Cd2+ effects on respiration and swelling of rat liver mitochondria were modified by monovalent cations.

Changes in Cd2+ effects on respiration of succinate-energized rat liver mitochondria were studied after replacement of 100 mM KCl in an incubation medium by equimolar amounts of NaCl or LiCl, or by 200 mM sucrose. In KCl medium, 2.5-10 microM Cd2+ decreased the state 3 and 2,4-dinitrophenol (DNP)-stimulated respiration of mitochondria, and increased their respiration in the state 4, however, 10-40 microM Cd2+ diminished the state 4 respiration. Compared to the experiments with KCl medium, it was demonstrated that Cd2+ effects on the mitochondrial respiration was increased in NaCl medium, decreased in sucrose medium, and unchanged in LiCl medium, except that 10-25 microM Cd2+ decreased the state 4 respiration of mitochondria in the same way as in the NaCl medium. Cd2+ (20 microM) stimulated an extensive swelling of nonenergized mitochondria incubated in 125 mM nitrate media, the effect being increased in the series of Li < Na < K < NH4. Swelling of succinate-energized mitochondria incubated in K-acetate medium was additionally stimulated by 10 microM Cd2+. The initially low swelling of succinate-energized mitochondria in the KCl medium increased with increase in Cd2+ concentrations in this medium. Differences found in the Cd2+ effects on respiration and on swelling of mitochondria incubated in the media used are discussed in terms of general ion permeabilities and differences in Cd2+ binding, its uptake, and interaction with respiratory enzymes.

Cuprous ions activate glibenclamide-sensitive potassium channel in liver mitochondria.

Cuprous ions at micromolar concentrations induced swelling of rat liver mitochondria in isotonic solutions of potassium thiocyanate and potassium acetate. The swelling in K-acetate in the presence of the protonophore [corrected] carbonyl cyanide m-chloropenylhydrazone was partly inhibited by glibenclamide. In K(+)-containing media, Cu+ collapsed the mitochondrial membrane potential formed by operation of the respiratory chain with succinate or tetramethyl p-phenylenediamine + ascorbate as substrates or by the proton-pumping ATPase. In contrast, in K(+)-free media, isotonic sucrose or choline chloride, but not in NaC1, Cu+ induced a transient potassium gradient potential. These results indicate that cuprous ions at low concentrations, apart from promoting the electroneutral K+/H+ exchange, facilitate the uniport of K+, presumably by activating the mitochondrial potassium channel sensitive to glibenclamide.

Anion-dependent transport of thallous ions through human erythrocyte membrane.

Undirectional fluxes of 204Tl+ through the human red blood cell membrane were measured. The inward rate coefficient measured in a K(+)-free saline was 15.6 +/- 0.6 hr-1. The influx of Tl+ could be partially inhibited with 0.1 mM ouabain (by 28%), 0.1 mM DIDS (by 50%) or 1 mM furosemide (by 51%). The inhibitory effects of ouabain and DIDS or furosemide were additive. Half-maximal responses were seen at 0.72 microM and 0.22 mM concentrations of DIDS and furosemide, respectively. A similar action of these blockers on Tl+ influx was observed in the erythrocytes incubated in MgCl2-sucrose media. The outward rate coefficient of 204Tl was also inhibited by DIDS and furosemide (by 65 and 52%, respectively). Rate coefficients of 204Tl influx and efflux decreased significantly in the red cells exposed to Cl(-)-free media (NaNO3 or Mg(NO3)2-sucrose). Under these conditions addition of DIDS and furosemide led to only a small inhibition of Tl+ fluxes. There was a linear increase in Tl+ influx with rising of external Cl- concentration within 80-155 mM or HCO3- concentration from 20 to 40 mM when the sum of anions was kept constant (155 mM) with NO3-. The HCO3(-)-stimulated Tl+ influx was completely blocked by 0.05 mM DIDS but only 67% by 1 mM furosemide. The present study provides direct evidence for the occurrence of Cl- (HCO3-)-dependent, DIDS-sensitive movement of Tl+ across the human erythrocyte membrane in both directions. Under physiological conditions, about half of net Tl+ fluxes occurs due to an anion exchange mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Highly selective blockade of the frog skin sodium channels by monovalent copper cations.

