An analysis of the binding of fluorescence probes in mitochondrial systems.

Measurements of the binding of the fluorescent probes 8-anilinonaphthalene-1-sulfonate (ANS) and ethidium ions to whole and disruped mitochondria and submitochondrial particles suggest that the inner mitochondrial membrane is freely permeable to the two probes. Equations relating the binding of permeant probes to the electro-chemical balance across the membrane of vesicular systems are derived and these equations used to analyze Scatchard plots of the binding of the two probes to energized and nonenergized mitochondria and EDTA particles.     

The mechanism of mitochondrial swelling. V. Permeability of mitochondria to alkali metal salts of strong acid anions

Mitochondria do not swell appreciably when suspended in media containing the chlorides or bromides of alkali metal or ammonium ions. On the other hand, extensive swelling takes place when mitochondria are suspended in ammonium or sodium acetate. These findings have been widely interpreted to mean that the mitochondrial membrane is impermeable to chloride and bromide ions. However, the resistance of the mitochondria to volume changes is not necessarily a valid criteria of impermeability to a given ion pair. Such a conclusion presumes the as yet untested assumptions that (1) permeability to the ion pair is pair is always the rate-limiting step in swelling, and (2) permeability to the ion pair is equivalent to the driving force for water influx. We have conducted experiments addressed to the question of mitochondrial permeability by methods (tracer exchange diffusion) which are independent of volume changes. Our findings indicate that the mitochondrial membrane is very readily penetrated by alkali metal chloride and bromide salts. Further, we have concluded that the resistance to swelling in such media is associated with a lack of driving force.     

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 of²⁰⁴Tl (less than 100 µM) 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°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, 30 mM or more, distributes across the mitochondrial inner membrane both in response to electrical fields and to pH. In energized mitochondria the uptake of Tl⁺ would occur electrophoretically, while Tl⁺/H⁺ exchange would constitute a leak. In the presence of NO ₃ ^– , the movements of Tl⁺ are determined by that of NO ₃ ^– , 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⁺.     

Rapid release of Mg(2+) from liver mitochondria by nonesterified long-chain fatty acids in alkaline media

Long-chain fatty acids induce a rapid release of Mg(2+) from both energized and nonenergized rat liver mitochondria suspended at pH 8 in isotonic saline but not sucrose media. The effect is observed only with fatty acids that possess protonophoric activity. The most active saturated fatty acids are myristic and palmitic, while the most active unsaturated acids are oleic, linolenic, and arachidonic. The rate of Mg(2+) release drastically decreases with decreasing medium pH to 7.2-7.6. However, at those pH values this rate is doubled by energization of mitochondria with respiratory substrates. Mg(2+) release is accompanied by cyclosporin A-insensitive large-amplitude swelling of mitochondria. This swelling is similar to that produced by the divalent metal ionophore A23187 and is interpreted as being due to activation of the inner membrane anion channel, the K(+) uniporter, and the K(+)/H(+) exchanger. In energized mitochondria, both swelling and Mg(2+) release are blocked by the exogenous K(+)/H(+) exchanger nigericin. It is proposed that fatty acids under conditions of alkaline mitochondrial matrix activate latent Mg(2+)-sensitive ion-conducting pathways in the inner mitochondrial membrane, which mediate swelling and Mg(2+) release. It is hypothesized that fatty acids activate an intrinsic Mg(2+)/H(+) exchanger that is related to, or identical with, the K(+)/H(+) exchanger.     

An analysis of the binding of fluorescence probes in mitochondrial systems

Summary Measurements of the binding of the fluorescent probes 8-anilinonaphthalene-1-sulfonate (ANS) and ethidium ions to whole and disrupted mitochondria and submitochondrial particles suggest that the inner mitochondrial membrane is freely permeable to the two probes. Equations relating the binding of permeant probes to the electro-chemical balance across the membrane of vesicular systems are derived and these equations used to analyze Scatchard plots of the binding of the two probes to energized and nonenergized mitochondria and EDTA particles.     

