporting lipophilic cations against their electrochemical gradient. Stationary-state distribution of TPMP+ between energized mitochondria and medium cannot be used for the membrane potential determination.

Unidirectional fluxes of tracers 137Cs+ (+valinomycin) and 204Tl+ (+nonactin) in mitochondria maintained under stationary state at 0 degree C have been studied. The tracer uptake in energized mitochondria (Q infinity Cmit/Cmed = Kin/kout) was about 10 times as high compared to that in a deenergized state. Energization caused a 5-10 fold increases of the influx rate constant kin, while the efflux rate constant, kout, decreased only by 1.5-2 times of less. There was a positive correlation between Q infinity and kin. No correlation could be found between Q infinity and kout. The results obtained suggest that under the experimental conditions studied the energization did not increase substantially the membrane potential expected to retard the efflux of permeant cations. In the presence of ionophores the monovalent cations are supposed to be accumulated in mitochondria against the electrochemical gradient.

Transport of monovalent thallium ions in bacterial cells was studied. An energy-dependent transport of T1+ against electrochemical gradient into the cells of S. faecalis and Micrococcus lysodeikticus, according to the Michaelis-Menten kinetics was observed. T1+, being a K+ analog, is involved into active K+ transport. Unlike K+, T1+ readily penetrates bacterial membranes, reaching the level of stationary distribution between the cells and the medium. This permits to use T1+ as a penetrating cation to study the mechanism of potassium transport in bacteria without the use of ionophores, which can destroy the integrity of cell membranes.

The energy-dependent accumulation of triphenylmethylphosphonium (TPMP+) in rat liver mitochondria at 0 degrees C is less than that at a room temperature only by 30-50%, while the cation penetration rate drastically decreases thus making possible an accurate studying of the transport kinetics. It is shown that K+-diffusion potential accelerates the influx of TPMP+ and decreases the efflux rate in full agreement with the expected behaviour of a penetrating cation diffusing along or against the electrical field. On the contrary, the energization of mitochondria, which has been generally believed to generate a metabolic membrane potential, caused only a stimulation of the influx of TPMP+ without affecting the efflux rate. The TPMP+ efflux half-time was found to be about 3 minutes independently of the energetic state of mitochondria. This independence indicates that the energization is not accompanied by a rise of the membrane potential. The accumulation of TPMP+ by the energized mitochondria must be considered as an active process transporting lipophilic cations against the electrochemical gradient. It follows that the steady state distribution of TPMP+ cannot be a measure of the membrane potential in mitochondria.