.5 mM Cu+ added to the mucosal side of frog skin caused rapid reversible inhibition of short-circuit current while no effect of Cu+ could be observed at the serosal side. In both cases Cu2+ was reduced to Cu+ by adding 10 mM ascorbic acid. Cu+ being similar to Na+ both in charge and crystal radius (0.096 and 0.095 nm, respectively) appears to block Na+ channels in the apical membrane. Cu2+ being of a smaller size (crystal radius 0.072 nm) was ineffective at the mucosal side causing only a rather slow irreversible inhibition of Na+ transport when added to the serosal bathing solution.

Potassium transport in lamprey (Lampetra fluviatilis) erythrocytes: evidence for K+ channels.

1. Unidirectional K+ (86Rb) influx in lamprey red blood cells was studied under different conditions. 2. The influx of 86Rb was markedly inhibited by 1 mM Ba2+ when cells were incubated in saline containing 4 mM K+. In K(+)-free media, the influx rate constant of 86Rb was lower, and 1 mM Ba2+ had no blocking effect. 3. Treatment of the red cells with 0.1 mM ouabain in the absence of external K+ resulted in the appearance of the component of 86Rb influx inhibited by 1 mM Ba2+, quinine, TEA or amiloride. 4. Similar results were obtained in red cells incubated in Na(+)-free media MgCl2-sucrose. 5. The results obtained provide evidence for the existence of K+ channels in the red cell membrane of the lamprey. Under physiological conditions (in the presence of 4 mM K+) the total rate constant for the 86Rb influx in erythrocytes was about 1.9/hr, including ouabain-sensitive (0.6/hr), Ba(2+)-sensitive (1.1/hr) and residual (0.2/hr) components.

Cadmium as a tool for studying calcium-dependent cation permeability of the human red blood cell membrane.

Three Ca(2+)-dependent procedures known to increase cation permeability of red blood cell membranes were tested with Cd2+ ions which equal Ca2+ ions both in their charge and the crystal radius, 1. Increase of non-selective permeability for monovalent cations by incubating the red cells in a Ca(2+)-free sucrose medium. Addition of Cd2+ to the suspension of leaky cells failed to restore the initial impermeability of the red cell membrane while a repairing effect of Ca2+ was evident both in the presence and absence of Cd2+. Thus, in low electrolyte medium, Cd2+ could neither mimic Ca2+, nor prevent the latter from interacting with membrane structures which control cation permeability. 2. Increase of the K(+)-selective permeability by propranolol plus Ca2+. Cd2+ added to a Ca(2+)-free Ringer type medium containing propranolol enhanced K+ permeability similar to that obtained with Ca2+. No changes of membrane permeability could be detected in the presence of 0.5 mmol/l Cd2+ in absence of propranolol. The Cd(2+)-stimulated K+ channels were different from those induced by Ca2+. They proved to be insensitive to quinine, exhibited a low K+/Na+ selectivity, and showed no tendency to self-inactivation. 3. Stimulation of K+ permeability by electron donors plus Ca2+. Substitution of Ca2+ by Cd2+ yielded results similar to those obtained with propranolol. The ability of Cd2+ to overtake the role of Ca2+ appears to depend on the system studied. It supplies information allowing to distinguish between the diverse Ca(2+)-dependent systems in cell membranes.

Interaction of Cu+ with mitochondria.