Adenine nucleotide translocase as a site of regulation by ADP of the rat liver mitochondria permeability to H+ and K+ lons

In the presence of oligomycin ADP inhibits the osmotic swelling of the nonenergized rat liver mitochondria in the NH₄NO₃ medium. With the energized mitochondria ADP enhances contraction of the mitochondria swollen in the NH₄NO₃ medium. Carboxyatractyloside and atractyloside abolish or prevent the effects of ADP. The direct measurements of the proton conductance of rat liver mitochondria shows that the inhibitory action of ADP + oligomycin on the H⁺ permeability does not depend on the energization of mitochondria. In these experiments the local anesthetic nupercaine and ADP additively inhibit the inner membrane conductance for protons, but carboxyatractyloside abolishes only the effect of ADP. In the presence of oligomycin ADP also inhibits the osmotic swelling of the nonenergized liver mitochondria in the KNO₃ medium, and the energy-dependent swelling of rat liver mitochondria in the medium with K⁺ ions and P_i. The inhibition by ADP of the membrane passive permeability for K⁺ is also sensitive to carboxyatractyloside. It is concluded that rat liver mitochondria possess an ADP-regulated channel for H⁺ and K⁺. The properties of this pathway for protons and potassium ions favor the idea that ADP regulates the mitochondrial permeability via adenine nucleotide translocase. It is assumed that the adenine nucleotides carrier should operate according to the “gated pore” mechanism.     

Long-chain fatty acid-promoted swelling of mitochondria: further evidence for the protonophoric effect of fatty acids in the inner mitochondrial membrane

Swelling of non-respiring rat liver mitochondria suspended in isotonic potassium acetate at pH 6.5-7.4 in the presence of valinomycin was promoted by long-chain fatty acids, such as myristate, indicating a protonophoric mechanism. This swelling was partly inhibited by inhibitors or substrates of mitochondrial anion carriers. The results show that the fatty acid cycling mechanism responsible for uncoupling of oxidative phosphorylation can also operate in the direction opposite to that originally proposed [Skulachev, V.P. (1991) FEBS Lett. 294, 158-162], i.e. the inwardly directed transfer of the fatty acid anion accompanied by outwardly directed free passage of undissociated fatty acid. They also extend the list of mitochondrial anion carriers, that are involved in this process, over the mono- and tricarboxylate transporters. At pH 8, myristate, but not the synthetic protonophore, p-trifluoromethoxycarbonyl-cyanide phenylhydrazone, induced mitochondrial swelling in both potassium acetate and KCl media, that did not require the presence of valinomycin. This indicates that, at alkaline pH, myristate facilitates permeation of the inner mitochondrial membrane to monovalent cations and, possibly, activates the inner membrane anion channel.     

Effective diameter of pores in rat liver mitochondria inner membrane and selectivity of transport of monovalent cations with a submicromolar concentration of Ca2+ ions or A23187 and EDTA]

Studies of swelling of rat liver mitochondria in isoosmotic solutions of nonelectrolytes in the presence of respiration inhibitors revealed that submicromolar concentrations of Ca2+ increase the diameter of pores in the inner mitochondrial membrane--from 5.5-6.0 A (10(-8) M Ca2+) up to 7.5 A (3 x 10(-7) M Ca2+) and 8.0-8.5 A (6 x 10(-7) M Ca2+); these increases are prevented by cyclosporin A. The inner mitochondrial membrane with an effective pore diameter of 7.5 A is readily permeable for potassium but not for sodium ions, although with an increase in the effective pore diameter up to 8.0-8.5 A the selectivity of the K(+)-Na+ channel decreases. A conclusion is drawn that in the presence of submicromolar concentrations of Ca2+ the conductivity of the cyclosporin-sensitive pore for monovalent cations increases in the following order: K > Na > Li.     