The uptake of Cu+ by rat liver mitochondria is rapid and extensive. Respiration is stimulated by 10 microM Cu+ then inhibited and the inhibition could not be relieved with uncoupling agents. Collapse of the membrane potential is induced by 5-10 microM Cu+. These effects are partially inhibited by radical scavengers indicating the involvement of radical production in these events. Reduction of the GSH content and production of peroxidation products by higher amounts of Cu+ was also demonstrated. Swelling of non-respiring rat liver and heart mitochondria in sodium or lithium acetate was used to study effects of Cu+ on the Na+/H+ exchanger. Swelling is stimulated by 5-100 microM Cu+. In the presence of a radical scavenger the swelling is reduced. In sodium nitrate media diltiazem-sensitive stimulated swelling is observed. Amiloride was found to inhibit Cu(+)-induced efflux of Ca2+. At high concentrations of Cu+, a general increase in permeability was the dominant feature.

Thallium and rubidium permeability of human and rat erythrocyte membrane.

Transport of Tl+ and Rb+ in human and rat erythrocytes was investigated in the presence of ouabain. The chloride-dependent cotransport of Tl+, Rb+ and Na+ was precluded by replacement of Cl- by NO3-. The inward and outward rate constants for the residual fluxes of the cations were determined by measuring the transport of 204Tl and 86Rb in double label experiments. The rate of passive transport of Tl+ exceeded that of Rb+ by one-two orders of magnitude in human as well as rat erythrocytes. The membrane barrier which contributes to the maintenance of ion gradients was shown not to be a barrier for Tl+ which easily penetrates the membrane by an unknown mechanism. In rat erythrocytes the barrier for Rb+ was 10-15 times weaker than that in human red blood cells, while the corresponding ratio of rat/human Tl+ permeabilities was about 1.8-2.0. It follows that Tl+ permeability is only slightly affected by factors modifying the permeability to alkali cations. The increase of temperature from 20 degrees to 37 degrees C resulted in a three-fourfold stimulation of the passive transport of Tl+ both in human and rat erythrocytes. The movement of Tl+ and Rb+ through the erythrocyte membrane differed substantially from their diffusion along the excitable membrane channels characterized both by poor Tl+/K+ selectivity and weak temperature dependence.

Interaction between propranolol and electron donors in altering the calcium ion-dependent potassium ion-permeability of the human red blood cell membrane.

Both propranolol and the electron donors ascorbate plus phenazine methosulfate increase the K+-permeability of the red blood cell membrane. The present investigation examined whether these effects were additive. Contrary to expectations, propranolol added after electron donors sharply inhibited the K+ (86Rb) efflux induced by such donors, without forming new K4 channels analogous to those induced by propranolol in intact red blood cells. The inhibitory effect of propranolol may be due to generalized disturbances of membrane structures necessary for the functioning of the K+ channels organized in the presence of reducing agents. In contrast, the electron donors added after propranolol caused a further stimulation of the 86Rb loss from the propranolol-treated red cells. The combined effect of these drugs therefore depends on the order of their addition. The possible mechanism of their interaction is briefly discussed.

The effect of the energy state of mitochondria on the kinetics of unidirectional cation fluxes.

Unidirectional fluxes of triphenylmethylphosphonium and of Cs+ as its valinomycin complex were studied using trace concentrations of the cations. The rate constants of influx and efflux were estimated mainly at 0 degrees C from the uptake kinetics in respiring mitochondria and the in/out ratios in the steady state. The efflux rate constants in the energized state were also measured after dilution of the mitochondrial suspension in the steady state, and in deenergized mitochondria from the efflux rates of cations after inhibition of respiration. It was found that the energy state of mitochondria had little effect on the rate constants of efflux, while the rate of influx was strongly stimulated by respiration. The former finding is not readily explained by the classical chemiosmotic theory, since a transmembrane potential, negative on the inside, formed on energization would be expected to strongly inhibit the efflux of cations. The data may be explained by a pump-and-leak model in which localized electrical fields in hydrophobic domains of the membrane are coupled to the pumping of hydrophobic cations against an electrochemical gradient, while leaks would effect efflux.