A comparative study of the effects of Pr<Superscript>3+</Superscript> and La<Superscript>3+</Superscript> ions on calcium dependent processes in frog cardiac muscle and rat heart mitochondria

The inotropic effect of Pr³⁺ and La³⁺ ions on the heart muscle of frog Rana ridibunda, as well as the influence of the ions on respiration, swelling, and the potential (ΔΨ_mito) on the inner membrane of Ca²⁺- loaded rat heart mitochondria, energized by glutamate and malate or succinate in the presence of rotenone were studied. It was found that 2 mM Pr³⁺ in Ringer’s solution reduces the force of spontaneous contractions and those induced by electrical stimulation in the heart; it had a negative chronotropic effect, decreasing the frequency of spontaneous contractions. Pr³⁺ and La³⁺ prevented a decrease in the 2,4-dinitrophenol (DNP)- uncoupled respiration of energized rat heart mitochondria, swelling of these organelles in salt media, and a reduction in ΔΨ_mito on the inner mitochondrial membrane that were induced by Ca²⁺ ions. Retardation by Pr³⁺ and La³⁺ ions of these calcium-induced effects may suggest that in the inner mitochondrial membrane these metals inhibit the opening of the mitochondrial permeability transition pore caused by Ca²⁺ overload of mitochondria. The data we obtained are important for a better understanding of the mechanisms of the damaging action of rare-earth elements on Ca²⁺-dependent processes in the vertebrate myocardium.     

Inorganic phosphate stimulates the toxic effects of Tl+ in rat liver mitochondria

We studied action of inorganic phosphate (P(i)) on toxic effects of Tl+ in isolated rat liver mitochondria. This is a convenient model to study the toxicity of heavy metals. P(i) 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 P(i). The mitochondrial permeability transition pore in the medium containing Ca2+, TlNO3, and nitrates of univalent cations was distinctly stimulated by P(i). However, P(i) 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 P(i). We suggested that stimulation by P(i) 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 P(i) and Ca2+.     

Effect of various ions, pH, and osmotic pressure on oxidation of elemental sulfur by Thiobacillus thiooxidans.

The oxidation of elemental sulfur by Thiobacillus thiooxidans was studied at pH 2.3, 4.5, and 7.0 in the presence of different concentrations of various anions (sulfate, phosphate, chloride, nitrate, and fluoride) and cations (potassium, sodium, lithium, rubidium, and cesium). The results agree with the expected response of this acidophilic bacterium to charge neutralization of colloids by ions, pH-dependent membrane permeability of ions, and osmotic pressure.     

Effects of the local anesthetic bupivacaine on oxidative phosphorylation in mitochondria. Change from decoupling to uncoupling by formation of a leakage type ion pathway specific for H+ in cooperation with hydrophobic anions

The effects of the local anesthetic bupivacaine on the oxidative phosphorylation in rat liver mitochondria were examined. Bupivacaine caused a maximum of about 7-fold stimulation of state 4 respiration at about 3 mM, released oligomycin-inhibited state 3 respiration, and activated ATPase to a similar extent to that by the weakly acidic uncoupler SF 6847. These effects were greatly enhanced by the addition of certain hydrophobic anions such as 1-anilino-8-naphthalenesulfonate, tetraphenyl borate, and picrate. In the absence of these anions, bupivacaine did not increase the proton conductance in either energized or nonenergized mitochondrial membranes or in artificial bilayer lipid membranes and did not have any effect on the proton motive force. However, it greatly enhanced the proton conductivity of these membrane systems and collapsed the proton motive force in the presence of hydrophobic anions. The results of noise analysis of artificial lipid bilayer membranes indicated that an ion pair complex of bupivacaine with hydrophobic anions formed a leakage-type ion pathway. Thus it is concluded that bupivacaine acts as a decoupler in the absence of added hydrophobic anions but in cooperation with certain anions as an uncoupler of oxidative phosphorylation due to formation of a H(+)-specific pathway in the membranes.     