Uptake of thallous ions by mitochondria is stimulated by nonactin but not by respiration alone.

Nonrespiring rat-liver mitochondria swell in media containing high concentrations of thallous nitrate, indicating passive penetration of Tl+. This swelling could be further stimulated by 10 nM or more nonactin while even 1 microM valinomycin was without effect. Nonactin was also much more potent than valinomycin in stimulating swelling of respiring mitochondria in the presence of thallous acetate. It is evident that nonactin acts as a potent ionophore of Tl+ able to promote both the passive and energized uptake of Tl+ in mitochondria. The distribution of Tl+, present in trace concentrations below 1 mM, was measured during energisation by respiration both in the presence and absence of ionophores. Respiration induced net uptake of Tl+ only in the presence of ionophores, though Tl+ as a permeant cation was expected to sense respiration-induced changes in the membrane potential. The data may be interpreted as indicating that no transmembrane potential is formed upon energisation, but localized fields, which are able to interact with the lipophilic ionophore complexes of Tl+, but not with the hydrophilic cation Tl+. This interpretation is valid only if thermodynamic equilibrium has been reached.

Mechanism of mitochondrial transport of thallous ions.

Rat liver mitochondria were found to swell under nonenergized conditions when suspended in media containing 30-40 mM TINO3. Respiration on succinate caused a rapid contraction of mitochondria swollen under nonenergized conditions. In the presence of thallous acetate, there was a rapid initial swelling under nonenergized conditions until a plateau was reached; respiration on succinate then caused a further swelling. Trace amounts of 204Tl (less than 100 microM) equilibrated fairly rapidly across the mitochondrial membrane. The influx of Tl+ was able to promote the decay not only of a valinomycin-induced K+-diffusion potential but also of respiration-generated fields in the inner membrane in accordance with the electrophoretic nature of Tl+ movement. Efflux of Tl+ showed a half-time of about 10 sec at 20 degrees C and was not affected appreciably by the energy state. Efflux was retarded by Mg2+ and by lowering the temperature. The data indicate that Tl+ when present at high concentrations, 30mM or more, distributes across the mitochondrial inner membrane both in response to electrical fields and to delta pH. In energized mitochondria the uptake of Tl+ would occur electrophoretically, while Tl+/H+ exchange would constitute a leak. In the presence of NO3-, the movements of Tl+ are determined by that of NO3-, indicating short-range coupling of electrical forces. At low concentrations of Tl+, 5 mM or less, there was no indication of a Tl+/H+ exchange, which appears to be induced by high concentrations of Tl+.

Effect of membrane potential on the passive transport of Tl+ in human red blood cells.

It was earlier found that Tl+ can easily penetrate the red cell membrane. The main finding of this work is that Tl+ can be used for studying alterations in the membrane potential of human red blood cells exposed to various experimental conditions. It was shown that after inhibiting active transport by ouabain, both the rate of trans-membrane movement and the cell/medium distribution of Tl+ were in a good agreement with the expected changes in membrane potential. Alterations in membrane potential were induced by modifying the cation permeability of the red cell membrane and by varying the cation concentration gradient across the membrane, which were achieved: 1) by incubation in an electrolyte-free sucrose solution. 2) by addition of valinomycin or 3) by addition of propranolol. Changes in cation permeability were followed by means of 86Rb tracer. Hyperpolarization of the red cell membrane led to accelerated influx and retarded efflux of Tl+. The opposite effect was obtained by depolarization. Quantification of the results was made using the Nernst equation and the cell/medium concentration ratio of Tl+ at equilibrium. The calculations show that the membrane potential of the propranolol-treated cells increased to about -20 mV, negative inside. The mechanism of the propranolol effect is briefly discussed.

Effect of extracellular potassium on the loss of potassium from human red blood cells treated with propranolol.