On the stabilization by fixation of configurational states in beef heart mitochondria

The energized configuration of the cristal membrane of beef heart mitochondria can be maintained only as long as oxygen is available for electron transfer. When the oxygen supply is exhausted, the membrane undergoes a transition to the nonenergized configuration. Since the exhaustion of the available oxygen supply is complete in 5–20 sec, it is impossible to apply the method of sedimenting the mitochondria prior to fixation for studying the energized configurational states of mitochondria. The direct addition of glutaraldehyde followed by osmium tetroxide to the mitochondrial suspension is the most effective way of freezing the configurational state of the cristal membrane. Fixation with glutaraldehyde appears to be complete within 1–2 sec even at 0°. Osmium tetroxide alone can also freeze the energized configuration by fixation but the concentration of the fixative is critical. The problem of capturing the configurational state applies not only to energized transitions (nonenergized to energized) but also to nonenergized transitions (orthodox to aggregated). The freezing by fixation of the cristal membrane in the aggregated configuration is best accomplished by the sequential use of glutaraldehyde and osmium tetroxide. When the levels of glutaraldehyde and osmium tetroxide are respectively too low or too high, the mitochondrion will undergo a transition from the aggregated to the orthodox configuration before fixation is complete. Light-scattering studies provide an independent method for monitoring configurational changes in mitochondria; these light-scattering measurements confirm that the conditions for fixation which lead to stabilization of the energized state as judged by electron microscopy, also show maintenance of configuration as judged by absence of light-scattering changes after the fixatives are introduced. Reagents used in negative staining will induce the geometrical form of the energized configuration of the mitochondrion even under nonenergizing conditions. These reagents are thus unsuitable for use in studies of configurational transitions in mitochondria.     

Understanding ion transport by quantification of mitochondrial swelling

Suspensions of mitochondria are turbid and scatter light. An increase in the matrix volume (swelling) due to the influx of permeable solutes results in a decrease in the amount of light scattered. This property can be used to study solute fluxes across the mitochondrial inner membrane. A rapid method for isolating mitochondria is presented along with three swelling experiments using energized and non-energized mitochondria to illustrate ion transport across energy transducing membranes.     

Effects of Tl<Superscript>+</Superscript> on ion permeability,membrane potential and respiration of isolated rat liver mitochondria

It is known that permeability of the inner mitochondrial membrane is low to most univalent cations (K⁺, Na⁺, H⁺) but high to Tl⁺. Swelling, state 4, state 3, and 2,4-dinitrophenol (DNP)-stimulated respiration as well as the membrane potential (ΔΨ_mito) of rat liver mitochondria were studied in media containing 0–75 mM TlNO₃ either with 250 mM sucrose or with 125 mM nitrate salts of other monovalent cations (KNO₃, or NaNO₃, or NH₄NO₃). Tl⁺ increased permeability of the inner mitochondrial membrane to K⁺, Na⁺, and H⁺, that was manifested as stimulation of the swelling of nonenergized and energized mitochondria as well as via an increase of state 4 and dissipation of ΔΨ_mito. These effects of Tl⁺ increased in the order of sucrose <K⁺ <Na⁺ ≤ NH₄⁺. They were stimulated by inorganic phosphate and decreased by ADP, Mg²⁺, and cyclosporine A. Contraction of energized mitochondria, swollen in the nitrate media, was markedly inhibited by quinine. It suggests participation of the mitochondrial K⁺/H⁺ exchanger in extruding of Tl⁺-induced excess of univalent cations from the mitochondrial matrix. It is discussed that Tl⁺ (like Cd²⁺ and other heavy metals) increases the ion permeability of the inner membrane of mitochondria regardless of their energization and stimulates the mitochondrial permeability transition pore in low conductance state. The observed decrease of state 3 and DNP-stimulated respiration in the nitrate media resulted from the mitochondrial swelling rather than from an inhibition of respiratory enzymes as is the case with the bivalent heavy metals.     