The Ca++-dependent propranolol-induced increase of K+ permeability of human red blood cells, well documented in previous studies, was found to depend on extracellular K+. This was shown by studying the passive transport of 86Rb and the loss of bulk cellular K+ in both K+-free and K+-containing media. The maximal effect of propranolol was achieved with 5-10 mM K+ in incubation media. The external K+ could be substituted with Tl+, but not with Na+. When added after propranolol, extracellular K+ failed to initiate the effect of propranolol on membrane permeability. The cell/medium distribution of permeant 204Tl showed that the propranolol-induced increase of K+ permeability did not result in considerable hyperpolarization of the red blood cell membrane. The data obtained seem to be more consistent with a counter-transport model for explaining the propranolol effect than with a mechanism based on free diffusion of K+ through the membrane.

Electrophoretic transport of T1+ in mitochondria.

The distribution of T1+ between rat liver mitochondria and the medium was studied; millimolar or smaller concentrations of T1+ were labeled with 204T1. The T1+ distribution responded to transient diffusion potentials in a way that indicated electrophoretic movements of T1+. The diffusion potentials were induced by efflux of K+ in response to addition of valinomycin to nonrespiring mitochondria suspended in a medium with low concentrations of K+ or by efflux of H+ induced by making the medium more alkaline in the presence of a protonophorous (proton-conducting) uncoupling agent. Changes in membrane potential induced by valinomycin were followed with the aid of safranine. T1+ brought about collapse of the diffusion potential. It is concluded that T1+ is able to penetrate the mitochondrial membrane electrophoretically.

Factors affecting the relative magnitudes of the ouabain-sensitive and the ouabain-insensitive fluxes of thallium ion in erythrocytes.

A maximal rate of the ouabain-sensitive 204-Tl influx in human erythrocytes can be attained at trace concentrations of Tl+ in Mg2+ isotonic media free of K+ and Na+. The maximal influx of Tl+ from isotonic Mg(NO3)2 at 20 degrees C and pH 7.4 was 0.45 mM.l(-1).h-1 with a Km of 0.025 mM. In contrast to the active influx of Tl+, the passive Tl+ fluxes were neither saturated nor influenced by external cations in the range of concentrations of Tl+ and K+ studied. The rate constants of Tl+ passive fluxes in human and cat erythrocytes can be related to pH by the equation log kin(OUT)= -A + B.pH, where A and B are empirical constants for particular conditions. The apparent activation energy was 16 and 11 kcal/mol in sulphate and nitrate media, respectively. Tl+ and the alkali metal cations seem to overcome a common barrier in the erythrocyte membrane. Nevertheless, the rate of the passive penetration of Tl+ is about two orders of magnitude faster than those of K+ or Rb+. An extra non-Coulombic interaction between Tl+ and membrane ligands appears to be involved providing an accumulation of Tl+ somewhere in the vicinity of the membrane barrier and increasing the diffusion fluxes of Tl+ in both directions.

Thallium inhibition of ouabain-sensitive sodium transport and of the (Na+ plus K+)-ATPase in human erythrocytes.

The influence of Tl+ on Na+ transport and on the ATPase activity in human erythrocytes was studied. 0.1-1.0 mM Tl+ added to a K+-free medium inhibited the ouabain-sensitive self-exchange of Na+ and activated both the ouabain-sensitive 22Na outward transport and the transport related ATPase. 5-10mM external Tl+ caused inhibition of the ouabain-sensitive 22Na efflux as well as the (Na+ plus Tl+)-ATPase. Competition between the internal Na+ and rapidly penetrating thallous ions at the inner Na+-specific binding sites of the erythrocyte membrane could account for the inhibitory effect of Tl+. An increase of the internal Na+ concentration in erythrocytes or in ghosts protected the system against the inhibitory effect of high concentration of Tl+. A protective effect of Na+ was also demonstrated on the (Na+ plus Tl+)-ATPase of fragmented erythrocyte membranes studied at various Na+ and Tl+ concentrations.