Interaction of electron leak and proton leak in respiratory chain of mitochondria——Proton leak induced by superoxide from an electron leak pathway of univalent reduction of oxygen

By incubating the isolated rat myocardial mitochondria with xanthine-xanthine oxidase, anexogenous superoxide (O2) generating system, and by ischemia-reperfusion procedure of isolated rat heart as an endogenous O2 generating system, it was found that both sources of O2 showed the same injurious effects on mitochondrial function resulting in (i) increasing proton leak rate, lowering proton pumping activity and Ht/2e ratio of respiratory chain, and (ii) decreasing transmembrane potential of energized mitochondria] inner membrane by succinate oxidation. The injurious effects of O2 on these mitochondrial bioenergitical parameters mentioned above exhibited a dosage- or reaction time-dependent mode. (X has no effects on the electron transfer activity and transmembrane potential of nonenergized mitochondria. Being a superoxide scavenger, 3, 4-dihydroxylphenyl lactate showed obvious protection effects against damage of both exogenous superoxide sources from xanthine-xanthine oxidase system and endogenous Or sou     

The effects of lactose on human erythrocytes

Human erythrocytes suspended in isotonic lactose solution lost potassium and continued to lose potassium even when resuspended in isotonic sodium chloride. The same phenomenon was observed when the cells were suspended in an isotonic solution of the sodium salt of glutamate, a nonpenetrating anion. The presence of 5 mEq per liter of sodium chloride in the lactose or sodium glutamate suspensions greatly reduced the initial potassium loss and the potassium loss when the cells were resuspended in sodium chloride solution. Salts of nonpenetrating anions were less or not effective in blocking lactose damage. The results indicate that absence of penetrating anions in the suspending media is the initiating condition of lactose damage. Chloride and consequently potassium are lost from the erythrocyte. Changes in cellular ionic pattern and/or changes in the cell membrane result in a nontransient damage manifested by continued potassium loss by lactose-treated cells resuspended in isotonic NaCl.     

Permeability Properties of the Inner Membrane of Mung Bean Mitochondria and Changes during Energization

The permeability properties of the inner membrane of mung bean mitochondria were studied by osmotic swelling techniques. Rapid mitochondrial swelling was observed in isotonic ammonium phosphate, which indicated that an active phosphate/hydroxyl antiporter was present. The phosphate carrier was specifically inhibited by sulfhydryl reagents. Mitochondria did not swell in isotonic ammonium salts of malate, succinate, or fumarate, either in the presence or absence of 10 millimolar phosphate. Additionally, no swelling was observed in ammonium citrate upon addition of malate plus phosphate. Consequently, no evidence was obtained with the osmotic swelling technique for a coupled exchange of phosphate for dicarboxylic acids across the membrane.     

Effect of Various Ions, pH, and Osmotic Pressure on Oxidation of Elemental Sulfur by Thiobacillus thiooxidans

The oxidation of elemental sulfur by Thiobacillus thiooxidans was studied at pH 2.3, 4.5, and 7.0 in the presence of different concentrations of various anions (sulfate, phosphate, chloride, nitrate, and fluoride) and cations (potassium, sodium, lithium, rubidium, and cesium). The results agree with the expected response of this acidophilic bacterium to charge neutralization of colloids by ions, pH-dependent membrane permeability of ions, and osmotic pressure.     

The inhibition of mitochondrial energized processes by fluorescein mercuric acetate

Fluorescein mercuric acetate (FMA) has been shown to be a potent inhibitor of energized processes in both beef heart mitochondria and ETP_H particles. FMA reacts preferentially with a small number of specific sulfur atoms and inhibits the phosphate-dependent configurational transition. FMA enhances the anaerobic to aerobic pH changes observed in intact mitochondria and submitochondrial particles, and also enhances nonenergized swelling in 0·15 M sodium or potassium chloride. The results are interpreted in terms of a model whereby FMA, in reacting with the mitochondrion, modifies its conformation. The resulting conformational changes which occur upon energization are therefore different from those conformational changes which would occur in the absence of FMA. The net result of this process is the inhibition of some processes (e.g., oxidative phosphorylation, ATP-³²P_i exchange, etc.) and the enhancement of other processes (the proton shift and nonenergized swelling in chloride salts).This work was supported in part by U.S. Public Health Service Program Project Grant GM-12847 and by a training grant GM-88, both from the National Institute of General Medical Sciences. Meat by-products were generously furnished by Oscar Mayer and Co., Madison, Wisconsin.